Joshua Munger

To fulfill the bioenergetic and biosynthetic demand of proliferation, T cells reprogram their metabolic pathways from fatty acid β-oxidation and pyruvate oxidation via the TCA cycle to the glycolytic, pentose-phosphate, and glutaminolytic pathways. Two of the top-ranked candidate transcription factors potentially responsible for the activation-induced T cell metabolic transcriptome, HIF1α and Myc, were induced upon T cell activation, but only the acute deletion of Myc markedly inhibited activation-induced glycolysis and glutaminolysis in T cells. Glutamine deprivation compromised activation-induced T cell growth and proliferation, and this was partially replaced by nucleotides and polyamines, implicating glutamine as an important source for biosynthetic precursors in active T cells. Metabolic tracer analysis revealed a Myc-dependent metabolic pathway linking glutaminolysis to the biosynthesis of polyamines. Therefore, a Myc-dependent global metabolic transcriptome drives metabolic reprogramming in activated, primary T lymphocytes. This may represent a general mechanism for metabolic reprogramming under patho-physiological conditions.

Munger et al. show that infection with human cytomegalovirus upregulates fatty acid biosynthesis and that pharmacological inhibition of this pathway inhibits replication of both this virus and influenza A. This approach, the first to reliably map major carbon fluxes in mammalian cells, extends the promise of metabolomics from diagnostic applications to identification of new therapeutic concepts. Viruses rely on the metabolic network of their cellular hosts to provide energy and building blocks for viral replication. We developed a flux measurement approach based on liquid chromatography–tandem mass spectrometry to quantify changes in metabolic activity induced by human cytomegalovirus (HCMV). This approach reliably elucidated fluxes in cultured mammalian cells by monitoring metabolome labeling kinetics after feeding cells 13C-labeled forms of glucose and glutamine. Infection with HCMV markedly upregulated flux through much of the central carbon metabolism, including glycolysis. Particularly notable increases occurred in flux through the tricarboxylic acid cycle and its efflux to the fatty acid biosynthesis pathway. Pharmacological inhibition of fatty acid biosynthesis suppressed the replication of both HCMV and influenza A, another enveloped virus. These results show that fatty acid synthesis is essential for the replication of two divergent enveloped viruses and that systems-level metabolic flux profiling can identify metabolic targets for antiviral therapy.

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting (13)C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments.

Mutations in isocitrate dehydrogenases IDH1 and IDH2 are common in human gliomas and acute myeloid leukaemias; here, mice that carry the IDH1(R132H) mutation are described, in a new model that should help in investigating the links between mutant IDH1 and leukaemia. Mutations in the IDH1 and IDH2 genes, which encode isocitrate dehydrogenases, are frequently found in human glioblastomas and acute myeloid leukaemias. These mutations drive the synthesis of the metabolite R-2-hydroxyglutarate (2HG), which inhibits enzymes that regulate levels of DNA and histone methylation. Here, Tak Mak and colleagues characterize conditional knock-in mice of the most common IDH1 mutation, IDH1–R132H, expressed in haematopoietic cells. The mutant mice have increased numbers of early haematopoietic progenitors and develop splenomegaly, anaemia and extramedullary haematopoiesis. Furthermore, cells exhibit changes in patterns of DNA and histone methylation that are similar to those observed in human IDH1/2-mutant acute myeloid leukaemias. This mouse model should be useful for the study of mechanistic links between mutant IDH1 and leukaemia. Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas1 and cytogenetically normal acute myeloid leukaemias (AML)2. These alterations are gain-of-function mutations in that they drive the synthesis of the ‘oncometabolite’ R-2-hydroxyglutarate (2HG)3. It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.

Viral replication requires energy and macromolecular precursors derived from the metabolic network of the host cell. Despite this reliance, the effect of viral infection on host cell metabolic composition remains poorly understood. Here we applied liquid chromatography-tandem mass spectrometry to measure the levels of 63 different intracellular metabolites at multiple times after human cytomegalovirus (HCMV) infection of human fibroblasts. Parallel microarray analysis provided complementary data on transcriptional regulation of metabolic pathways. As the infection progressed, the levels of metabolites involved in glycolysis, the citric acid cycle, and pyrimidine nucleotide biosynthesis markedly increased. HCMV-induced transcriptional upregulation of specific glycolytic and citric acid cycle enzymes mirrored the increases in metabolite levels. The peak levels of numerous metabolites during infection far exceeded those observed during normal fibroblast growth or quiescence, demonstrating that HCMV markedly disrupts cellular metabolic homeostasis and institutes its own specific metabolic program.

Numerous viruses modulate host-cell metabolic processes to ensure successful infection. The host-cell metabolic network contributes the energy, precursors, and specialized components necessary to produce infectious virions. Viruses deploy host-cell metabolic activities to organize viral maturation compartments. Metabolic control is a host–pathogen interaction that can sway the outcome of viral infection. Host cells possess the metabolic assets required for viral infection. Recent studies indicate that control of the host's metabolic resources is a core host–pathogen interaction. Viruses have evolved mechanisms to usurp the host's metabolic resources, funneling them towards the production of virion components as well as the organization of specialized compartments for replication, maturation, and dissemination. Consequently, hosts have developed a variety of metabolic countermeasures to sense and resist these viral changes. The complex interplay between virus and host over metabolic control has only just begun to be deconvoluted. However, it is clear that virally induced metabolic reprogramming can substantially impact infectious outcomes, highlighting the promise of targeting these processes for antiviral therapeutic development. Host cells possess the metabolic assets required for viral infection. Recent studies indicate that control of the host's metabolic resources is a core host–pathogen interaction. Viruses have evolved mechanisms to usurp the host's metabolic resources, funneling them towards the production of virion components as well as the organization of specialized compartments for replication, maturation, and dissemination. Consequently, hosts have developed a variety of metabolic countermeasures to sense and resist these viral changes. The complex interplay between virus and host over metabolic control has only just begun to be deconvoluted. However, it is clear that virally induced metabolic reprogramming can substantially impact infectious outcomes, highlighting the promise of targeting these processes for antiviral therapeutic development. Viruses are obligate parasites that depend on the host cell to provide the energy and molecular precursors necessary for successful infection. A wide variety of evolutionarily divergent viruses have evolved mechanisms that target the host cell metabolic network as part of their infectious programs, and virally induced metabolic activities are commonly exploited for therapeutic intervention. For example, numerous different nucleotide metabolic activities are targeted by a variety of pharmaceuticals to treat viral infections, including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), varicella-zoster Virus (VZV), and herpes simplex virus (HSV) (Table 1) [1Das K. Arnold E. HIV-1 reverse transcriptase and antiviral drug resistance. Part 1.Curr. Opin. Virol. 2013; 3: 111-118Crossref PubMed Scopus (118) Google Scholar, 2Menendez-Arias L. et al.Nucleoside/nucleotide analog inhibitors of hepatitis B virus polymerase: mechanism of action and resistance.Curr. Opin. Virol. 2014; 8: 1-9Crossref PubMed Scopus (124) Google Scholar, 3Lee W.A. Martin J.C. Perspectives on the development of acyclic nucleotide analogs as antiviral drugs.Antivir. Res. 2006; 71: 254-259Crossref PubMed Scopus (62) Google Scholar, 4Andrei G. et al.Novel inhibitors of human CMV.Curr. Opin. Investig. Drugs. 2008; 9: 132-145PubMed Google Scholar, 5Li H.C. Lo S.Y. Hepatitis C virus: Virology, diagnosis and treatment.World J. Hepatol. 2015; 7: 1377-1389Crossref PubMed Scopus (126) Google Scholar]. In recent years, the number of metabolic activities that have been found to be important for viral infection has expanded. Further, our understanding of the viral mechanisms through which viruses usurp cellular metabolic resources has increased. Many of these viral mechanisms stimulate nutrient uptake and catabolism to support the production of viral progeny. In addition to providing the energy and biomass necessary for turning cells into productive 'virus factories', new metabolic contributions to infection have emerged. These include small-molecule enzymatic activities that organize viral maturation compartments, synthesize specialized virion components, or regulate the immunological environment (Figure 1). Such virally induced metabolic changes do not go unnoticed by the host, but rather represent a major host–pathogen interaction that can sway infectious outcomes. Collectively, recent findings have made it clear that the landscape for metabolically targeted therapeutic intervention has expanded.Table 1Nucleoside/Nucleotide-Based TherapeuticsaHIV, human immunodeficiency virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HCMV, human cytomegalovirus; HSV, herpes simplex virus; VZV, varicella-zoster virus.VirusNucleoside/Nucleotide AnalogsHIVTenovovir; Emtricitabine; Zidovudine; Abacavir; LamivudineHBVTenovovir; Lamivudine; Entecavir; TelbivudineHCVSofosbuvir; RibavirinHCMVGanciclovir; CidofovirHSVAcyclovir; ValacyclovirVZVAcyclovir; Valacyclovira HIV, human immunodeficiency virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HCMV, human cytomegalovirus; HSV, herpes simplex virus; VZV, varicella-zoster virus. Open table in a new tab A wide variety of viruses activate glycolysis, which drives the production of energy in the form of ATP, NADH, and NADPH (Figure 2). Activated glycolysis also supplies the carbon necessary for the synthesis of numerous core biomolecules, including nucleotides, lipids, amino acids and carbohydrates (Figure 2). A number of DNA viruses induce glycolysis, including Kaposi's sarcoma-associated herpesvirus (KSHV) [6Yogev O. et al.Kaposi's sarcoma herpesvirus microRNAs induce metabolic transformation of infected cells.PLoS Pathog. 2014; 10: e1004400Crossref PubMed Scopus (96) Google Scholar], HCMV [7Munger J. et al.Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy.Nat. Biotechnol. 2008; 26: 1179-1186Crossref PubMed Scopus (518) Google Scholar], adenovirus [8Thai M. et al.Adenovirus E4ORF1-induced MYC activation promotes host cell anabolic glucose metabolism and virus replication.Cell Metab. 2014; 19: 694-701Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar], human papillomavirus (HPV) and Epstein–Barr virus (EBV) [9Xiao L. et al.Targeting Epstein–Barr virus oncoprotein LMP1-mediated glycolysis sensitizes nasopharyngeal carcinoma to radiation therapy.Oncogene. 2014; 33: 4568-4578Crossref PubMed Scopus (154) Google Scholar]. Multiple RNA viruses also activate glycolytic flux, including dengue [10Fontaine K.A. et al.Dengue virus induces and requires glycolysis for optimal replication.J. Virol. 2015; 89: 2358-2366Crossref PubMed Scopus (219) Google Scholar], hepatitis C (HCV), [11Diamond D.L. et al.Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics.PLoS Pathog. 2010; 6: e1000719Crossref PubMed Scopus (337) Google Scholar] and influenza A [12Ritter J. et al.Metabolic effects of influenza virus infection in cultured animal cells: Intra- and extracellular metabolite profiling.BMC Syst. Biol. 2010; 4: 61Crossref PubMed Scopus (169) Google Scholar]. Although there are some notable exceptions, such as herpes simplex virus-1 (HSV-1) and vaccinia virus [13Fontaine K.A. et al.Vaccinia virus requires glutamine but not glucose for efficient replication.J. Virol. 2014; 88: 4366-4374Crossref PubMed Scopus (122) Google Scholar, 14Vastag L. et al.Divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism.PLoS Pathog. 2011; 7: e1002124Crossref PubMed Scopus (266) Google Scholar], both the number and evolutionary diversity of viruses that target glycolysis speak to the broad importance of this pathway for viral infection. Recently, the specific viral mechanisms targeting glycolysis have begun to be elucidated. For instance, the HCV NS5A protein has been shown to bind and activate hexose kinase, a rate-controlling glycolytic enzyme [15Ramière C. et al.Hexokinase activity is increased by its interaction with Hepatitis C virus protein NS5A.J. Virol. 2014; 88: 3246-3254Crossref PubMed Scopus (92) Google Scholar]. KSHV employs specific viral microRNAs targeting known regulators of glucose metabolism and mitochondrial biogenesis to induce glycolytic activity [6Yogev O. et al.Kaposi's sarcoma herpesvirus microRNAs induce metabolic transformation of infected cells.PLoS Pathog. 2014; 10: e1004400Crossref PubMed Scopus (96) Google Scholar]. HCMV has been shown to induce the activity of the AMP-activated kinase (AMPK) [16McArdle J. et al.HCMV Targets the metabolic stress response through activation of AMPK whose activity is important for viral replication.PLoS Pathog. 2012; 8: e1002502Crossref PubMed Scopus (90) Google Scholar], which regulates numerous glycolytic activities [17Towler M.C. Hardie D.G. AMP-activated protein kinase in metabolic control and insulin signaling.Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1099) Google Scholar]. HCMV-mediated activation of AMPK was found to be necessary for glycolytic activation and high-titer infection [16McArdle J. et al.HCMV Targets the metabolic stress response through activation of AMPK whose activity is important for viral replication.PLoS Pathog. 2012; 8: e1002502Crossref PubMed Scopus (90) Google Scholar]. HCMV has also been shown to induce a pro-glycolytic transcriptional program through activation of a host transcription factor, chREBP, which induces the expression of numerous glycolytic enzymes, and is important for virally mediated activation of glycolysis [18Yu Y. et al.ChREBP, a glucose-responsive transcriptional factor, enhances glucose metabolism to support biosynthesis in human cytomegalovirus-infected cells.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 1951-1956Crossref PubMed Scopus (60) Google Scholar]. Glycolytic carbon can enter the tricarboxylic acid (TCA) cycle, the reactions of which provide additional energy as well as metabolic precursors that feed biosynthesis of amino acids and fatty acids (Figure 2). Many of the viruses that activate glycolysis also induce increased concentrations of TCA cycle components during infection. HCMV activates both the TCA cycle and glycolysis simultaneously, using glycolytic carbon to feed the TCA cycle and ultimately produce fatty acids that are important for infection [7Munger J. et al.Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy.Nat. Biotechnol. 2008; 26: 1179-1186Crossref PubMed Scopus (518) Google Scholar]. Similarly to HCMV, vaccinia virus induces increased glutamine catabolism [13Fontaine K.A. et al.Vaccinia virus requires glutamine but not glucose for efficient replication.J. Virol. 2014; 88: 4366-4374Crossref PubMed Scopus (122) Google Scholar], and both HCMV and vaccinia are dependent on increased glutamine catabolism for high-titer infection [13Fontaine K.A. et al.Vaccinia virus requires glutamine but not glucose for efficient replication.J. Virol. 2014; 88: 4366-4374Crossref PubMed Scopus (122) Google Scholar, 19Chambers J.W. et al.Glutamine metabolism is essential for human cytomegalovirus infection.J. Virol. 2010; 84: 1867-1873Crossref PubMed Scopus (185) Google Scholar] (Figure 2). In contrast, HSV-1 does not substantially impact glycolysis, but induces pyruvate carboxylation to anaplerotically replenish TCA cycle metabolites while diverting other TCA intermediates towards pyrimidine biosynthesis [14Vastag L. et al.Divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism.PLoS Pathog. 2011; 7: e1002124Crossref PubMed Scopus (266) Google Scholar]. In comparison to glycolysis, less is known about how various viral infections impact specific TCA cycle metabolic fluxes. Measuring TCA cycle metabolic activity is difficult due to the number of metabolic fluxes that flow into and out of the cycle. Additionally, many TCA intermediates are compartmentalized into both cytoplasmic and mitochondrial pools, further complicating metabolic inquiry. Mitochondrial physiology plays an important role in TCA cycle function, and while not covered in detail here, a number of viruses have been implicated in targeting mitochondrial dynamics (reviewed in [20Khan M. et al.Mitochondrial dynamics and viral infections: A close nexus.Biochim. Biophys. Acta. 2015; (Published online January 13 2015)https://doi.org/10.1016/j.bbamcr.2014.12.040Crossref Scopus (124) Google Scholar]). As stated above, KSHV encodes targeted microRNAs that negatively impact mitochondrial biogenesis and activity, potentially through an interaction with heat shock protein HSPA9 [21Yogev O. et al.Kaposi's sarcoma herpesvirus microRNAs induce metabolic transformation of infected cells.Plos Pathog. 2014; 10: e1004400Crossref PubMed Scopus (0) Google Scholar].HBV and HCV promote mitochondrial fission and mitophagy through dynamin-related protein 1 (Drp1), which downregulates apoptosis and may enhance viral persistence [22Kim S.J. et al.Hepatitis C virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 6413-6418Crossref PubMed Scopus (208) Google Scholar, 23Kim S.J. et al.Hepatitis B virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis.PLoS Pathog. 2013; 9: e1003722Crossref PubMed Scopus (226) Google Scholar]. The HCMV UL37 protein also disrupts the reticular mitochondrial network and blocks mitochondrial apoptotic signaling [24Arnoult D. et al.Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7988-7993Crossref PubMed Scopus (178) Google Scholar]. While many of these activities are anti-apoptotic, with clear pro-viral consequences, they would also be predicted to have substantial effects on mitochondrial metabolism, including TCA cycle activity and cellular energetics, which are likely also important for viral infection. However, it is largely unclear how the viral proteins that target mitochondria dynamics impact core mitochondrial metabolic function. TCA-cycle-derived citrate is transported out of the mitochondria to supply carbon for fatty acid biosynthesis, which ultimately supports lipid biosynthesis (Figure 2). A number of DNA viruses, including HCMV and vaccinia, induce lipid biosynthesis, which is necessary for their high-titer infections [7Munger J. et al.Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy.Nat. Biotechnol. 2008; 26: 1179-1186Crossref PubMed Scopus (518) Google Scholar, 25Spencer C.M. et al.Human cytomegalovirus induces the activity and expression of acetyl-coenzyme a carboxylase, a fatty acid biosynthetic enzyme whose inhibition attenuates viral replication.J. Virol. 2011; 85: 5814-5824Crossref PubMed Scopus (85) Google Scholar, 26Greseth M.D. Traktman P. De novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection.PLoS Pathog. 2014; 10: e1004021Crossref PubMed Scopus (96) Google Scholar]. HCMV activates this pathway in part by inducing the expression and activity of acetyl-CoA carboxylase (ACC1) [25Spencer C.M. et al.Human cytomegalovirus induces the activity and expression of acetyl-coenzyme a carboxylase, a fatty acid biosynthetic enzyme whose inhibition attenuates viral replication.J. Virol. 2011; 85: 5814-5824Crossref PubMed Scopus (85) Google Scholar], a rate-controlling enzyme of fatty acid biosynthesis. Inhibition of ACC1 or fatty acid synthase (FASN) attenuates HCMV replication at a late stage of infection without impacting viral protein accumulation [7Munger J. et al.Systems-level metabolic flux profiling identifies fatty acid synthesis as a target for antiviral therapy.Nat. Biotechnol. 2008; 26: 1179-1186Crossref PubMed Scopus (518) Google Scholar, 25Spencer C.M. et al.Human cytomegalovirus induces the activity and expression of acetyl-coenzyme a carboxylase, a fatty acid biosynthetic enzyme whose inhibition attenuates viral replication.J. Virol. 2011; 85: 5814-5824Crossref PubMed Scopus (85) Google Scholar]. The late timing of this defect is consistent with a role for fatty acid biosynthesis in viral assembly or envelopment. During HCMV infection, the induction of fatty acid biosynthetic enzymes is mediated by viral activation of the sterol regulatory element binding proteins (SREBPs) 1 and 2 [25Spencer C.M. et al.Human cytomegalovirus induces the activity and expression of acetyl-coenzyme a carboxylase, a fatty acid biosynthetic enzyme whose inhibition attenuates viral replication.J. Virol. 2011; 85: 5814-5824Crossref PubMed Scopus (85) Google Scholar, 27Yu Y. et al.Human cytomegalovirus infection induces adipocyte-like lipogenesis through activation of sterol regulatory element binding protein 1.J. Virol. 2012; 86: 2942-2949Crossref PubMed Scopus (57) Google Scholar], transcription factors that control the expression of fatty acid metabolic genes. Vaccinia infection is also sensitive to ACC1 or FASN inhibition. Pharmacological inhibition of either ACC or FASN strongly reduces viral titers and inhibits virion envelopment in a manner that could be partially rescued by the addition of exogenous palmitate, the key product of fatty acid biosynthesis [26Greseth M.D. Traktman P. De novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection.PLoS Pathog. 2014; 10: e1004021Crossref PubMed Scopus (96) Google Scholar]. Various RNA viruses also target fatty acid biosynthesis. Many studies have implicated the importance of lipid metabolism during HCV infection, which are summarized in more detail in the following reviews [28Herker E. Ott M. Unique ties between hepatitis C virus replication and intracellular lipids.Trends Endocrinol. Metab. 2011; 22: 241-248Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 29Syed G.H. et al.Hepatitis C virus hijacks host lipid metabolism.Trends Endocrinol. Metab. 2010; 21: 33-40Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar]. Rotavirus (RV) replication has also been shown to be susceptible to inhibitors targeting various lipid synthetic enzymes, and studies in the context of dengue virus infection show that the viral nonstructural protein 3 preferentially recruits and activates FASN at sites of viral replication [30Kim Y. et al.Novel triacsin C analogs as potential antivirals against rotavirus infections.Eur. J. Med. Chem. 2012; 50: 311-318Crossref PubMed Scopus (35) Google Scholar, 31Heaton N.S. et al.Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 17345-17350Crossref PubMed Scopus (403) Google Scholar]. De novo fatty acid biosynthesis and formation of lipid droplets have been found to promote tombusvirus and rhinovirus replication [32Xu K. Nagy P.D. Expanding use of multi-origin subcellular membranes by positive-strand RNA viruses during replication.Curr. Opin. Virol. 2014; 9: 119-126Crossref PubMed Scopus (50) Google Scholar]. In addition to targeting fatty acid biosynthesis, viruses have been found to target the reverse catabolic process, fatty acid oxidation. HCMV and the Japanese encephalitis virus (JEV) inhibit fatty acid oxidation by targeting the mitochondrial trifunctional protein (MTP) which catalyzes fatty acid oxidation [33Kao Y.T. et al.Japanese encephalitis virus nonstructural protein NS5 interacts with mitochondrial trifunctional protein and impairs fatty acid beta-oxidation.PLoS Pathog. 2015; 11: e1004750Crossref PubMed Scopus (35) Google Scholar, 34Seo J.Y. Cresswell P. Viperin regulates cellular lipid metabolism during human cytomegalovirus infection.PLoS Pathog. 2013; 9: e1003497Crossref PubMed Scopus (86) Google Scholar]. JEV inhibits MTP through its NS5 protein [33Kao Y.T. et al.Japanese encephalitis virus nonstructural protein NS5 interacts with mitochondrial trifunctional protein and impairs fatty acid beta-oxidation.PLoS Pathog. 2015; 11: e1004750Crossref PubMed Scopus (35) Google Scholar], while HCMV redirects the cellular viperin protein to inhibit MTP [34Seo J.Y. Cresswell P. Viperin regulates cellular lipid metabolism during human cytomegalovirus infection.PLoS Pathog. 2013; 9: e1003497Crossref PubMed Scopus (86) Google Scholar]. In contrast to these viruses that inhibit fatty acid oxidation, HCV infection requires fatty acid oxidation for its replication, which again highlights that certain aspects of viral metabolic manipulation are virus specific [35Rasmussen A.L. et al.Systems virology identifies a mitochondrial fatty acid oxidation enzyme, dodecenoyl coenzyme A delta isomerase, required for hepatitis C virus replication and likely pathogenesis.J. Virol. 2011; 85: 11646-11654Crossref PubMed Scopus (45) Google Scholar]. Combined, the data indicate that virally mediated manipulation of lipid metabolism is broadly important to viral infection, and further, that viruses have evolved diverse mechanisms to ensure that the host-cell lipid metabolic machinery is co-opted to support viral infection. As indicated above, a number of viruses induce and rely on host-cell lipid metabolism. In many cases, the exact contributions that specific lipid metabolic enzymes make towards viral infection are unclear; however, certain themes are emerging. Increasingly, it appears that viruses are targeting host-cell lipid-modifying enzymes as a means to organize viral assembly and maturation compartments (Figure 1). This theme is readily apparent in recent work on the replication complexes formed by plus-strand RNA viruses. Dengue virus, HCV, and rhinovirus all remodel the endoplasmic reticulum (ER) to generate sites for their replication using mechanisms that rely on host lipid biosynthesis. The dengue virus NSP3 protein specifically recruits fatty acid synthase to viral replication sites, which is critical for dengue replication [31Heaton N.S. et al.Dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 17345-17350Crossref PubMed Scopus (403) Google Scholar, 36Tang W.C. et al.Rab18 facilitates dengue virus infection by targeting fatty acid synthase to sites of viral replication.J. Virol. 2014; 88: 6793-6804Crossref PubMed Scopus (86) Google Scholar]. For HCV, phosphatidylinositol 4-kinase III alpha (PI4KA) is an important host factor for the formation of replication compartments [37Berger K.L. et al.Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication.J. Virol. 2011; 85: 8870-8883Crossref PubMed Scopus (155) Google Scholar]. HCV stimulates PI4KA activity, likely through its interaction with the HCV NS5A, which induces accumulation of phosphatidylinositol 4-phosphate (PI4P) in the ER and allows for productive replication [37Berger K.L. et al.Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication.J. Virol. 2011; 85: 8870-8883Crossref PubMed Scopus (155) Google Scholar]. Similarly, rhinovirus replication requires redistribution of PI4P and cholesterol in ER and Golgi membranes [38Roulin P.S. et al.Rhinovirus uses a phosphatidylinositol 4-phosphate/cholesterol counter-current for the formation of replication compartments at the ER-Golgi interface.Cell Host Microbe. 2014; 16: 677-690Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar]. Despite the similar lipid requirements of these related viruses, their respective ER-derived replication compartments are each unique in both conformation and size [39Chatel-Chaix L. Bartenschlager R. Dengue virus- and hepatitis C virus-induced replication and assembly compartments: the enemy inside--caught in the web.J. Virol. 2014; 88: 5907-5911Crossref PubMed Scopus (105) Google Scholar]. Further investigation may reveal virus-specific lipid requirements driving the formation of these structures that could be therapeutically exploitable [40Xu K. Nagy P.D. RNA virus replication depends on enrichment of phosphatidylethanolamine at replication sites in subcellular membranes.Proc. Natl. Acad. Sci. U.S.A. 2015; 112: E1782-E1791Crossref PubMed Scopus (97) Google Scholar, 41Barajas D. et al.Co-opted oxysterol-binding ORP and VAP proteins channel sterols to RNA virus replication sites via membrane contact sites.PLoS Pathog. 2014; 10: e1004388Crossref PubMed Scopus (97) Google Scholar]. Modulation of host-cell lipid metabolic activities also appears to be important for virion assembly. HCV relies on triglyceride (TAG) and cholesterol ester biosynthesis for viral assembly, as pharmaceutical inhibition of these pathways impairs this process, reducing virus infectivity [42Liefhebber J.M. et al.Modulation of triglyceride and cholesterol ester synthesis impairs assembly of infectious hepatitis C virus.J. Biol. Chem. 2014; 289: 21276-21288Crossref PubMed Scopus (38) Google Scholar]. TAG mediates the interaction of HCV nucleocapsid protein with lipid droplets and plays a critical role in the stability of HCV nucleocapsids [42Liefhebber J.M. et al.Modulation of triglyceride and cholesterol ester synthesis impairs assembly of infectious hepatitis C virus.J. Biol. Chem. 2014; 289: 21276-21288Crossref PubMed Scopus (38) Google Scholar]. Phosphatidylserine, a core phospholipid membrane constituent, has also been implicated as being important for viral infection. Ebola particles preferentially incorporate phosphatidylserine-rich membranes into their envelope [43Soni S.P. Stahelin R.V. The Ebola virus matrix protein VP40 selectively induces vesiculation from phosphatidylserine-enriched membranes.J. Biol. Chem. 2014; 289: 33590-33597Crossref PubMed Scopus (47) Google Scholar], and during enterovirus infection, specialized phosphatidylserine-rich vesicles facilitate mass transmission of multiple genomes to uninfected cells [44Chen Y.H. et al.Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses.Cell. 2015; 160: 619-630Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar]. It is apparent that virally mediated alteration of lipid-modifying metabolic activities to shape lipid compartments has emerged as an important component of diverse viral life cycles. It is becoming increasingly evident that, in addition to simply elevating the production of molecular precursors to support infection, viruses are targeting specific metabolic activities to individually tailor specialized virion components (Figure 1). For example, HCMV infection induces the expression of fatty acid elongases, which increase the concentrations of saturated very-long-chain fatty acids (VLCFAs). These VLCFAs are concentrated in the envelope of HCMV virions [45Koyuncu E. et al.Saturated very long chain fatty acids are required for the production of infectious human cytomegalovirus progeny.PLoS Pathog. 2013; 9: e1003333Crossref PubMed Scopus (78) Google Scholar, 46Purdy J.G. et al.Fatty acid elongase 7 catalyzes lipidome remodeling essential for human cytomegalovirus replication.Cell Rep. 2015; 10: 1375-1385Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar]. Pharmaceutical inhibition or RNAi-mediated knockdown of long-chain acyl-CoA synthetase or fatty acid elongase 7, key enzymes for biosynthesis of VLCFAs, attenuates HCMV replication [45Koyuncu E. et al.Saturated very long chain fatty acids are required for the production of infectious human cytomegalovirus progeny.PLoS Pathog. 2013; 9: e1003333Crossref PubMed Scopus (78) Google Scholar, 46Purdy J.G. et al.Fatty acid elongase 7 catalyzes lipidome remodeling essential for human cytomegalovirus replication.Cell Rep. 2015; 10: 1375-1385Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar]. While the exact contributions that VLFCAs make towards viral replication are unclear, it is likely that specific biophysical properties of VLCFAs contribute to aspects of virion maturation, stability, or transmission. Regardless, the findings that viral infection can selectively induce specific host-cell metabolic activities that are important for infection raise the possibility that such activities would make attractive targets for therapeutic intervention. In addition to tailoring fatty acid metabolic activities for the viral envelope, viruses target small-molecule metabolic activities to post-translationally modify viral proteins. Such modifications are diverse and include fatty-acid-based modifications such as myristoylation and palmitoylation. Myristate modification of HIV gag plays a crucial role in gag binding to the plasma membrane, an integral step that allows HIV to enter the cell [47Lalonde M.S. Sundquist W.I. How HIV finds the door.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 18631-18632Crossref PubMed Scopus (16) Google Scholar, 48Saad J.S. et al.Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11364-11369Crossref PubMed Scopus (450) Google Scholar, 49Tang C. et al.Entropic switch regulates myristate exposure in the HIV-1 matrix protein.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 517-522Crossref PubMed Scopus (276) Google Scholar]. Myristoylation of virion proteins is also important for HCMV infection. The addition of a myristoyl group to p

<h2>Summary</h2> Succinate accumulates during ischemia, and its oxidation at reperfusion drives injury. The mechanism of ischemic succinate accumulation is controversial and is proposed to involve reversal of mitochondrial complex II. Herein, using stable-isotope-resolved metabolomics, we demonstrate that complex II reversal is possible in hypoxic mitochondria but is not the primary succinate source in hypoxic cardiomyocytes or ischemic hearts. Rather, in these intact systems succinate primarily originates from canonical Krebs cycle activity, partly supported by aminotransferase anaplerosis and glycolysis from glycogen. Augmentation of canonical Krebs cycle activity with dimethyl-α-ketoglutarate both increases ischemic succinate accumulation and drives substrate-level phosphorylation by succinyl-CoA synthetase, improving ischemic energetics. Although two-thirds of ischemic succinate accumulation is extracellular, the remaining one-third is metabolized during early reperfusion, wherein acute complex II inhibition is protective. These results highlight a bifunctional role for succinate: its complex-II-independent accumulation being beneficial in ischemia and its complex-II-dependent oxidation being detrimental at reperfusion.

ABSTRACT In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

2-Hydroxyglutarate (2-HG) is an important epigenetic regulator, with potential roles in cancer and stem cell biology. The d-(R)-enantiomer (d-2-HG) is an oncometabolite generated from α-ketoglutarate (α-KG) by mutant isocitrate dehydrogenase, whereas l-(S)-2-HG is generated by lactate dehydrogenase and malate dehydrogenase in response to hypoxia. Because acidic pH is a common feature of hypoxia, as well as tumor and stem cell microenvironments, we hypothesized that pH may regulate cellular 2-HG levels. Herein we report that cytosolic acidification under normoxia moderately elevated 2-HG in cells, and boosting endogenous substrate α-KG levels further stimulated this elevation. Studies with isolated lactate dehydrogenase-1 and malate dehydrogenase-2 revealed that generation of 2-HG by both enzymes was stimulated severalfold at acidic pH, relative to normal physiologic pH. In addition, acidic pH was found to inhibit the activity of the mitochondrial l-2-HG removal enzyme l-2-HG dehydrogenase and to stimulate the reverse reaction of isocitrate dehydrogenase (carboxylation of α-KG to isocitrate). Furthermore, because acidic pH is known to stabilize hypoxia-inducible factor (HIF) and 2-HG is a known inhibitor of HIF prolyl hydroxylases, we hypothesized that 2-HG may be required for acid-induced HIF stabilization. Accordingly, cells stably overexpressing l-2-HG dehydrogenase exhibited a blunted HIF response to acid. Together, these results suggest that acidosis is an important and previously overlooked regulator of 2-HG accumulation and other oncometabolic events, with implications for HIF signaling. 2-Hydroxyglutarate (2-HG) is an important epigenetic regulator, with potential roles in cancer and stem cell biology. The d-(R)-enantiomer (d-2-HG) is an oncometabolite generated from α-ketoglutarate (α-KG) by mutant isocitrate dehydrogenase, whereas l-(S)-2-HG is generated by lactate dehydrogenase and malate dehydrogenase in response to hypoxia. Because acidic pH is a common feature of hypoxia, as well as tumor and stem cell microenvironments, we hypothesized that pH may regulate cellular 2-HG levels. Herein we report that cytosolic acidification under normoxia moderately elevated 2-HG in cells, and boosting endogenous substrate α-KG levels further stimulated this elevation. Studies with isolated lactate dehydrogenase-1 and malate dehydrogenase-2 revealed that generation of 2-HG by both enzymes was stimulated severalfold at acidic pH, relative to normal physiologic pH. In addition, acidic pH was found to inhibit the activity of the mitochondrial l-2-HG removal enzyme l-2-HG dehydrogenase and to stimulate the reverse reaction of isocitrate dehydrogenase (carboxylation of α-KG to isocitrate). Furthermore, because acidic pH is known to stabilize hypoxia-inducible factor (HIF) and 2-HG is a known inhibitor of HIF prolyl hydroxylases, we hypothesized that 2-HG may be required for acid-induced HIF stabilization. Accordingly, cells stably overexpressing l-2-HG dehydrogenase exhibited a blunted HIF response to acid. Together, these results suggest that acidosis is an important and previously overlooked regulator of 2-HG accumulation and other oncometabolic events, with implications for HIF signaling.

Metabolic reprogramming is critical to oncogenesis, but the emergence and function of this profound reorganization remain poorly understood. Here we find that cooperating oncogenic mutations drive large-scale metabolic reprogramming, which is both intrinsic to cancer cells and obligatory for the transition to malignancy. This involves synergistic regulation of several genes encoding metabolic enzymes, including the lactate dehydrogenases LDHA and LDHB and mitochondrial glutamic pyruvate transaminase 2 (GPT2). Notably, GPT2 engages activated glycolysis to drive the utilization of glutamine as a carbon source for TCA cycle anaplerosis in colon cancer cells. Our data indicate that the Warburg effect supports oncogenesis via GPT2-mediated coupling of pyruvate production to glutamine catabolism. Although critical to the cancer phenotype, GPT2 activity is dispensable in cells that are not fully transformed, thus pinpointing a metabolic vulnerability specifically associated with cancer cell progression to malignancy.

Human immunodeficiency virus type 1 (HIV-1) infects both activated CD4+ T cells and macrophages. We tested if liquid chromatography–tandem mass spectrometry (LC–MS/MS) technology can monitor metabolic alterations induced by HIV-1 in the infected cells. Here we monitored glucose uptake and conducted LC–MS/MS-based metabolomic analysis in HIV-1 infected primary human CD4+ T cells and a macrophage model system: differentiated U1 (HIV-1 producing) and differentiated U937 (control) cells. HIV-1 infected CD4+ T cells have higher glucose uptake and increases in several metabolite pool sizes, whereas HIV-1 producing macrophages had substantial reductions in glucose uptake and steady state glycolytic intermediates. This data suggests that the two HIV-1 target cell types exhibit very different metabolic outcomes during viral production. This study also validates the LC–MS/MS technology as an effective metabolomic approach to monitor various metabolic alterations made by HIV-1 infection.

We have previously reported that human cytomegalovirus (HCMV) infection induces large-scale changes to host cell glycolytic, nucleic acid, and phospholipid metabolism. Here we explore the viral mechanisms involved in fatty acid biosynthetic activation. Our results indicate that HCMV targets ACC1, the rate-limiting enzyme of fatty acid biosynthesis, through multiple mechanisms. HCMV infection was found to activate ACC1 expression, increasing the abundance of both ACC1 mRNA and protein. Viral gene expression but not viral DNA replication was found to be necessary for HCMV-mediated induction of ACC1 levels. HCMV infection was also found to increase the proteolytic processing of SREBP-2, a transcription factor whose proteolytic cleavage is known to activate a variety of phospholipid metabolic genes. Processing of SREBP-2 was found to be dependent on mTOR activity; pharmaceutical inhibition of mTOR blocked HCMV-induced SREBP-2 processing and prevented the induction of fatty acid biosynthesis and ACC1 expression. Independent of the increases in ACC1 expression, HCMV infection also induced ACC1's enzymatic activity. Inhibition of ACC1 through either RNA interference (RNAi) or inhibitor treatment was found to attenuate HCMV replication, and HCMV replication was sensitive to ACC1 inhibition even at the later stages of infection, suggesting a late role for fatty acid biosynthesis during HCMV replication. These findings indicate that HCMV infection actively modulates numerous functional aspects of a key metabolic regulatory enzyme that is important for high-titer viral replication.

Human Cytomegalovirus (HCMV) infection induces several metabolic activities that have been found to be important for viral replication. The cellular AMP-activated protein kinase (AMPK) is a metabolic stress response kinase that regulates both energy-producing catabolic processes and energy-consuming anabolic processes. Here we explore the role AMPK plays in generating an environment conducive to HCMV replication. We find that HCMV infection induces AMPK activity, resulting in the phosphorylation and increased abundance of several targets downstream of activated AMPK. Pharmacological and RNA-based inhibition of AMPK blocked the glycolytic activation induced by HCMV-infection, but had little impact on the glycolytic pathway of uninfected cells. Furthermore, inhibition of AMPK severely attenuated HCMV replication suggesting that AMPK is an important cellular factor for HCMV replication. Inhibition of AMPK attenuated early and late gene expression as well as viral DNA synthesis, but had no detectable impact on immediate-early gene expression, suggesting that AMPK activity is important at the immediate early to early transition of viral gene expression. Lastly, we find that inhibition of the Ca²⁺-calmodulin-dependent kinase kinase (CaMKK), a kinase known to activate AMPK, blocks HCMV-mediated AMPK activation. The combined data suggest a model in which HCMV activates AMPK through CaMKK, and depends on their activation for high titer replication, likely through induction of a metabolic environment conducive to viral replication.

Earlier studies have shown that the d120 mutant of herpes simplex virus 1, which lacks both copies of the alpha4 gene, induces apoptosis in all cell lines tested. In some cell lines d120-induced apoptosis, manifested by the release of cytochrome c, activation of caspase 3, and fragmentation of cellular DNA, is blocked by the overexpression of Bcl-2. In these cells viral protein kinase U(S)3 delivered in trans blocks apoptosis induced by the mutant virus at a premitochondrial stage. We report that the U(S)3 protein kinase targets the pro-apoptotic BAD member of the Bcl-2 family. Specifically, the U(S)3 protein kinase mediates a posttranslational modification of BAD and blocks its cleavage, which is reported to activate apoptosis. Thus, U(S)3 protein kinase is the sole viral protein required to block activation of caspase 3, prevent cleavage of poly(ADP-ribose) polymerase, and block fragmentation of cellular DNA induced by BAD.

Viruses depend on the host cell to provide the energy and biomolecular subunits necessary for production of viral progeny. We have previously reported that human cytomegalovirus (HCMV) infection induces dramatic changes to central carbon metabolism, including glycolysis, the tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, and nucleotide biosynthesis. Here, we explore the mechanisms involved in HCMV-mediated glycolytic activation. We find that HCMV virion binding and tegument protein delivery are insufficient for HCMV-mediated activation of glycolysis. Viral DNA replication and late-gene expression, however, are not required. To narrow down the list of cellular pathways important for HCMV-mediated [corrected] activation of glycolysis, we utilized pharmaceutical inhibitors to block pathways reported to be both involved in metabolic control and activated by HCMV infection. We find that inhibition of calmodulin-dependent kinase kinase (CaMKK), but not calmodulin-dependent kinase II (CaMKII) or protein kinase A (PKA), blocks HCMV-mediated activation of glycolysis. HCMV infection was also found to target calmodulin-dependent kinase kinase 1 (CaMKK1) expression, increasing the levels of CaMKK1 mRNA and protein. Our results indicate that inhibition of CaMKK has a negligible impact on immediate-early-protein accumulation yet severely attenuates production of HCMV viral progeny, reduces expression of at least one early gene, and blocks viral DNA replication. Inhibition of CaMKK did not affect the glycolytic activation induced by another herpes virus, herpes simplex virus type 1 (HSV-1). Furthermore, inhibition of CaMKK had a much smaller impact on HSV-1 replication than on that of HCMV. These data suggest that the role of CaMKK during the viral life cycle is, in this regard, HCMV specific. Taken together, our results suggest that CaMKK is an important factor for HCMV replication and HCMV-mediated glycolytic activation.

Significance Viruses use the host cell to provide the energy and molecular subunits to assemble viral progeny. The progeny of a variety of viral families possess envelope glycoproteins that are essential for viral infection. The production of these functional glycoproteins requires an ample supply of UDP–sugar subunits that serve as the substrates for glycosylation reactions. Our results indicate that human cytomegalovirus induces a viral metabolic program that activates pyrimidine biosynthesis to drive UDP–sugar biosynthesis. This metabolic activation is important for viral protein glycosylation and high-titer viral replication. Further, our results suggest that this metabolic link between pyrimidine and UDP–sugar biosynthesis is shared between evolutionarily diverse viral families, which may provide novel avenues for antiviral therapeutic intervention.

Human Cytomegalovirus (HCMV) infection induces several metabolic activities that are essential for viral replication. Despite the important role that this metabolic modulation plays during infection, the viral mechanisms involved are largely unclear. We find that the HCMV UL38 protein is responsible for many aspects of HCMV-mediated metabolic activation, with UL38 being necessary and sufficient to drive glycolytic activation and induce the catabolism of specific amino acids. UL38's metabolic reprogramming role is dependent on its interaction with TSC2, a tumor suppressor that inhibits mTOR signaling. Further, shRNA-mediated knockdown of TSC2 recapitulates the metabolic phenotypes associated with UL38 expression. Notably, we find that in many cases the metabolic flux activation associated with UL38 expression is largely independent of mTOR activity, as broad spectrum mTOR inhibition does not impact UL38-mediated induction of glycolysis, glutamine consumption, or the secretion of proline or alanine. In contrast, the induction of metabolite concentrations observed with UL38 expression are largely dependent on active mTOR. Collectively, our results indicate that the HCMV UL38 protein induces a pro-viral metabolic environment via inhibition of TSC2.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals.

Earlier studies have shown that the d120 mutant of herpes simplex virus 1, which lacks both copies of the alpha4 gene, induces caspase-3-dependent apoptosis in HEp-2 cells. Apoptosis was also induced by the alpha4 rescuant but was blocked by the complementation of rescuant with a DNA fragment encoding the U(S)3 protein kinase (R. Leopardi and B. Roizman, Proc. Natl. Acad. Sci. USA 93:9583-9587, 1996, and R. Leopardi, C. Van Sant, and B. Roizman, Proc. Natl. Acad. Sci. USA 94:7891-7896, 1997). To investigate its role in the apoptotic cascade, the U(S)3 open reading frame was cloned into a baculovirus (Bac-U(S)3) under the control of the human cytomegalovirus immediate-early promoter. We report the following. (i) Bac-U(S)3 blocks processing of procaspase-3 to active caspase. Procaspase-3 levels remained unaltered if superinfected with Bac-U(S)3 at 3 h after d120 mutant infection, but significant amounts of procaspase-3 remained in cells superinfected with Bac-Us3 at 9 h postinfection with d120 mutant. (ii) The U(S)3 protein kinase blocks the proapoptotic cascade upstream of mitochondrial involvement inasmuch as Bac-U(S)3 blocks release of cytochrome c in cells infected with the d120 mutant. (iii) Concurrent infection of HEp-2 cells with Bac-U(S)3 and the d120 mutant did not alter the pattern of accumulation or processing of ICP0, -22, or -27, and therefore U(S)3 does not appear to block apoptosis by targeting these proteins.

Arenaviruses merit significant interest as important human pathogens, since several of them cause severe hemorrhagic fever disease that is associated with high morbidity and significant mortality. Currently, there are no FDA-licensed arenavirus vaccines available, and current antiarenaviral therapy is limited to an off-labeled use of the nucleoside analog ribavirin, which has limited prophylactic efficacy. The pyrimidine biosynthesis inhibitor A3, which was identified in a high-throughput screen for compounds that blocked influenza virus replication, exhibits a broad-spectrum antiviral activity against negative- and positive-sense RNA viruses, retroviruses, and DNA viruses. In this study, we evaluated the antiviral activity of A3 against representative Old World (lymphocytic choriomeningitis virus) and New World (Junin virus) arenaviruses in rodent, monkey, and human cell lines. We show that A3 is significantly more efficient than ribavirin in controlling arenavirus multiplication and that the A3 inhibitory effect is in part due to its ability to interfere with viral RNA replication and transcription. We document an additive antiarenavirus effect of A3 and ribavirin, supporting the potential combination therapy of ribavirin and pyrimidine biosynthesis inhibitors for the treatment of arenavirus infections.

Abstract In addition to dopaminergic neuron loss, it is clear that Parkinson disease includes other pathological changes, including loss of additional neuronal populations. As a means of addressing multiple pathological changes with a single therapeutically‐relevant approach, we employed delayed transplantation of a unique class of astrocytes, GDA s BMP , that are generated in vitro by directed differentiation of glial precursors. GDA s BMP produce multiple agents of interest as treatments for PD and other neurodegenerative disorders, including BDNF , GDNF , neurturin and IGF 1. GDA s BMP also exhibit increased levels of antioxidant pathway components, including levels of NADPH and glutathione. Delayed GDA BMP transplantation into the 6‐hydroxydopamine lesioned rat striatum restored tyrosine hydroxylase expression and promoted behavioral recovery. GDA BMP transplantation also rescued pathological changes not prevented in other studies, such as the rescue of parvalbumin + GABA ergic interneurons. Consistent with expression of the synaptic modulatory proteins thrombospondin‐1 and 2 by GDA s BMP , increased expression of the synaptic protein synaptophysin was also observed. Thus, GDA s BMP offer a multimodal support cell therapy that provides multiple benefits without requiring prior genetic manipulation.

Viral infection frequently triggers activation of host innate immune pathways that attempt to limit viral spread. The NF-κB pathway is a critical component that governs this response. We have found that the human cytomegalovirus (HCMV) U(L)26 protein antagonizes NF-κB activation. Upon infection, an HCMV strain lacking the U(L)26 gene (ΔU(L)26) induced the nuclear translocation of the NF-κB RelB subunit and activated expression and secretion of interleukin-6 (IL-6), an NF-κB target gene. The ΔU(L)26 mutant was also more sensitive to challenge with tumor necrosis factor alpha (TNF-α), a canonical NF-κB inducer. Further, expression of U(L)26 in the absence of other viral proteins blocked NF-κB activation induced by either TNF-α treatment or infection with Sendai virus (SeV). Our results indicate that U(L)26 expression is sufficient to block TNF-α-induced NF-κB nuclear translocation and IκB degradation. Last, U(L)26 blocks TNF-α-induced IκB-kinase (IKK) phosphorylation, a key step in NF-κB activation. Combined, our results indicate that U(L)26 is part of a viral program to antagonize innate immunity through modulation of NF-κB signaling.The NF-κB signaling pathway regulates innate immunity, an integral host process that limits viral pathogenesis. Viruses have evolved mechanisms to modulate NF-κB signaling to ensure their replication. HCMV is a major cause of birth defects and disease in immunosuppressed populations. HCMV is known to actively target the NF-κB pathway, which is important for HCMV infection. Our results indicate that the HCMV U(L)26 gene is a key modulator of NF-κB pathway activity. We find the U(L)26 gene is both necessary and sufficient to block NF-κB activation upon challenge with antiviral cytokines. Further, U(L)26 attenuates the phosphorylation and activation of a key NF-κB activating kinase complex, IKK. Our study provides new insight into how HCMV targets the NF-κB pathway. Given its importance to viral infection, the mechanisms through which viruses target the NF-κB pathway highlight areas of vulnerability that could be therapeutically targeted to attenuate viral replication.

Ischemic preconditioning (IPC) protects tissues such as the heart from prolonged ischemia-reperfusion (IR) injury. We previously showed that the lysine deacetylase SIRT1 is required for acute IPC, and has numerous metabolic targets. While it is known that metabolism is altered during IPC, the underlying metabolic regulatory mechanisms are unknown, including the relative importance of SIRT1. Thus, we sought to test the hypothesis that some of the metabolic adaptations that occur in IPC may require SIRT1 as a regulatory mediator. Using both ex-vivo-perfused and in-vivo mouse hearts, LC–MS/MS based metabolomics and 13C-labeled substrate tracing, we found that acute IPC altered several metabolic pathways including: (i) stimulation of glycolysis, (ii) increased synthesis of glycogen and several amino acids, (iii) increased reduced glutathione levels, (iv) elevation in the oncometabolite 2-hydroxyglutarate, and (v) inhibition of fatty-acid dependent respiration. The majority (83%) of metabolic alterations induced by IPC were ablated when SIRT1 was acutely inhibited with splitomicin, and a principal component analysis revealed that metabolic changes in response to IPC were fundamentally different in nature when SIRT1 was inhibited. Furthermore, the protective benefit of IPC was abrogated by eliminating glucose from perfusion media while sustaining normal cardiac function by burning fat, thus indicating that glucose dependency is required for acute IPC. Together, these data suggest that SIRT1 signaling is required for rapid cardioprotective metabolic adaptation in acute IPC.

The phagocytosis of apoptotic cells (efferocytosis) shifts macrophages to an anti-inflammatory state through a set of still poorly understood soluble and cell-bound signals. Apoptosis is a common feature of inflamed tissues, and efferocytosis by tissue macrophages is thought to promote the resolution of inflammation. However, it is not clear how the exposure of tissue macrophages to inflammatory cues (e.g., PAMPs, DAMPs) in the early stages of inflammation affects immune outcomes of macrophage-apoptotic cell interactions occurring at later stages of inflammation. To address this, we used low-dose endotoxin conditioning (LEC, 1 ng/ml LPS 18 h) of mouse resident peritoneal macrophages (RPMФ) to model the effects of suboptimal (i.e., non-tolerizing), antecedent TLR activation on macrophage inflammatory responses to apoptotic cells. Compared with unconditioned macrophages (MФ), LEC-MФ showed a significant enhancement of apoptotic cell-driven suppression of many inflammatory cytokines (e.g., TNF, MIP-1β, MCP-1). We then found that enzymatic depletion of adenosine or inhibition of the adenosine receptor A2a on LEC-MФ abrogated apoptotic cell suppression of TNF, and this suppression was entirely dependent on the ecto-enzyme CD73 (AMP→adenosine) but not CD39 (ATP→AMP), both of which are highly expressed on RPMФ. In addition to a requirement for CD73, we also show that Adora2a levels in macrophages are a critical determinant of TNF suppression by apoptotic cells. LEC treatment of RPMФ led to a ~3-fold increase in Adora2a and a ~28-fold increase in adenosine sensitivity. Moreover, in RAW264.7 cells, ectopic expression of both A2a and CD73 was required for TNF suppression by apoptotic cells. In mice, mild, TLR4-dependent inflammation in the lungs and peritoneum caused a rapid increase in macrophage Adora2a and Adora2b levels, and CD73 was required to limit neutrophil influx in this peritonitis model. Thus immune signaling via the CD73–A2a axis in macrophages links early inflammatory events to subsequent immune responses to apoptotic cells.

Viruses are parasites that depend on the host cell’s metabolic resources to provide the energy and molecular building blocks necessary for the production of viral progeny. It has become increasingly clear that viruses extensively modulate the cellular metabolic network to support productive infection. Here, we review the numerous ways through which human cytomegalovirus (HCMV) modulates cellular metabolism, highlighting known mechanisms of HCMV-mediated metabolic manipulation and identifying key outstanding questions that remain to be addressed.

Despite producing enormous amounts of cytoplasmic DNA, poxviruses continue to replicate efficiently by deploying an armory of proteins that counter host antiviral responses at multiple levels. Among these, poxvirus protein F17 dysregulates the host kinase mammalian target of rapamycin (mTOR) to prevent the activation of stimulator of interferon genes (STING) expression and impair the production of interferon-stimulated genes (ISGs). However, the host DNA sensor(s) involved and their impact on infection in the absence of F17 remain unknown. Here, we show that cyclic-di-GMP-AMP (cGAMP) synthase (cGAS) is the primary sensor that mediates interferon response factor (IRF) activation and ISG responses to vaccinia virus lacking F17 in both macrophages and lung fibroblasts, although additional sensors also operate in the latter cell type. Despite this, ablation of ISG responses through cGAS or STING knockout did not rescue defects in late-viral-protein production, and the experimental data pointed to other functions of mTOR in this regard. mTOR adjusts both autophagic and protein-synthetic processes to cellular demands. No significant differences in autophagic responses to wild-type or F17 mutant viruses could be detected, with autophagic activity differing across cell types or states and exhibiting no correlations with defects in viral-protein accumulation. In contrast, results using transformed cells or altered growth conditions suggested that late-stage defects in protein accumulation reflect failure of the F17 mutant to deregulate mTOR and stimulate protein production. Finally, rescue approaches suggest that phosphorylation may partition F17's functions as a structural protein and mTOR regulator. Our findings reveal the complex multifunctionality of F17 during infection.IMPORTANCE Poxviruses are large, double-stranded DNA viruses that replicate entirely in the cytoplasm, an unusual act that activates pathogen sensors and innate antiviral responses. In order to replicate, poxviruses therefore encode a wide range of innate immune antagonists that include F17, a protein that dysregulates the kinase mammalian target of rapamycin (mTOR) to suppress interferon-stimulated gene (ISG) responses. However, the host sensor(s) that detects infection in the absence of F17 and its precise contribution to infection remains unknown. Here, we show that the cytosolic DNA sensor cGAS is primarily responsible for activating ISG responses in biologically relevant cell types infected with a poxvirus that does not express F17. However, in line with their expression of ∼100 proteins that act as immune response and ISG antagonists, while F17 helps suppress cGAS-mediated responses, we find that a critical function of its mTOR dysregulation activity is to enhance poxvirus protein production.

An earlier report showed that the U(S)3 protein kinase blocked the apoptosis induced by the herpes simplex virus 1 (HSV-1) d120 mutant at a premitochondrial stage. Further studies revealed that the kinase also blocks programmed cell death induced by the proapoptotic protein BAD. Here we report the effects of the U(S)3 protein kinase on the function and state of a murine BAD protein. Specifically, (i) in uninfected cells, BAD was processed by at least two proteolytic cleavages that were blocked by a general caspase inhibitor. The untreated transduced cells expressed elevated caspase 3 activity. (ii) In cells cotransduced with the U(S)3 protein kinase, the BAD protein was not cleaved and the caspase 3 activity was not elevated. (iii) Inasmuch as the U(S)3 protein kinase blocked the proapoptotic activity and cleavage of a mutant (BAD3S/A) in which the codons for the regulatory serines at positions 112, 136, and 155 were each replaced with alanine codons, the U(S)3 protein kinase does not act by phosphorylation of these sites nor was the phosphorylation of these sites required for the antiapoptotic function of the U(S)3 protein kinase. (iv) The U(S)3 protein kinase did not enable the binding of the BAD3S/A mutant to the antiapoptotic proteins 14-3-3. Finally, (v) whereas cleavage of BAD at ASP56 and ASP61 has been reported and results in the generation of a more effective proapoptotic protein with an M(r) of 15,000, in this report we also show the existence of a second caspase-dependent cleavage site most likely at the ASP156 that is predicted to inactivate the proapoptotic activity of BAD. We conclude that the primary effect of U(S)3 was to block the caspases that cleave BAD at either residue 56 or 61 predicted to render the protein more proapoptotic or at residue 156, which would inactivate the protein.

As essential components of the host's innate immune response, NFκB and interferon signaling are critical determinants of the outcome of infection. Over the past 25 years, numerous Human Cytomegalovirus (HCMV) genes have been identified that antagonize or modulate the signaling of these pathways. Here we review the biology of the HCMV factors that alter NFκB and interferon signaling, including what is currently known about how these viral genes contribute to infection and persistence, as well as the major outstanding questions that remain.

The human cytomegalovirus UL26 open reading frame encodes proteins of 21 and 27 kDa that result from the use of two different in-frame initiation codons. The UL26 protein is a constituent of the virion and thus is delivered to cells upon viral entry. We have characterized a mutant of human cytomegalovirus in which the UL26 open reading frame has been deleted. The UL26 deletion mutant has a profound growth defect, the magnitude of which is dependent on the multiplicity of infection. Two very early defects were discovered. First, even though they were present in normal amounts within mutant virions, the UL99-coded pp28 and UL83-coded pp65 tegument proteins were present in reduced amounts at the earliest times assayed within newly infected cells; second, there was a delay in immediate-early mRNA and protein accumulation. Further analysis revealed that although wild-type levels of the pp28 tegument protein were present in UL26 deletion mutant virions, the protein was hypophosphorylated. We conclude that the UL26 protein influences the normal phosphorylation of at least pp28 in virions and possibly additional tegument proteins. We propose that the hypophosphorylation of tegument proteins causes their destabilization within newly infected cells, perhaps disrupting the normal detegumentation process and leading to a delay in the onset of immediate-early gene expression.

ABSTRACT In contrast to many viruses, human cytomegalovirus (HCMV) is unable to productively infect most cancer-derived cell lines. The mechanisms of this restriction are unclear. To explore this issue, we tested whether defined oncogenic alleles, including the simian virus 40 (SV40) T antigen (TAg) and oncogenic H-Ras, inhibit HCMV infection. We found that expression of SV40 TAg blocks HCMV infection in human fibroblasts, whereas the replication of a related herpesvirus, herpes simplex virus 1 (HSV-1), was not impacted. The earliest restriction of HCMV infection involves a block of viral entry, as TAg expression prevented the nuclear delivery of viral DNA and pp65. Subsequently, we found that TAg expression reduces the abundance of platelet-derived growth factor receptor α (PDGFRα), a host protein important for HCMV entry. Viral entry into TAg-immortalized fibroblasts could largely be rescued by PDGFRα overexpression. Similarly, PDGFRα overexpression in HeLa cells markedly increased the levels of HCMV gene expression and DNA replication. However, the robust production of viral progeny was not restored by PDGFRα overexpression in either HeLa cells or TAg-immortalized fibroblasts, suggesting additional restrictions associated with transformation and TAg expression. In TAg-expressing fibroblasts, expression of the immediate early 2 (IE2) protein was not rescued to the same extent as that of the immediate early 1 (IE1) protein, suggesting that TAg expression impacts the accumulation of major immediate early (MIE) transcripts. Transduction of IE2 largely rescued HCMV gene expression in TAg-expressing fibroblasts but did not rescue the production of infectious virions. Collectively, our data indicate that oncogenic alleles induce multiple restrictions to HCMV replication. IMPORTANCE HCMV cannot replicate in most cancerous cells, yet the causes of this restriction are not clear. The mechanisms that restrict viral replication in cancerous cells represent viral vulnerabilities that can potentially be exploited therapeutically in other contexts. Here we found that SV40 T antigen-mediated transformation inhibits HCMV infection at multiple points in the viral life cycle, including through inhibition of proper viral entry, normal expression of immediate early genes, and viral DNA replication. Our results suggest that the SV40 T antigen could be a valuable tool to dissect cellular activities that are important for successful infection, thereby potentially informing novel antiviral development strategies. This is an important consideration, given that HCMV is a leading cause of birth defects and causes severe infection in immunocompromised individuals.

ABSTRACT Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type virus blocks the execution of the cell death program triggered by expression of viral genes, by the Fas and tumor necrosis factor pathways, or by nonspecific stress agents. In particular, an earlier report from this laboratory showed that the mutant virus d 120 lacking the genes encoding infected cell protein 4 (ICP4), the major regulatory protein of the virus, induces a caspase-3-independent pathway of apoptosis in human SK-N-SH cells. Here we report that the pathway of apoptosis induced by the d 120 mutant in human HEp-2 cells is caspase dependent. Specifically, in HEp-2 cells infected with d 120, (i) a broad-range inhibitor of caspase activity, z-vad-FMK, efficiently blocked DNA fragmentation, (ii) cytochrome c was released into the cytoplasm, (iii) caspase-3 was activated inasmuch as poly(ADP-ribose) polymerase was cleaved, and (iv) chromatin condensation and fragmentation of cellular DNA were observed. In parallel studies, HEp-2 cells were transfected with a plasmid encoding human Bcl-2 and a clone (VAX-3) expressing high levels of Bcl-2 was selected. This report shows that Bcl-2 blocked all of the manifestations associated with programmed cell death caused by infection with the d 120 mutant. Consistent with their resistance to programmed cell death, VAX-3 cells overproduced infected cell protein 0 (ICP0). An unexpected observation was that ICP0 encoded by the d 120 mutant accumulated late in infection in small, quasi-uniform vesicle-like structures in all cell lines tested. Immunofluorescence-based colocalization studies indicated that these structures were not mitochondria or components of the endoplasmic reticulum or the late endosomal compartment. These studies affirm the conclusion that HSV can induce programmed cell death at multiple steps in the course of its replication, that the d 120 mutant can induce both caspase-dependent and -independent pathways of programmed cell death, and that virus-induced stimuli of programmed cell death may differ with respect to the pathway that they activate.

The coding domain of the herpes simplex virus type 1 (HSV-1) alpha22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and U(S)1.5, a protein colinear with the carboxyl-terminal domain of ICP22. In HSV-1-infected cells, ICP22 and U(S)1.5 are extensively modified by the U(L)13 and U(S)3 viral protein kinases. In this report, we show that in contrast to other viral proteins defined by their properties as alpha proteins, U(S)1.5 becomes detectable and accumulated only at late times after infection. Moreover, significantly more U(S)1.5 protein accumulated in cells infected with a mutant lacking the U(L)13 gene than in cells infected with wild-type virus. To define the role of viral protein kinases on the accumulation of U(S)1.5 protein, rabbit skin cells or Vero cells were exposed to recombinant baculoviruses that expressed U(S)1.5, U(L)13, or U(S)3 proteins under a human cytomegalovirus immediate-early promoter. The results were as follows. (i) Accumulation of the U(S)1.5 protein was reduced by concurrent expression of the U(L)13 protein kinase and augmented by concurrent expression of the U(S)3 protein kinase. The magnitude of the reduction or increase in the accumulation of the U(S)1.5 protein was cell type dependent. The effect of U(L)13 kinase appears to be specific inasmuch as it did not affect the accumulation of glycoprotein D in cells doubly infected by recombinant baculoviruses expressing these genes. (ii) The reduction in accumulation of the U(S)1.5 protein was partially due to proteasome-dependent degradation. (iii) Both U(S)1.5 and U(L)13 proteins activated caspase 3, indicative of programmed cell death. (iv) Concurrent expression of the U(S)3 protein kinase blocked activation of caspase 3. The results are concordant with those published elsewhere (J. Munger and B. Roizman, Proc. Natl. Acad. Sci. USA 98:10410-10415, 2001) that the U(S)3 protein kinase can block apoptosis by degradation or posttranslational modification of BAD.

We evaluated cellular metabolism profiles of HIV-1 and HIV-2 infected primary human monocyte-derived macrophages (MDMs). First, HIV-2 GL-AN displays faster production kinetics and greater amounts of virus as compared to HIV-1s: YU-2, 89.6 and JR-CSF. Second, quantitative LC–MS/MS metabolomics analysis demonstrates very similar metabolic profiles in glycolysis and TCA cycle metabolic intermediates between HIV-1 and HIV-2 infected macrophages, with a few notable exceptions. The most striking metabolic change in MDMs infected with HIV-2 relative to HIV-1-infected MDMs was the increased levels of quinolinate, a metabolite in the tryptophan catabolism pathway that has been linked to HIV/AIDS pathogenesis. Third, both HIV-1 and HIV-2 infected MDMs showed elevated levels of ribose-5-phosphate, a key metabolic component in nucleotide biosynthesis. Finally, HIV-2 infected MDMs display increased dNTP concentrations as predicted by Vpx-mediated SAMHD1 degradation. Collectively, these data show differential metabolic changes during HIV-1 and HIV-2 infection of macrophages.

Human Cytomegalovirus (HCMV) is an opportunistic pathogen that causes substantial disease in neonates and immunocompromised individuals. Reverse genetic analysis of the HCMV genome is a powerful tool to dissect the roles that various viral genes play during infection. However, genetic engineering of HCMV is hampered by both the large size of the HCMV genome and HCMV's slow replication cycle. Currently, most laboratories that genetically engineer HCMV employ Bacterial Artificial Chromosome (BAC) mediated recombineering, which is a relatively lengthy process. We explored an alternative method of producing recombinant HCMV using the CRISPR/Cas9 system. We employed both homologous recombination (HR) and Non-homologous end-joining (NHEJ)-based methods, and find that each approach is capable of efficiently mutating the HCMV genome, with optimal efficiencies of 42% and 81% respectively. Our results suggest that CRISPR-mediated genomic engineering of HCMV is competitive with BAC-mediated recombineering and provide a framework for using CRISPR/Cas9 for mutational analysis of the HCMV genome.

Calcium signaling and the AMP-activated protein kinase (AMPK) signaling network broadly regulate numerous aspects of cell biology. Human Cytomegalovirus (HCMV) infection has been found to actively manipulate the calcium-AMPK signaling axis to support infection. Many HCMV genes have been linked to modulating calcium signaling, and HCMV infection has been found to be reliant on calcium signaling and AMPK activation. Here, we focus on the cell biology of calcium and AMP-activated protein kinase (AMPK) signaling and what is currently known about how HCMV modulates these pathways to support HCMV infection and potentially contribute to oncomodulation.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wildtype human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

Mitochondria play a vital role in cellular metabolism and energetics and support normal cardiac function. Disrupted mitochondrial function and homeostasis cause a variety of heart diseases. Fam210a (family with sequence similarity 210 member A), a novel mitochondrial gene, is identified as a hub gene in mouse cardiac remodeling by multi-omics studies. Human FAM210A mutations are associated with sarcopenia. However, the physiological role and molecular function of FAM210A remain elusive in the heart. We aim to determine the biological role and molecular mechanism of FAM210A in regulating mitochondrial function and cardiac health in vivo .Tamoxifen-induced αMHCMCM -driven conditional knockout of Fam210a in the mouse cardiomyocytes induced progressive dilated cardiomyopathy and heart failure, ultimately causing mortality. Fam210a deficient cardiomyocytes exhibit severe mitochondrial morphological disruption and functional decline accompanied by myofilament disarray at the late stage of cardiomyopathy. Furthermore, we observed increased mitochondrial reactive oxygen species production, disturbed mitochondrial membrane potential, and reduced respiratory activity in cardiomyocytes at the early stage before contractile dysfunction and heart failure. Multi-omics analyses indicate that FAM210A deficiency persistently activates integrated stress response (ISR), resulting in transcriptomic, translatomic, proteomic, and metabolomic reprogramming, ultimately leading to pathogenic progression of heart failure. Mechanistically, mitochondrial polysome profiling analysis shows that FAM210A loss of function compromises mitochondrial mRNA translation and leads to reduced mitochondrial encoded proteins, followed by disrupted proteostasis. We observed decreased FAM210A protein expression in human ischemic heart failure and mouse myocardial infarction tissue samples. To further corroborate FAM210A function in the heart, AAV9-mediated overexpression of FAM210A promotes mitochondrial-encoded protein expression, improves cardiac mitochondrial function, and partially rescues murine hearts from cardiac remodeling and damage in ischemia-induced heart failure.These results suggest that FAM210A is a mitochondrial translation regulator to maintain mitochondrial homeostasis and normal cardiomyocyte contractile function. This study also offers a new therapeutic target for treating ischemic heart disease.Mitochondrial homeostasis is critical for maintaining healthy cardiac function. Disruption of mitochondrial function causes severe cardiomyopathy and heart failure. In the present study, we show that FAM210A is a mitochondrial translation regulator required for maintaining cardiac mitochondrial homeostasis in vivo . Cardiomyocyte-specific FAM210A deficiency leads to mitochondrial dysfunction and spontaneous cardiomyopathy. Moreover, our results indicate that FAM210A is downregulated in human and mouse ischemic heart failure samples and overexpression of FAM210A protects hearts from myocardial infarction induced heart failure, suggesting that FAM210A mediated mitochondrial translation regulatory pathway can be a potential therapeutic target for ischemic heart disease.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wild-type human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

Viral disruption of innate immune signaling is a critical determinant of productive infection. The Human Cytomegalovirus (HCMV) UL26 protein prevents anti-viral gene expression during infection, yet the mechanisms involved are unclear. We used TurboID-driven proximity proteomics to identify putative UL26 interacting proteins during infection to address this issue. We find that UL26 forms a complex with several immuno-regulatory proteins, including several STAT family members and various PIAS proteins, a family of E3 SUMO ligases. Our results indicate that UL26 prevents STAT phosphorylation during infection and antagonizes transcriptional activation induced by either interferon alpha (IFNA) or tumor necrosis factor alpha (TNFalpha). Additionally, we find that the inactivation of PIAS1 sensitizes cells to inflammatory stimulation, resulting in an anti-viral transcriptional environment that mirrors DeltaUL26 infection. Further, PIAS1 is important for HCMV cell-to-cell spread, which depends on the presence of UL26, suggesting that the UL26-PIAS1 interaction is vital for modulating intrinsic anti-viral defense.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wildtype human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

Abstract Glutathione (GSH), the most abundant antioxidant, scavenges free radicals and prevents the detrimental effects of oxidative stress. As a result, GSH levels are associated with protection from stress-related conditions, such as aging and cancer. Given this rationale, antioxidants are routinely consumed by the public, mainly because they are viewed as “cure-alls” and almost uniformly beneficial to a range of diseases. This is an oversimplification, as the relationship between antioxidants and diseases is much more complex. Recent evidence has implicated GSH in protecting cancer cells from oxidative stress and promoting their survival. Therefore, understanding GSH metabolism in vivo and its intricate crosstalk with other cellular pathways is vital to improving the efficacy of therapies for diseases, including cancer. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance via the LXR/SREBP signaling pathway, as well as by repressing NRF2, a transcription factor induced by oxidative stress. Overall, these findings suggest an essential function for GSH in maintaining circulating lipids levels and lipid depots, possibly as a mechanism to prevent lipid peroxidation and redox imbalances. Notably, the accumulation of oxidized lipids propagates oxidative stress and associated cell death phenotypes (i.e., ferroptosis). This also highlights a potentially novel mechanism by which GSH regulates lipid production and suggests a possible link between oxidative stress (caused by GSH deficiency) and cachexia (commonly characterized by loss of fat and lean mass), a disorder linked to cancer but poorly understood. Consequently, by elucidating the contributions of GSH to lipid homeostasis, we can better understand the basic biology surrounding GSH and obtain critical insight into potentially improving the treatment of cancer-related cachexia. Citation Format: Gloria Asantewaa, Emily T. Tuttle, Nathan P. Ward, Yun P. Kang, Yumi Kim, Madeline Kavanagh, Aaron R. Huber, Joshua Munger, Benjamin Cravatt, Calvin L. Cole, Gina M. DeNicola, Isaac S. Harris. Glutathione supports lipid abundance in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB292.

Viral disruption of innate immune signaling is a critical determinant of productive infection. The Human Cytomegalovirus (HCMV) UL26 protein prevents anti-viral gene expression during infection, yet the mechanisms involved are unclear. We used TurboID-driven proximity proteomics to identify putative UL26 interacting proteins during infection to address this issue. We find that UL26 forms a complex with several immuno-regulatory proteins, including several STAT family members and various PIAS proteins, a family of E3 SUMO ligases. Our results indicate that UL26 prevents STAT phosphorylation during infection and antagonizes transcriptional activation induced by either interferon α (IFNA) or tumor necrosis factor α (TNFα). Additionally, we find that the inactivation of PIAS1 sensitizes cells to inflammatory stimulation, resulting in an anti-viral transcriptional environment similar to ΔUL26 infection. Further, PIAS1 is important for HCMV cell-to-cell spread, which depends on the presence of UL26, suggesting that the UL26-PIAS1 interaction is vital for modulating intrinsic anti-viral defense.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wild-type human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

SUMMARY The emergence of SARS-CoV-2 virus has resulted in a worldwide pandemic, but an effective antiviral therapy has yet to be discovered. To improve treatment options, we conducted a high-throughput drug repurposing screen to uncover compounds that block the viral activity of SARS-CoV-2. A minimally pathogenic human betacoronavirus (OC43) was used to infect physiologically-relevant human pulmonary fibroblasts (MRC5) to facilitate rapid antiviral discovery in a preclinical model. Comprehensive profiling was conducted on more than 600 compounds, with each compound arrayed at 10 dose points (ranging from 20 μM to 1 nM). Our screening revealed several FDA-approved agents that act as novel antivirals that block both OC43 and SARS-CoV-2 viral replication, including lapatinib, doramapimod, and 17-AAG. Importantly, lapatinib inhibited SARS-CoV-2 replication by over 50,000-fold without any toxicity and at doses readily achievable in human tissues. Further, both lapatinib and doramapimod could be combined with remdesivir to dramatically improve antiviral activity in cells. These findings reveal novel treatment options for people infected with SARS-CoV-2 that can be readily implemented during the pandemic.

Human Cytomegalovirus (HCMV) infection modulates cellular metabolism to support viral replication. Calcium/calmodulin-dependent kinase kinase (CaMKK) and AMP-activated protein kinase (AMPK) regulate metabolic activation and have been found to be important for successful HCMV infection. Here, we explored the contributions that specific CaMKK isoforms and AMPK subunit isoforms make toward HCMV infection. Our results indicate that various CaMKK and AMPK isoforms contribute to infection in unique ways. For example, CaMKK1 is important for HCMV infection at a low multiplicity of infection, but is dispensable for AMPK activation at the earliest times of infection, which our data suggest is more reliant on CaMKK2. Our results also indicate that HCMV specifically induces the expression of the non-ubiquitous AMPKa2 catalytic subunit, found to be important for both HCMV-mediated glycolytic activation and high titer infection. Further, we find that AMPK-mediated glycolytic activation is important for infection, as overexpression of GLUT4, the high capacity glucose transporter, partially rescues viral replication in the face of AMPK inhibition. Collectively, our data indicate that HCMV infection selectively induces the expression of specific metabolic regulatory kinases, relying on their activity to support glycolytic activation and productive infection.IMPORTANCE Viruses are obligate parasites that depend on the host cell to provide the energy and molecular building blocks to mass produce infectious viral progeny. The processes that govern viral modulation of cellular resources have emerged as critical for successful infection. Here, we find that HCMV depends on two kinase isoforms to support infection, CaMKK1 and AMPKa2. We find that HCMV specifically induces expression of the AMPKa2 subunit to induce metabolic activation and drive robust viral replication. These results suggest that HCMV has evolved mechanisms to target specific metabolic regulatory kinase subunits to support productive infection, thereby providing insight into how HCMV hijacks cellular metabolism for its replication, and sheds light on potential viral therapeutic vulnerabilities.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals.

Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals.

Astroglial dysfunction plays an important role in neurodegenerative diseases otherwise attributed to neuronal loss of function. Here we focus on the role of astroglia in ataxia–telangiectasia (A–T), a disease caused by mutations in the ataxia–telangiectasia mutated ( ATM ) gene. A hallmark of A–T pathology is progressive loss of cerebellar neurons, but the mechanisms that impact neuronal survival are unclear. We now provide a possible mechanism by which A–T astroglia affect the survival of cerebellar neurons. As astroglial functions are difficult to study in an in vivo setting, particularly in the cerebellum where these cells are intertwined with the far more numerous neurons, we conducted in vitro coculture experiments that allow for the generation and pharmacological manipulation of purified cell populations. Our analyses revealed that cerebellar astroglia isolated from Atm mutant mice show decreased expression of the cystine/glutamate exchanger subunit xCT, glutathione (GSH) reductase, and glutathione‐S‐transferase. We also found decreased levels of intercellular and secreted GSH in A–T astroglia. Metabolic labeling of l ‐cystine, the major precursor for GSH, revealed that a key component of the defect in A–T astroglia is an impaired ability to import this rate‐limiting precursor for the production of GSH. This impairment resulted in suboptimal extracellular GSH supply, which in turn impaired survival of cerebellar neurons. We show that by circumventing the xCT‐dependent import of l ‐cystine through addition of N ‐acetyl‐ l ‐cysteine (NAC) as an alternative cysteine source, we were able to restore GSH levels in A–T mutant astroglia providing a possible future avenue for targeted therapeutic intervention. GLIA 2016;64:227–239

Modulation of cellular antiviral signaling is a key determinant of viral pathogenesis. Human cytomegalovirus (HCMV) is a significant source of morbidity in neonates and the immunosuppressed that contains many genes that modulate antiviral signaling, yet how these genes contribute to shaping the host cell’s transcriptional response to infection is largely unclear. Our results indicate that the HCMV U L 26 protein is critical in preventing the establishment of a broad cellular proinflammatory transcriptional environment. Further, we find that the host gene IKKβ is an essential determinant governing the host cell’s antiviral transcriptional response. Given their importance to viral pathogenesis, continuing to elucidate the functional interactions between viruses and the cellular innate immune response could enable the development of therapeutic strategies to limit viral infection.

The human cytomegalovirus (HCMV) U(L)26 gene encodes a virion protein that is important for high titer viral replication. To identify specific domains within the U(L)26 protein that contribute to viral infection, we created a panel of site-directed U(L)26 mutant viruses and assessed their impact on phenotypes attributed to U(L)26. We find that the C-terminal 38 amino acids of the U(L)26 protein are absolutely necessary for U(L)26 function. A stop-insertion mutant that produced a truncated U(L)26 protein lacking this region behaved identically to U(L)26-null viruses. This included reduced accumulation of IE1 protein at early time points, smaller plaque size, reduced virion stability, and growth with similarly attenuated kinetics. This C-terminal truncation decreased the amount of U(L)26 packaged into the virion resulting in reduced delivery of U(L)26 to newly infected cells. Further, this C-terminal truncated U(L)26 exhibited substantially reduced nuclear localization compared to wildtype U(L)26. Translation of U(L)26 mRNA is initiated from two separate in frame methionines that give rise to a long and a short isoform of U(L)26. We find that the N-terminal 34 amino acids, which are unique to the long isoform of U(L)26, are also important for the function of the U(L)26 protein. A viral mutant that produces only the short isoform of U(L)26 and lacks these N-terminal 34 amino acids exhibits delayed IE1 accumulation, and demonstrates intermediate defects in viral plaque size, virion stability and viral growth kinetics. Ablation of the short U(L)26 isoform in the presence of the long U(L)26 isoform did not impact any of the in vitro phenotypes tested. These experiments highlight important domains within the U(L)26 protein that contribute to HCMV infection.

Viruses depend on cellular metabolic resources to supply the energy and biomolecular building blocks necessary for their replication. Human cytomegalovirus (HCMV), a leading cause of birth defects and morbidity in immunosuppressed individuals, induces numerous metabolic activities that are important for productive infection. However, many of the mechanisms through which these metabolic activities are induced and how they contribute to infection are unclear. We find that HCMV infection of fibroblasts induces a neuronal gene signature as well as the expression of several metabolic enzyme isoforms that are typically expressed in other tissue types. Of these, the most substantially induced glycolytic gene was the neuron-specific isoform of enolase 2 (ENO2). Induction of ENO2 expression is important for HCMV-mediated glycolytic activation as well as for the virally induced remodeling of pyrimidine-sugar metabolism, which provides the glycosyl subunits necessary for protein glycosylation. Inhibition of ENO2 expression or activity reduced uridine diphosphate (UDP)-sugar pools, attenuated the accumulation of viral glycoproteins, and induced the accumulation of noninfectious viral particles. In addition, our data indicate that the induction of ENO2 expression depends on the HCMV UL38 protein. Collectively, our data indicate that HCMV infection induces a tissue atypical neuronal glycolytic enzyme to activate glycolysis and UDP-sugar metabolism, increase the accumulation of glycosyl building blocks, and enable the expression of an essential viral glycoprotein and the production of infectious virions.

Earlier reports have shown that the d120 mutant of herpes simplex virus 1 lacking both copies of the gene encoding the infected cells protein No. 4 (ICP4) induces apoptosis in a variety of cell lines. The programmed cell death induced by this mutant is blocked by overexpression of Bcl-2 or by transduction of infected cells with the gene encoding the viral U(S)3 protein kinase. HEp-2 cells infected with the d120 mutant express predominantly alpha proteins. Studies on these proteins revealed the accumulation of a M(r) 37,500 protein that reacted with antibody directed against the carboxyl-terminal domain of ICP22. We report that the M(r) 37,500 protein is a product of the proteolytic cleavage of ICP22 by a caspase activated by the d120 mutant. Thus the accumulation of the M(r) 37,500 protein was blocked in cells transduced with the U(S)3 protein kinase, in cells overexpressing Bcl-2, or in infected cells treated with the general caspase inhibitor zVAD-fmk. Exposure of ICP22 made in wild-type virus-infected cells to caspase 3 yielded two polypeptides, of which one could not be differentiated from the M(r) 37,500 protein with respect to electrophoretic mobility. We conclude that the cellular apoptotic response targets at least one viral protein for destruction.

Cytokines induce an anti-viral state, yet many of the functional determinants responsible for limiting viral infection are poorly understood. Here, we find that TNFα induces significant metabolic remodeling that is critical for its anti-viral activity. Our data demonstrate that TNFα activates glycolysis through the induction of hexokinase 2 (HK2), the isoform predominantly expressed in muscle. Further, we show that glycolysis is broadly important for TNFα-mediated anti-viral defense, as its inhibition attenuates TNFα's ability to limit the replication of evolutionarily divergent viruses. TNFα was also found to modulate the metabolism of UDP-sugars, which are essential precursor substrates for glycosylation. Our data indicate that TNFα increases the concentration of UDP-glucose, as well as the glucose-derived labeling of UDP-glucose and UDP-N-acetyl-glucosamine in a glycolytically-dependent manner. Glycolysis was also necessary for the TNFα-mediated accumulation of several glycosylated anti-viral proteins. Consistent with the importance of glucose-driven glycosylation, glycosyl-transferase inhibition attenuated TNFα's ability to promote the anti-viral cell state. Collectively, our data indicate that cytokine-mediated metabolic remodeling is an essential component of the anti-viral response.

Abstract Aims Mitochondria play a vital role in cellular metabolism and energetics and support normal cardiac function. Disrupted mitochondrial function and homeostasis cause a variety of heart diseases. Fam210a (family with sequence similarity 210 member A), a novel mitochondrial gene, is identified as a hub gene in mouse cardiac remodelling by multi-omics studies. Human FAM210A mutations are associated with sarcopenia. However, the physiological role and molecular function of FAM210A remain elusive in the heart. We aim to determine the biological role and molecular mechanism of FAM210A in regulating mitochondrial function and cardiac health in vivo. Methods and results Tamoxifen-induced αMHCMCM-driven conditional knockout of Fam210a in the mouse cardiomyocytes induced progressive dilated cardiomyopathy and heart failure, ultimately causing mortality. Fam210a deficient cardiomyocytes exhibit severe mitochondrial morphological disruption and functional decline accompanied by myofilament disarray at the late stage of cardiomyopathy. Furthermore, we observed increased mitochondrial reactive oxygen species production, disturbed mitochondrial membrane potential, and reduced respiratory activity in cardiomyocytes at the early stage before contractile dysfunction and heart failure. Multi-omics analyses indicate that FAM210A deficiency persistently activates integrated stress response, resulting in transcriptomic, translatomic, proteomic, and metabolomic reprogramming, ultimately leading to pathogenic progression of heart failure. Mechanistically, mitochondrial polysome profiling analysis shows that FAM210A loss of function compromises mitochondrial mRNA translation and leads to reduced mitochondrial-encoded proteins, followed by disrupted proteostasis. We observed decreased FAM210A protein expression in human ischaemic heart failure and mouse myocardial infarction tissue samples. To further corroborate FAM210A function in the heart, AAV9-mediated overexpression of FAM210A promotes mitochondrial-encoded protein expression, improves cardiac mitochondrial function, and partially rescues murine hearts from cardiac remodelling and damage in ischaemia-induced heart failure. Conclusion These results suggest that FAM210A is a mitochondrial translation regulator to maintain mitochondrial homeostasis and normal cardiomyocyte contractile function. This study also offers a new therapeutic target for treating ischaemic heart disease.

Innate immune signaling is a critical defense against viral infection and represents a central host-virus interaction that frequently determines the outcomes of infections. NF-κB signaling is an essential component of innate immunity that is extensively modulated by HCMV, a significant cause of morbidity in neonates and immunosuppressed individuals. However, the roles that various facets of NF-κB signaling play during HCMV infection have remained elusive. We find that the two major regulatory kinases in this pathway, IKKα and IKKβ, limit the initiation of infection, viral replication, and cell-to-cell spread. In addition, our results indicate that these kinases contribute differently to the host cell response to infection in the absence of a virally encoded NF-κB inhibitor, U L 26. Given the importance of NF-κB in viral infection, elucidating the contributions of various NF-κB constituents to infection is an essential first step toward the possibility of targeting this pathway therapeutically.

Abstract Human cytomegalovirus (HCMV) modulates cellular metabolism to support productive infection, and the HCMV U L 38 protein drives many aspects of this HCMV-induced metabolic program. However, it remains to be determined whether virally-induced metabolic alterations might induce novel therapeutic vulnerabilities in virally infected cells. Here, we explore how HCMV infection and the U L 38 protein modulate cellular metabolism and how these changes alter the response to nutrient limitation. We find that expression of U L 38, either in the context of HCMV infection or in isolation, sensitizes cells to glucose limitation resulting in cell death. This sensitivity is mediated through U L 38’s inactivation of the TSC complex subunit 2 (TSC2) protein, a central metabolic regulator that possesses tumor-suppressive properties. Further, expression of U L 38 or the inactivation of TSC2 results in anabolic rigidity in that the resulting increased levels of fatty acid biosynthesis are insensitive to glucose limitation. This failure to regulate fatty acid biosynthesis in response to glucose availability sensitizes cells to glucose limitation, resulting in cell death unless fatty acid biosynthesis is inhibited. These experiments identify a regulatory circuit between glycolysis and fatty acid biosynthesis that is critical for cell survival upon glucose limitation and highlight a metabolic vulnerability associated with viral infection and the inactivation of normal metabolic regulatory controls. Importance Viruses modulate host cell metabolism to support the mass production of viral progeny. For Human Cytomegalovirus, we find that the viral U L 38 protein is critical for driving these pro-viral metabolic changes. However, our results indicate that these changes come at a cost, as U L 38 induces an anabolic rigidity that leads to a metabolic vulnerability. We find that U L 38 decouples the link between glucose availability and fatty acid biosynthetic activity. Normal cells respond to glucose limitation by down-regulating fatty acid biosynthesis. Expression of U L 38 results in the inability to modulate fatty acid biosynthesis in response to glucose limitation, which results in cell death. We find this vulnerability in the context of viral infection, but this linkage between fatty acid biosynthesis, glucose availability, and cell death could have broader implications in other contexts or pathologies that rely on glycolytic remodeling, for example, oncogenesis.

Raf1 is a key player in growth factor receptor signaling, which has been linked to multiple viral infections, including Human Cytomegalovirus (HCMV) infection. Although HCMV remains latent in most individuals, it can cause acute infection in immunocompromised populations such as transplant recipients, neonates, and cancer patients. Current treatments are suboptimal, highlighting the need for novel treatments. Multiple points in the growth factor signaling pathway are important for HCMV infection, but the relationship between HCMV and Raf1, a component of the mitogen-activated protein kinase (MAPK) cascade, is not well understood. The AMP-activated protein kinase (AMPK) is a known regulator of Raf1, and AMPK activity is both induced by infection and important for HCMV replication. Our data indicate that HCMV infection induces AMPK-specific changes in Raf1 phosphorylation, including increasing phosphorylation at Raf1-Ser621, a known AMPK phospho-site, which results in increased binding to the 14-3-3 scaffolding protein, an important aspect of Raf1 activation. Inhibition of Raf1, either pharmacologically or via shRNA or CRISPR-mediated targeting, inhibits viral replication and spread in both fibroblasts and epithelial cells. Collectively, our data indicate that HCMV infection and AMPK activation modulate Raf1 activity, which are important for viral replication.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wildtype human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

ABSTRACT Interactions between acute myeloid leukemia (AML) and the hematopoietic bone marrow microenvironment (BMME) are critical to leukemia progression and chemoresistance. We measured elevated extracellular metabolites in the BMME of AML patients, including lactate. Lactate has been implicated in solid tumors for inducing suppressive tumor-associated macrophages, and correlates with poor prognosis. We describe a role of lactate in the polarization of leukemia-associated macrophages (LAMs), using a murine model of blast crisis chronic myelogenous leukemia (bcCML). Elevated lactate also diminished the function of hematopoietic progenitors and stromal support in vitro . Mice genetically lacking the lactate receptor GPR81 were used to demonstrate lactate-GPR81 signaling as a mechanism of both the polarization of LAMs and the direct support of leukemia cells. We report microenvironmental lactate as a critical driver of AML-induced BMME dysfunction and leukemic progression, thus identifying GPR81 as an exciting and novel therapeutic target for the treatment of this devastating disease.

ABSTRACT Human cytomegalovirus (HCMV) modulates cellular metabolism to support productive infection, and the HCMV U L 38 protein drives many aspects of this HCMV-induced metabolic program. However, it remains to be determined whether virally induced metabolic alterations might induce novel therapeutic vulnerabilities in virally infected cells. Here, we explore how HCMV infection and the U L 38 protein modulate cellular metabolism and how these changes alter the response to nutrient limitation. We find that expression of U L 38, either in the context of HCMV infection or in isolation, sensitizes cells to glucose limitation resulting in cell death. This sensitivity is mediated through U L 38’s inactivation of the TSC complex subunit 2 (TSC2) protein, a central metabolic regulator that possesses tumor-suppressive properties. Furthermore, expression of U L 38 or the inactivation of TSC2 results in anabolic rigidity, in that the resulting increased levels of fatty acid biosynthesis are insensitive to glucose limitation. This failure to regulate fatty acid biosynthesis in response to glucose availability sensitizes cells to glucose limitation, resulting in cell death unless fatty acid biosynthesis is inhibited. These experiments identify a regulatory circuit between glycolysis and fatty acid biosynthesis that is critical for cell survival upon glucose limitation and highlight a metabolic vulnerability associated with viral infection and the inactivation of normal metabolic regulatory controls. IMPORTANCE Viruses modulate host cell metabolism to support the mass production of viral progeny. For human cytomegalovirus, we find that the viral U L 38 protein is critical for driving these pro-viral metabolic changes. However, our results indicate that these changes come at a cost, as U L 38 induces an anabolic rigidity that leads to a metabolic vulnerability. We find that U L 38 decouples the link between glucose availability and fatty acid biosynthetic activity. Normal cells respond to glucose limitation by down-regulating fatty acid biosynthesis. Expression of U L 38 results in the inability to modulate fatty acid biosynthesis in response to glucose limitation, which results in cell death. We find this vulnerability in the context of viral infection, but this linkage between fatty acid biosynthesis, glucose availability, and cell death could have broader implications in other contexts or pathologies that rely on glycolytic remodeling, for example, oncogenesis.

Oncogenesis is frequently accompanied by the activation of specific metabolic pathways. One such pathway is fatty acid biosynthesis, whose induction is observed upon transformation of a wide variety of cell types. Here, we explored how defined oncogenic alleles, specifically the simian virus 40 (SV40) T antigens and oncogenic Ras(12V), affect fatty acid metabolism. Our results indicate that SV40/Ras(12V)-mediated transformation of fibroblasts induces fatty acid biosynthesis in the absence of significant changes in the concentration of fatty acid biosynthetic enzymes. This oncogene-induced activation of fatty acid biosynthesis was found to be mammalian target of rapamycin (mTOR) dependent, as it was attenuated by rapamycin treatment. Furthermore, SV40/Ras(12V)-mediated transformation induced sensitivity to treatment with fatty acid biosynthetic inhibitors. Pharmaceutical inhibition of acetyl-coenzyme A (CoA) carboxylase (ACC), a key fatty acid biosynthetic enzyme, induced caspase-dependent cell death in oncogene-transduced cells. In contrast, isogenic nontransformed cells were resistant to fatty acid biosynthetic inhibition. This oncogene-induced sensitivity to fatty acid biosynthetic inhibition was independent of the cells' growth rates and could be attenuated by supplementing the medium with unsaturated fatty acids. Both the activation of fatty acid biosynthesis and the sensitivity to fatty acid biosynthetic inhibition could be conveyed to nontransformed breast epithelial cells through transduction with oncogenic Ras(12V). Similar to what was observed in the transformed fibroblasts, the Ras(12V)-induced sensitivity to fatty acid biosynthetic inhibition was independent of the proliferative status and could be attenuated by supplementing the medium with unsaturated fatty acids. Combined, our results indicate that specific oncogenic alleles can directly confer sensitivity to inhibitors of fatty acid biosynthesis.Viral oncoproteins and cellular mutations drive the transformation of normal cells to the cancerous state. These oncogenic alterations induce metabolic changes and dependencies that can be targeted to kill cancerous cells. Here, we find that the cellular transformation resulting from combined expression of the SV40 early region with an oncogenic Ras allele is sufficient to induce cellular susceptibility to fatty acid biosynthetic inhibition. Inhibition of fatty acid biosynthesis in these cells resulted in programmed cell death, which could be rescued by supplementing the medium with nonsaturated fatty acids. Similar results were observed with the expression of oncogenic Ras in nontransformed breast epithelial cells. Combined, our results suggest that specific oncogenic alleles induce metabolic dependencies that can be exploited to selectively kill cancerous cells.

Rationale: Nicotinamide adenine dinucleotide (NAD + ) is a substrate for sirtuin (SIRT) lysine deacylases. Stimulation of SIRT1 is cardioprotective against ischemia-reperfusion (IR) injury, prompting interest in orally-available NAD + precursors such as nicotinamide mononucleotide (NMN) as potential cardioprotective agents. While the biological activity of NMN has been largely attributed to SIRT stimulation, NMN effects on metabolism, and any role these may play in cardioprotection, are less well understood. Objective: To investigate potential non-SIRT mechanisms for NMN cardioprotection, with a focus on metabolism. Methods & Results: NMN was protective in perfused mouse hearts (post-IR functional recovery: NMN 42±7% vs. vehicle 11±3%). However, protection was insensitive to the SIRT1 inhibitor splitomicin (recovery 47±8%), and NMN did not impact lysine acetylation in the cytosol where cardiac SIRT1 is located, thus suggesting NMN does not stimulate cardiac SIRT1 activity. Surprisingly, NMN was not protective in hearts perfused without glucose (palmitate as fuel source; recovery 11±4%). Since glycolysis requires NAD + , and is associated with some cardioprotective paradigms, we hypothesized NMN protection may be due to glycolytic stimulation. In primary cardiomyocytes, NMN induced cytosolic and extracellular acidification, and enhanced lactate generation, indicative of increased glycolysis. Finally, since extension of ischemic acidosis into early reperfusion (i.e., acid post-conditioning) is cardioprotective, we hypothesized that NMN delivery at reperfusion may protect, and indeed this was the case (recovery 39±8%). Conclusions: The acute cardioprotective benefit of NMN is mediated via glycolytic stimulation, and this effect of NMN may be worthy of investigation in other situations where NAD + precursor supplements are of therapeutic interest.

Abstract Human cytomegalovirus (HCMV) poses serious threats to newborns and immune-compromised individuals. In contrast to most viruses studied, HCMV is unable to replicate in cancerous cells, but the mechanisms of this restriction are unclear. We explored these mechanisms by determining whether defined oncogenic alleles including the simian virus 40 (SV40) T antigens and oncogenic HRas G12V could inhibit HCMV replication. Our results show that the expression of SV40 T antigens blocks HCMV replication. In contrast, the replication of a related herpes virus, herpes simplex virus I, is not impacted by T antigen expression. The earliest restriction of HCMV infection involves a block of viral entry, as T antigen expression prevents viral tegument protein delivery. Subsequently we find that T antigen expression reduces the accumulation of platelet-derived growth factor receptor (PDGFR) alpha, a host protein that facilitates HCMV entry. Viral entry, gene expression and DNA replication could be partially rescued by PDGFR alpha over-expression. However, production of infectious viral progeny is not restored by PDGFR alpha over-expression in T antigen-expressing fibroblasts. Analysis of viral gene expression indicates that the accumulation of immediate early 2 (IE2), the major viral transcriptional activator, is inhibited by T antigen expression. Restoration of IE2 expression markedly increases downstream HCMV gene expression but not virion production, suggesting additional viral restrictions induced by T antigen. Similarly, PDGFR alpha over-expression in Hela cells markedly increases HCMV gene expression and DNA replication but not virion production. Combined, our results indicate that oncogenic alleles induce multiple restrictions to HCMV infection. Citation Format: Shihao Xu, Xenia Schafer, Joshua Munger. Expression of oncogenic alleles induces multiple blocks to HCMV infection. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A47.

ABSTRACT 2-hydroxyglutarate (2-HG) is an important epigenetic regulator, with potential roles in cancer and stem cell biology. The D (R) enantiomer (D-2-HG) is an oncometabolite generated from αketoglutarate (α-KG) by mutant isocitrate dehydrogenase (ICDH), while L (S) 2-HG is generated by lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in response to hypoxia. Since acidic pH is a common feature of hypoxia, as well as tumor and stem cell microenvironments, we hypothesized that pH may regulate cellular 2-HG levels. Herein we report that cytosolic acidification under normoxia moderately elevated 2-HG in cells, and boosting endogenous substrate α-KG levels further stimulated this elevation. Studies with isolated LDH-1 and MDH-2 revealed that generation of 2-HG by both enzymes was stimulated several-fold at acidic pH, relative to normal physiologic pH. In addition, acidic pH was found to inhibit the activity of the mitochondrial L-2-HG removal enzyme L-2-HG dehydrogenase, and to stimulate the reverse reaction of ICDH (carboxylation of αKG to isocitrate). Furthermore, since acidic pH is known to stabilize hypoxia-inducible factor (HIF), and 2-HG is a known inhibitor of HIF prolyl hydroxylases, we hypothesized that 2-HG may be required for acid-induced HIF stabilization. Accordingly, cells stably over-expressing L-2HGDH exhibited a blunted HIF response to acid. Together these results suggest that acidosis is an important and previously overlooked regulator of 2-HG accumulation and other oncometabolic events, with implications for HIF signaling.

Viral oncoproteins and cellular mutations drive malignant transformation. These oncogenic alterations induce metabolic changes and dependencies that can be targeted to kill cancerous cells. In my first project, we find that oncogenic Ras expression activates fatty acid biosynthesis and confers sensitivity to fatty acid biosynthetic inhibition in human fibroblasts. In addition, we find that a human breast cancer cell line harboring an oncogenic Ras mutation is more sensitive to fatty acid biosynthetic inhibition relative to a non-transformed human breast epithelial cell line (MCF10A). To further explore the impact of specific oncogenic alleles on epithelial cells, we created isogenic MCF10A cells expressing oncogenic Ras. Our data show that oncogenic Ras expression increases fatty acid biosynthesis and sensitizes MCF10A cells to fatty acid biosynthetic inhibition without increasing cellular proliferation. Together, our results indicate that oncogenic Ras confers sensitivity to fatty acid biosynthetic inhibition in human fibroblasts and epithelial cells. This oncogene-induced sensitivity may make an attractive target for therapeutic intervention. In my second project, we investigated the mechanisms that restrict human cytomegalovirus (HCMV) replication in cancerous cells. These mechanisms of viral restriction represent vulnerabilities that could be therapeutically exploited in other contexts. We explored these mechanisms by determining whether defined oncogenic alleles could inhibit HCMV replication. We find that expression of the SV40 T antigens (TAg) blocks HCMV infection in human fibroblasts. The earliest restriction of HCMV infection involves a block of viral entry. Subsequently, we found that TAg expression reduces the abundance of platelet-derived growth factor receptor α (PDGFRα), a host protein important for HCMV entry. Viral entry into TAg-immortalized fibroblasts could largely be rescued by PDGFRα over-expression. However, robust production of viral progeny was not restored by PDGFRα over-expression in TAg-immortalized fibroblasts. In TAg-expressing fibroblasts, the immediate early 2 (IE2) protein was not rescued to the same extent as the immediate early 1 protein. Transduction of IE2 largely rescued HCMV gene expression in TAg-expressing fibroblasts, but did not rescue virion production. Collectively, our data indicate that oncogenic alleles induce multiple restrictions to HCMV replication, the mechanisms of which may provide novel strategies to limit HCMV infection.

Abstract Oncogenesis induces profound changes to the cellular metabolic network. A number of these cancer-cell metabolic phenotypes have been found to be important for in vivo tumorigenesis. We have utilized liquid chromatography tandem mass spectrometry (LC-MS/MS) to globally profile the metabolic network of a genetically-defined experimental model of oncogenesis in an attempt to identify novel metabolic activities induced by transformation. Furthermore, we sought to delineate how common oncogenic mutations contribute to specific metabolic phenotypes. We have analyzed how dominant negative p53 and constitutively active Ras expression, either independently or in concert, impacts the metabolic activity of non-transformed parental colon crypt epithelial cells. In addition to measuring steady-state metabolite concentrations, we have employed isotope tracer analysis to measure the impact of oncogenic mutation on metabolic pathway kinetics. We find that combined p53 and Ras oncogenic mutation synergistically activates numerous aspects of central carbon metabolism including glycolysis, glutamine catabolism, and pyrimidine and purine biosynthesis. Our data indicate that many well-defined oncogenic metabolic phenotypes are the result of a cooperative effect of combinatorial mutation highlighting the importance of identifying the interaction between oncogenic signal transduction and metabolic regulation for understanding the etiology of the cancer-cell metabolic state. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1249. doi:10.1158/1538-7445.AM2011-1249

ABSTRACT Stimulation of the cytosolic NAD + dependent deacetylase SIRT1 is cardioprotective against ischemia-reperfusion (IR) injury. NAD + precursors including nicotinamide mononucleotide (NMN) are thought to induce cardioprotection via SIRT1. Herein, while NMN protected perfused hearts against IR (functional recovery: NMN 42±7% vs. vehicle 11±3%), this protection was insensitive to the SIRT1 inhibitor splitomicin (recovery 47±8%). Although NMN-induced cardioprotection was absent in Sirt3 -/- hearts (recovery 9±5%), this was likely due to enhanced baseline injury in Sirt3 -/- (recovery 6±2%), since similar injury levels in WT hearts also blunted the protective efficacy of NMN. Considering alternative cardiac effects of NMN, and the requirement of glycolysis for NAD + , we hypothesized NMN may confer protection via direct stimulation of cardiac glycolysis. In primary cardiomyocytes, NMN induced cytosolic and extracellular acidification and elevated lactate. In addition, [U- 13 C]glucose tracing in intact hearts revealed that NMN stimulated glycolytic flux. Consistent with a role for glycolysis in NMN-induced protection, hearts perfused without glucose (palmitate as fuel source), or hearts perfused with galactose (no ATP from glycolysis) exhibited no benefit from NMN (recovery 11±4% and 15±2% respectively). Acidosis during early reperfusion is known to be cardioprotective (i.e., acid post-conditioning), and we also found that NMN was cardioprotective when delivered acutely at reperfusion (recovery 39±8%). This effect of NMN was not additive with acidosis, suggesting overlapping mechanisms. We conclude that the acute cardioprotective benefits of NMN are mediated via glycolytic stimulation, with the downstream protective mechanism involving enhanced ATP synthesis during ischemia and/or enhanced acidosis during reperfusion.

Abstract Cytokines induce an anti-viral state, yet many of the functional determinants responsible for limiting viral infection are poorly understood. Here, we find that TNFα induces significant metabolic remodeling that is critical for its anti-viral activity. Our data demonstrate that TNFα activates glycolysis through the induction of muscle-specific hexokinase (HK2). Further, we show that glycolysis is broadly important for TNFα-mediated anti-viral defense, as its inhibition attenuates TNFα’s ability to limit the replication of evolutionarily divergent viruses. Stable-isotope tracing revealed that TNFα-mediated glycolytic activation promotes the biosynthesis of UDP-sugars (essential precursors of protein glycosylation) and that inhibition of glycolysis prevents the accumulation of several glycosylated anti-viral proteins. Consistent with the importance of glucose-driven glycosylation, glycosyl-transferase inhibition also attenuated TNFα’s ability to promote the anti-viral cell state. Collectively, our data indicate that cytokine-mediated metabolic remodeling is an essential component of the anti-viral response.

Abstract Viruses depend on cellular metabolic resources to supply the energy and biomolecular building blocks necessary for their replication. Human Cytomegalovirus (HCMV), a leading cause of birth defects and morbidity in immunosuppressed individuals, induces numerous metabolic activities that are important for productive infection. However, many of the mechanisms through which these metabolic activities are induced and how they contribute to infection are unclear. We find that HCMV infection of fibroblasts induces a neuronal gene signature, as well as the expression of several metabolic enzyme isoforms that are typically expressed in other tissue types. Of these, the most substantially induced gene was the neuron-specific isoform of enolase (ENO2). Induction of ENO2 expression is important for HCMV-mediated glycolytic activation, as well as for the virally-induced remodeling of pyrimidine-sugar metabolism, which provides the glycosyl subunits necessary for protein glycosylation. Inhibition of ENO2 expression or activity reduced UDP-sugar pools, attenuated the accumulation of viral glycoproteins, and induced the accumulation of non-infectious viral particles. In addition, our data indicate that the induction of ENO2 expression depends on the HCMV U L 38 protein. Collectively, our data indicate that HCMV infection induces a tissue atypical neuronal glycolytic enzyme to activate glycolysis and UDP-sugar metabolism to provide the glycosyl building blocks necessary for viral protein glycosylation and the production of infectious virions. Significance Statement Viruses are obligate parasites that obtain energy and mass from their host cell. Control over the metabolic resources of the cell has emerged as an important host-pathogen interaction that can determine infectious outcomes. We find that the Human Cytomegalovirus (HCMV), a major cause of birth defects and morbidity in immunosuppressed patient populations, induces a neuronal gene signature in fibroblasts including the expression of neuronal-specific enolase (ENO2). Our data indicate that ENO2 is important for HCMV-mediated metabolic remodeling including glycolytic activation and the production of pyrimidine sugars, as well as for viral infectivity. These findings indicate that viruses are capable of tapping into alternative tissue-specific metabolic programs to support infection, highlighting an important viral mechanism of metabolic modulation.

Leukemia drives dysfunction of the hematopoietic bone marrow microenvironment (BMME), however, the mechanisms are poorly understood. Loss of normal hematopoiesis accounts for much of the morbidity and mortality (~29% survival at 5 years) of acute myeloid leukemia (AML). To define extracellular metabolomic changes in the bone marrow during disease, we assayed global metabolite levels using liquid chromatography coupled with mass spectrometry (LC-MS). This unbiased analysis demonstrated that lactate levels are elevated in the bone marrow supernatant of AML patients at diagnosis compared to healthy controls (mmol/L = 3.62 vs 1.31, p < 0.05, n = 5). Therefore, we have focused on the effects of lactate in the hematopoietic BMME and on AML cells. We hypothesized that lactate secreted to the BMME by AML cells contributes to bone marrow dysfunction and leukemia progression including loss of normal hematopoiesis. To test this, we used both: (i) a murine model of blast crisis chronic myelogenous leukemia (bcCML), which recapitulates the metabolomic analysis of the human AML BMME, and (ii) transgenic mice with a global knockout of the G-protein-coupled lactate receptor GPR81 that do not display hematopoietic defects. Bone marrow support for hematopoiesis was assayed by utilizing cocultures of murine hematopoietic stem and progenitor cells (HSPCs) maintained in vitro by a monolayer of mesenchymal stem cells (MSCs) and macrophages. Exposure to physiologically-relevant elevated lactate levels resulted in a significant reduction in the ability of HSPCs to form colonies in methylcellulose-containing media (CFU-C =12.17 vs 1.167, p < 0.05, n = 3). Next, we wanted to establish which critical HSPC-supportive cells in the BMME are altered by excess lactate. Elevated lactate levels reduced murine MSC colony-forming ability (area in pixels = 330146 at 0 mmol/L vs 146325-18502 at 10-15 mmol/L, p < 0.05, n = 3). In bcCML, leukemia-associated macrophages (LAMs) were found to overexpress the mannose receptor CD206, and GPR81 signaling contributed to this phenotype in vivo (CD206 MFI = 5784 vs 2633, p < 0.05, n = 3). The contribution of lactate signaling to this phenotype was confirmed by lactate treatment of wild-type or GPR81-/- bone-marrow-derived macrophages in vitro. These data together suggest that lactate regulates multiple components of the HSPC niche to drive leukemia-associated hematopoietic dysfunction. To determine if LAMs adversely affect hematopoiesis, the HSPC support assays were repeated but with the addition of equal numbers of LAMs or normal macrophages to the coculture. Addition of LAMs reduced HSPC colony-forming ability compared to normal macrophages (mean fold-change CFU-C normalized to non-leukemic control = 1.00 vs 0.63, p < 0.0001, n = 4). Furthermore, this effect is reduced when LAMs from bcCML in GPR81-/- mice are added, compared to LAMs from bcCML in wt mice (mean fold-change CFU-C normalized to non-leukemic control = 0.69 vs 0.78, p < 0.05, n = 2). This suggests that GPR81 signaling in the BMME polarizes macrophages to a LAM phenotype that has reduced support for hematopoiesis. Finally, leukemic progression was substantially reduced by mid-stage disease when bcCML was initiated using GPR81-/- leukemic cells (% leukemic cells in bone = 45.54 vs 11.75, p < 0.05, n = 5). To assay the involvement of GPR81 signaling on cancer stem cell ability, serial passaging of bcCML cells in methylcellulose-containing media was performed. The number of viable passages was reduced in GPR81-/- bcCML cells compared to wt bcCML cells suggesting that GPR81-/- cells have less repopulating ability, and that GPR81 functions to maintain an LSC phenotype during AML. This research investigates the role of lactate as a critical driver of AML-induced BMME dysfunction and leukemic progression, thus identifying GPR81 as an exciting and novel therapeutic target for the treatment of this devastating disease. Furthermore, as lactate production is a hallmark of cancer, this mechanism is potentially applicable to multiple malignancies with bone marrow involvement including additional types of leukemia as well as bone marrow metastases of solid tumors.

Viral genes and their products are the critical determinants of viral infection. Human cytomegalovirus (HCMV) encodes many gene products whose roles during viral infection have not been assessed. Elucidation of the contributions that various HCMV gene products make to infection provides insight into the infectious program, which could potentially be used to limit HCMV-associated morbidity, a major issue during congenital infection and in immunosuppressed populations.

The malaria causing Plasmodium parasite is a major public health threat. Plasmodium vivax (P vivax) is the cause of uncomplicated malaria (UCM). Platelets are the cellular mediators of thrombosis and are also the most numerous immune cells in the blood, and a first responder to infections. Thrombocytopenia is a frequent complication of malaria, and a decrease in platelet count is a negative predictor of disease outcome. Malaria infection elicits a strong interferon gamma (IFNγ) response. IFNγ is a potent inducer of indoleamine 2,3-dioxygenase (IDO1) the rate-limiting enzyme that catalyzes the first step in Tryptophan (Trp) metabolism in the kynurenine (Kyn) pathway, shunting Trp away from serotonin production. Trp metabolism may be altered in malaria infection as a means to regulate immunometabolic responses, but the mechanisms remain unknown. Our platelet RNA-sequencing data from P vivax infected humans and from P yoelii infected mice showed increased expression of genes related to Trp metabolism, including IDO1 . The role for platelets in metabolic pathway regulation is poorly explored in general, but particularly in infectious diseases. We introduce a novel idea that platelets participate in immunometabolism to infection. Using complementary experimental approaches such as liquid chromatography-mass spectrometry, ELISA, PCR, western blot, and flow cytometry, we test the hypothesis that platelets are a source of IDO1 in UCM malaria, and thrombocytopenia results in IDO1 depletion and immune dysregulation. We have discovered a role for platelets in Trp metabolic pathway regulation and that platelet regulated immune responses to malaria infection are in part dependent on the Trp metabolic pathways. During P yoelii infection there is a depletion of Trp, and increased Kyn metabolites, as well as decreased plasma serotonin. Platelet transfusions to infected mice can increase Kyn. Understanding the interplay between platelets and immunometabolic pathways may provide a better understanding of the impact of thrombocytopenia in diseases beyond malaria, and provide a means to improve malaria infection responses as well as improved platelet-directed therapeutics in many hematological, metabolic, and immune diseases .