R. Blake Richardson

The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2.

The endoplasmic reticulum (ER) is an architecturally diverse organelle that serves as a membrane source for the replication of multiple viruses. Flaviviruses, including yellow fever virus, West Nile virus, dengue virus and Zika virus, induce unique single-membrane ER invaginations that house the viral replication machinery1. Whether this virus-induced ER remodelling is vulnerable to antiviral pathways is unknown. Here, we show that flavivirus replication at the ER is targeted by the interferon (IFN) response. Through genome-scale CRISPR screening, we uncovered an antiviral mechanism mediated by a functional gene pairing between IFI6 (encoding IFN-α-inducible protein 6), an IFN-stimulated gene cloned over 30 years ago2, and HSPA5, which encodes the ER-resident heat shock protein 70 chaperone BiP. We reveal that IFI6 is an ER-localized integral membrane effector that is stabilized through interactions with BiP. Mechanistically, IFI6 prophylactically protects uninfected cells by preventing the formation of virus-induced ER membrane invaginations. Notably, IFI6 has little effect on other mammalian RNA viruses, including the related Flaviviridae family member hepatitis C virus, which replicates in double-membrane vesicles that protrude outwards from the ER. These findings support a model in which the IFN response is armed with a membrane-targeted effector that discriminately blocks the establishment of virus-specific ER microenvironments that are required for replication. Flavivirus replication at the endoplasmic reticulum (ER) is targeted by the interferon response through blocking of the formation of virus-induced ER membrane invaginations by the interferon-stimulated gene IFI6, encoding an ER-localized integral membrane effector.

The recent Zika virus (ZIKV) outbreak in the Western hemisphere is associated with severe pathology in newborns, including microcephaly and brain damage. The mechanisms underlying these outcomes are under intense investigation. Here, we show that a 2015 ZIKV isolate replicates in multiple cell types, including primary human fetal neural progenitors (hNPs). In immortalized cells, ZIKV is cytopathic and grossly rearranges endoplasmic reticulum membranes similar to other flaviviruses. In hNPs, ZIKV infection has a partial cytopathic phase characterized by cell rounding, pyknosis, and activation of caspase 3. Despite notable cell death, ZIKV did not activate a cytokine response in hNPs. This lack of cell intrinsic immunity to ZIKV is consistent with our observation that virus replication persists in hNPs for at least 28 days. These findings, supported by published fetal neuropathology, establish a proof-of-concept that neural progenitors in the developing human fetus can be direct targets of detrimental ZIKV-induced pathology.

Interferons (IFNs) contribute to cell-intrinsic antiviral immunity by inducing hundreds of interferon-stimulated genes (ISGs). In a screen to identify antiviral ISGs, we unexpectedly found that LY6E, a member of the LY6/uPAR family, enhanced viral infection. Here, we show that viral enhancement by ectopically expressed LY6E extends to several cellular backgrounds and affects multiple RNA viruses. LY6E does not impair IFN antiviral activity or signaling, but rather promotes viral entry. Using influenza A virus as a model, we narrow the enhancing effect of LY6E to uncoating after endosomal escape. Diverse mammalian orthologs of LY6E also enhance viral infectivity, indicating evolutionary conservation of function. By structure-function analyses, we identify a single amino acid in a predicted loop region that is essential for viral enhancement. Our study suggests that LY6E belongs to a class of IFN-inducible host factors that enhance viral infectivity without suppressing IFN antiviral activity.

Autophagy, a process of degradation that occurs via the lysosomal pathway, has an essential role in multiple aspects of immunity, including immune system development, regulation of innate and adaptive immune and inflammatory responses, selective degradation of intracellular microorganisms, and host protection against infectious diseases1,2. Autophagy is known to be induced by stimuli such as nutrient deprivation and suppression of mTOR, but little is known about how autophagosomal biogenesis is initiated in mammalian cells in response to viral infection. Here, using genome-wide short interfering RNA screens, we find that the endosomal protein sorting nexin 5 (SNX5)3,4 is essential for virus-induced, but not for basal, stress- or endosome-induced, autophagy. We show that SNX5 deletion increases cellular susceptibility to viral infection in vitro, and that Snx5 knockout in mice enhances lethality after infection with several human viruses. Mechanistically, SNX5 interacts with beclin 1 and ATG14-containing class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), increases the lipid kinase activity of purified PI3KC3-C1, and is required for endosomal generation of phosphatidylinositol-3-phosphate (PtdIns(3)P) and recruitment of the PtdIns(3)P-binding protein WIPI2 to virion-containing endosomes. These findings identify a context- and organelle-specific mechanism-SNX5-dependent PI3KC3-C1 activation at endosomes-for initiation of autophagy during viral infection.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro , and in the Syrian golden hamster model in vivo . In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants.SARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

Flaviviruses such as Zika virus and West Nile virus have the potential to cause severe neuropathology if they invade the central nervous system. The type I interferon response is well characterized as contributing to control of flavivirus-induced neuropathogenesis. However, the interferon-stimulated gene (ISG) effectors that confer these neuroprotective effects are less well studied. Here, we used an ISG expression screen to identify Shiftless (SHFL, C19orf66) as a potent inhibitor of diverse positive-stranded RNA viruses, including multiple members of the Flaviviridae (Zika, West Nile, dengue, yellow fever, and hepatitis C viruses). In cultured cells, SHFL functions as a viral RNA-binding protein that inhibits viral replication at a step after primary translation of the incoming genome. The murine ortholog, Shfl, is expressed constitutively in multiple tissues, including the central nervous system. In a mouse model of Zika virus infection, Shfl-/- knockout mice exhibit reduced survival, exacerbated neuropathological outcomes, and increased viral replication in the brain and spinal cord. These studies demonstrate that Shfl is an important antiviral effector that contributes to host protection from Zika virus infection and virus-induced neuropathological disease.

ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 57 (ORF57)-encoded protein (Mta) is a multifunctional regulator of viral gene expression. ORF57 is essential for viral replication, so elucidation of its molecular mechanisms is important for understanding KSHV infection. ORF57 has been implicated in nearly every aspect of viral gene expression, including transcription, RNA stability, splicing, export, and translation. Here we demonstrate that ORF57 interacts with the KSHV K-bZIP protein in vitro and in cell extracts from lytically reactivated infected cells. To further test the biological relevance of the interaction, we performed a chromatin immunoprecipitation and microarray (ChIP-chip) analysis using anti-ORF57 antibodies and a KSHV tiling array. The results revealed four specific areas of enrichment, including the ORF4 and K8 (K-bZIP) promoters, as well as oriLyt, all of which interact with K-bZIP. In addition, ORF57 associated with DNA corresponding to the PAN RNA transcribed region, a known posttranscriptional target of ORF57. All of the peaks were RNase insensitive, demonstrating that ORF57 association with the viral genome is unlikely to be mediated exclusively by an RNA tether. Our data demonstrate that ORF57 associates with the viral genome by using at least two modes of recruitment, and they suggest that ORF57 and K-bZIP coregulate viral gene expression during lytic infection.

Nature 505, 691–695 (2014); doi:10.1038/nature12862 In this Letter, we carried out bioinformatic analyses on interferon-stimulated gene screening data sets for multiple viruses, including a data set for West Nile virus (WNV) (Supplementary Table 8 in ref. 1). We recently discovered that the WNV-GFP stock used in our 2011 study 1 was actually Venezuelan equine encephalitis virus (VEEV-GFP).

Microgravity conditions associated with space flight have been shown to cause immune deficiencies in a number of ways, such as decreasing hematopoietic differentiation, cytokine production, and lymphocyte proliferation1. Space flight has also been shown to cause changes in DNA fragmentation and changes in lymphoid organ size in mice models2. Research on immunology in spaceflight became a prevalent topic when NASA published results of a significantly high re-emergence of latent viruses in astronauts returning from space missions, with additional studies showing decreases in cytokine activation and virus-specific T cells3, 4. The evidence for immune system impairment in microgravity conditions is particularly concerning given the plans for longer duration spaceflights for humans. Further research needs to be conducted to better understand the effect of microgravity on immune function. This proposed experiment will investigate the effect of microgravity on adaptive immune response to viral infection by inoculating mice with a virus and studying the immune responses of mice in simulated microgravity conditions. It is hypothesized that the mice in the simulated microgravity condition will show a lower antibody response to LCMV than the control group. Data will be obtained through the use of plaque assays, intracellular cytokine staining, tetramer staining, and ELISAs. Based on previous findings, is anticipated that the hypothesis will be supported; however, aberrant results would still provide insight to the role of microgravity on immune function and response. Introduction The first landing on the moon was in 1969, and NASA proposes to have humans land on Mars by 2030. Outer space is an exciting scientific frontier to explore, but the factors associated with spaceflights of any duration can lead to significant impairment of health. Unsurprisingly, leaving the earth’s orbit brings about a number of unfamiliar circumstances to the individuals who venture out of the atmosphere. The rocket’s takeoff results in a massive exposure to G forces, and the missions themselves tend to result in severe sleep deprivation, nutritional deficiencies, and anxiety or depression1. In addition, research shows that the immune system suffers from the microgravity conditions experienced. Microgravity refers to conditions where the force of gravity is so low that feelings of weightlessness occur. Gravity is generally measured by the speed at which an object would fall in free fall. On Earth, gravity is 9.807m/s2. This value significantly decreases once Earth’s atmosphere is exited—on Mars the force of gravity is 3.711m/s2, and on the moon, gravity is 1.622m/s2 (5). These significant decreases in gravity lead to observable impairment in immune function for a number of species. Drosophila melanogaster, the common fruit fly, showed a complete inability to activate Toll-mediated responses to fungal infections. The stress of microgravity on the body resulted in a faulty transcriptional response. Interestingly, hypergravity conditions, where the force of gravity was higher than experienced on earth, actually improved the signaling strength of Toll-responses, resulting in a faster clearing of the fungal infection6. In mice models, a number of immune changes have been observed. Lymphocyte proliferation is decreased due to a downregulation of T-cell activation markers CD25, CD69, and CD71. Decreased lymphocyte function and proliferation, especially for CD4 T-cells, occurred in a time-dependent manner with exposure to microgravity conditions7. Microgravity causes a significant unloading of mammalian tissues, which decreases tissue growth and regeneration. As a result, mice showed decreased hematopoiesis due to down-regulation of gene-expression markers for early mesenchymal and hematopoietic differentiation by at least two-fold. Cultures of bone marrow cells after microgravity conditions showed increase of mesenchymal differentiation to mineralized bone nodules, whereas hemotopoietic differentiation primarily resulted in osteoclasts. This indicates an increase of undifferentiated progenitor cells after microgravity8. Mice also show a decrease in lymphoid organ size relative to their body mass, with a 13-day spaceflight showing a significant decrease in spleen size. Thymus size was not significantly affected, but an increase in DNA fragmentation in the thymus was observed. T-cell and cancer gene expression markers were highly altered, with 30 out of 84 T-cell genes altered, and 15 out of 84 cancer related genes altered2. This evidence suggests that there could be an increased risk of infection and cancer development associated with spaceflight. IFNγ production is of particular importance, as it is the first response to viral infections. Rodents showed a decrease in IFNγ production in response to T-cell mitogen ConA9. The decrease in IFNγ present in mice causes Swiss/Webster mice that are normally immune to the D variant of encephalomyocarditis to become unusually susceptible in microgravity conditions10. A further investigation of rodent models also indicated an overproduction of cytokines IL-6 and IL-10, and a decrease in TNFα in microgravity conditions11. Humans also show an impaired immune response during microgravity conditions experienced during spaceflight. Astronauts show decreased functionality of their monocytes, with reduced ability to engulf E. coli, induce oxidative burst or degranulate. In addition, responses to gram-negative LPS endotoxins have decreased responsiveness12. These alterations in immunity are likely due to decreased expression of CD14 and increased expression of TLR4, due to LPS responsiveness depending on the association of the CD14-TLR4 myeloid differentiation protein 2 complex1. Cytotoxicity of non-MHC-restricted killer cells was found to be depressed in spaceflight as well, with a 40% decrease in lytic activity after landing13. Cytokine expression changes have also been observed in astronauts, suggesting further indication of decreased natural immune response. Astronauts returning from a 7day spaceflight showed low NK cell activity and decreased IFN secretion, which would reduce the ability to respond to viral infections14. The study of microgravity conditions on immune function became an especially prevalent topic when NASA observed a higher re-emergence of latent viruses in astronauts on short-duration space flights of 12 to 16 days. Astronauts showed increased re-emergence of Epstein-Barr virus, cytomegalovirus, herpes simplex I, and varicella virus3. NASA conducted further studies to investigate the alarming immune deficiencies in astronauts with simulated microgravity experiments. Research participants were found to have a decrease in activation of CD8+, CD69+, and virus-specific T-cells for both CMV and EBV4. Research on immune function in microgravity conditions is highly relevant due to NASA’s plans to send humans to Mars by mid 2030s—this mission would be a duration of a minimum of 520 days15. Additionally, bacteria actually survive better in microgravity conditions, leaving humans at an even greater risk for infections. Bacteria are found to proliferate faster, improve gene expression and pathogenesis, and require higher doses of antibiotics to kill. The increased radiation in space also subjects bacteria to higher levels of mutations, which could lead to increased virulence1. Spaceflight also includes inherent risks that could increase susceptibility to infections, such as the emotional stress due to confinement or fear of failure. Air, food, water, and waste are all recycled on board the space crafts, and the confinement of the passengers has been shown to increases in the transfer of microorganisms16, 17. The role of gravity on immune cells needs to be established before humans are sent on space missions lasting such an exorbitant amount of time, especially considering the evidence of decreased immune function in shortduration spaceflights. Further research could definitively establish a connection between mechanical unloading of tissues negatively impacting hematopoiesis, cytokine production, and adaptive and innate immune responses. Despite the compelling research suggesting significant immune impairment in microgravity conditions, gaps in scientific knowledge exist on the adaptive immune response to specific viral infections in microgravity conditions. This research will investigate the hypothesis that adaptive immune responses will be deficient in microgravity conditions when exposed to viral infection. It is suspected that mice subjected to microgravity conditions will show a lower antibody response of IFNγ and TNF at 30 days than control groups.