Janet L. Douglas

ABSTRACT The primary roles attributed to the human immunodeficiency virus type 1 (HIV-1) Vpu protein are the degradation of the viral receptor CD4 and the enhancement of virion release. With regard to CD4 downregulation, Vpu has been shown to act as an adapter linking CD4 with the ubiquitin-proteasome machinery via interaction with the F-box protein βTrCP. To identify additional cellular βTrCP-dependent Vpu targets, we performed quantitative proteomics analyses using the plasma membrane fraction of HeLa cells expressing either wild-type Vpu or a Vpu mutant (S52N/S56N) that does not bind βTrCP. One cellular protein, BST-2 (CD317), was consistently underrepresented in the membrane proteome of cells expressing wild-type Vpu compared to the proteome of cells expressing the Vpu mutant. To verify the biological relevance of this phenotype for HIV pathogenesis, we showed that in T cells infected with HIV-1, BST-2 downregulation occurred in a Vpu-dependent manner. Recently, BST-2 has been identified as the interferon-inducible cellular factor Tetherin, which restricts HIV virion release in the absence of Vpu. We address here the unresolved mechanism of Vpu-mediated BST-2 downregulation. Our data show that the presence of wild-type Vpu reduced cell surface and total steady-state BST-2 levels, whereas that of the mutant Vpu had no effect. In addition, treatment of cells with the lysosome acidification inhibitor concanamycin A, but not treatment with the proteasome inhibitor MG132, reduced BST-2 downregulation by wild-type Vpu, thereby suggesting that the presence of Vpu leads to the degradation of BST-2 via an endosome-lysosome degradation pathway. The importance of βTrCP in this process was confirmed by demonstrating that in the absence of βTrCP, BST-2 levels were restored despite the presence of Vpu. Taken together, these data support the hypothesis that, in similarity to its role in CD4 degradation, Vpu acts as an adapter molecule linking BST-2 to the cellular ubiquitination machinery via βTrCP. However, in contrast to the proteasome-dependent degradation of CD4, which occurs in the endoplasmic reticulum, Vpu appears to interact with BST-2 in the trans-Golgi network or in early endosomes, leading to lysosomal degradation of BST-2. Via this action, Vpu could counter the tethering function of BST-2, resulting in enhanced HIV-1 virion release. Interestingly, although HIV-2 does not express Vpu, an isolate known to exhibit enhanced viral egress can downregulate surface BST-2 by an as-yet-unknown mechanism that does not appear to involve degradation. Understanding the molecular mechanisms of both Vpu-dependent and -independent mediated antagonism of BST-2 will be critical for therapeutic strategies that exploit this novel viral function.

K3/MIR1 and K5/MIR2 of Kaposi's sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.

The interferon-induced BST-2 protein has the unique ability to restrict the egress of HIV-1, Kaposi's sarcoma-associated herpesvirus (KSHV), Ebola virus, and other enveloped viruses. The observation that virions remain attached to the surface of BST-2-expressing cells led to the renaming of BST-2 as "tetherin". However, viral proteins such as HIV-1 Vpu, simian immunodeficiency virus Nef, and KSHV K5 counteract BST-2, thereby allowing mature virions to readily escape from infected cells. Since the anti-viral function of BST-2 was discovered, there has been an explosion of research into several aspects of this intriguing interplay between host and virus. This review focuses on recent work addressing the molecular mechanisms involved in BST-2 restriction of viral egress and the species-specific countermeasures employed by various viruses.

ABSTRACT Human respiratory syncytial virus (RSV) is a major cause of respiratory tract infections worldwide. Several novel small-molecule inhibitors of RSV have been identified, but they are still in preclinical or early clinical evaluation. One such inhibitor is a recently discovered triphenol-based molecule, VP-14637 (ViroPharma). Initial experiments suggested that VP-14637 acted early and might be an RSV fusion inhibitor. Here we present studies demonstrating that VP-14637 does not block RSV adsorption but inhibits RSV-induced cell-cell fusion and binds specifically to RSV-infected cells with an affinity corresponding to its inhibitory potency. VP-14637 is capable of specifically interacting with the RSV fusion protein expressed by a T7 vaccinia virus system. RSV variants resistant to VP-14637 were selected; they had mutations localized to two distinct regions of the RSV F protein, heptad repeat 2 (HR2) and the intervening domain between heptad repeat 1 (HR1) and HR2. No mutations arose in HR1, suggesting a mechanism other than direct disruption of the heptad repeat interaction. The F proteins containing the resistance mutations exhibited greatly reduced binding of VP-14637. Despite segregating with the membrane fraction following incubation with intact RSV-infected cells, the compound did not bind to membranes isolated from RSV-infected cells. In addition, binding of VP-14637 was substantially compromised at temperatures of ≤22°C. Therefore, we propose that VP-14637 inhibits RSV through a novel mechanism involving an interaction between the compound and a transient conformation of the RSV F protein.

GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] is a novel ribose-modified human immunodeficiency virus type 1 (HIV-1) nucleotide reverse transcriptase (RT) inhibitor (NRTI) selected from a series of nucleoside phosphonate analogs for its favorable in vitro biological properties including (i) a low potential for mitochondrial toxicity, (ii) a minimal cytotoxicity in renal proximal tubule cells and other cell types, (iii) synergy in combination with other antiretrovirals, and (iv) a unique resistance profile against multiple NRTI-resistant HIV-1 strains. Notably, antiviral resistance analysis indicated that neither the K65R, L74V, or M184V RT mutation nor their combinations had any effect on the antiretroviral activity of GS-9148. Viruses carrying four or more thymidine analog mutations showed a substantially smaller change in GS-9148 activity relative to that observed with most marketed NRTIs. GS-9131, an ethylalaninyl phosphonoamidate prodrug designed to maximize the intracellular delivery of GS-9148, is a potent inhibitor of multiple subtypes of HIV-1 clinical isolates, with a mean 50% effective concentration of 37 nM. Inside cells, GS-9131 is readily hydrolyzed to GS-9148, which is further phosphorylated to its active diphosphate metabolite (A. S. Ray, J. E. Vela, C. G. Boojamra, L. Zhang, H. Hui, C. Callebaut, K. Stray, K.-Y. Lin, Y. Gao, R. L. Mackman, and T. Cihlar, Antimicrob. Agents Chemother. 52:648-654, 2008). GS-9148 diphosphate acts as a competitive inhibitor of RT with respect to dATP (K(i) = 0.8 muM) and exhibits low inhibitory potency against host polymerases including DNA polymerase gamma. Oral administration of GS-9131 to beagle dogs at a dose of 3 mg/kg of body weight resulted in high and persistent levels of GS-9148 diphosphate in peripheral blood mononuclear cells (with a maximum intracellular concentration of >9 microM and a half-life of >24 h). This favorable preclinical profile makes GS-9131 an attractive clinical development candidate for the treatment of patients infected with NRTI-resistant HIV.

ABSTRACT Here we present data on the mechanism of action of VP-14637 and JNJ-2408068 (formerly R-170591), two small-molecule inhibitors of respiratory syncytial virus (RSV). Both inhibitors exhibited potent antiviral activity with 50% effective concentrations (EC 50 s) of 1.4 and 2.1 nM, respectively. A similar inhibitory effect was observed in a RSV-mediated cell fusion assay (EC 50 = 5.4 and 0.9 nM, respectively). Several drug-resistant RSV variants were selected in vitro in the presence of each compound. All selected viruses exhibited significant cross-resistance to both inhibitors and contained various single amino acid substitutions in two distinct regions of the viral F protein, the heptad repeat 2 (HR2; mutations D486N, E487D, and F488Y), and the intervening domain between HR1 and HR2 (mutation K399I and T400A). Studies using [ 3 H]VP-14637 revealed a specific binding of the compound to RSV-infected cells that was efficiently inhibited by JNJ-2408068 (50% inhibitory concentration = 2.9 nM) but not by the HR2-derived peptide T-118. Further analysis using a transient T7 vaccinia expression system indicated that RSV F protein is sufficient for this interaction. F proteins containing either the VP-14637 or JNJ-2408068 resistance mutations exhibited greatly reduced binding of [ 3 H]VP-14637. Molecular modeling analysis suggests that both molecules may bind into a small hydrophobic cavity in the inner core of F protein, interacting simultaneously with both the HR1 and HR2 domains. Altogether, these data indicate that VP-14637 and JNJ-2408068 interfere with RSV fusion through a mechanism involving a similar interaction with the F protein.

The transmembrane ubiquitin ligase K5/MIR2 of Kaposi sarcoma herpesvirus (KSHV) mediates internalization and lysosomal degradation of glycoproteins involved in antigen presentation and co-stimulation. In endothelial cells (ECs), K5 additionally reduced expression of CD31/platelet-endothelial cell adhesion molecule (PECAM), an adhesion molecule regulating cell-cell interactions of ECs, platelets, monocytes, and T cells. K5 also reduced EC migration, a CD31-dependent process. Unlike other K5 substrates, both newly synthesized and pre-existing CD31 molecules were targeted by K5. K5 was transported to the cell surface and ubiquitinated pre-existing CD31, resulting in endocytosis and lysosomal degradation. In the endoplasmic reticulum, newly synthesized CD31 was degraded by proteasomes, which required binding of phosphofurin acidic cluster sorting protein-2 (PACS-2) to acidic residues in the carboxyterminal tail of K5. Thus, CD31, a novel target of K5, is efficiently removed from ECs by a dual degradation mechanism that is regulated by the subcellular sorting of the ubiquitin ligase. K5-mediated degradation of CD31 is likely to affect EC function in KS tumors.

Like the other more well-characterized post-translational modifications (phosphorylation, methylation, acetylation, acylation, etc.), the attachment of the 76 amino acid ubiquitin (Ub) protein to substrates has been shown to govern countless cellular processes. As obligate intracellular parasites, viruses have evolved the capability to commandeer many host processes in order to maximize their own survival, whether it be to increase viral production or to ensure the long-term survival of latently infected host cells. The first evidence that viruses could usurp the Ub system came from the DNA tumor viruses and Adenoviruses, each of which use Ub to dysregulate the host cell cycle (Scheffner et al., 1990; Querido et al., 2001). Today, the list of viruses that utilize Ub includes members from almost every viral class, encompassing both RNA and DNA viruses. Among these, there are examples of Ub usage at every stage of the viral life cycle, involving both ubiquitination and de-ubiquitination. In addition to viruses that merely modify the host Ub system, many of the large DNA viruses encode their own Ub modifying machinery. In this review, we highlight the latest discoveries regarding the myriad ways that viruses utilize Ub to their advantage.

Retroviruses have been extensively used in the development of gene transfer systems. Recently, there has been a great deal of interest in the use of lentiviruses for gene transfer because they infect nondividing cells. Human immunodeficiency virus (HIV) has been the lentivirus most often used for this purpose, but its genomic complexity and limited tropism present some challenges to the establishment of efficient gene transfer systems. In this paper we present data showing intrinsic differences between the infectivity of wild-type HIV and HIV particles pseudotyped with heterologous envelope glycoproteins. Interestingly, HIV pseudotypes with envelope glycoproteins from the amphotropic murine leukemia virus or the vesicular stomatitis virus (VSV) are 3 and 40 times more infectious than wild-type HIV, respectively. In addition, we show that the reliance on Nef expression for maximal infectivity of HIV particles is dependent on the path of virus entry. The dependence on Nef for higher infectivity is greater for amphotropic pseudotypes and wild-type HIV than for VSV-G pseudotypes. We conclude that VSV-G pseudotypes of HIV vectors are an excellent choice for gene transfer purposes and Nef-mediated viral infectivity enhancement is affected by virus entry pathway.

GS-9148 (2′-Fd4AP, 4) has been identified as a nucleoside phosphonate reverse transcriptase (RT) inhibitor with activity against wild-type HIV (EC50 = 12 μM). Unlike many clinical RT inhibitors, relevant reverse transcriptase mutants (M184V, K65R, 6-TAMs) maintain a susceptibility to 2′-Fd4AP that is similar to wild-type virus. The 2′-fluorine group was rationally designed into the molecule to improve the selectivity profile and in preliminary studies using HepG2 cells, compound 4 showed no measurable effect on mitochondrial DNA content indicating a low potential for mitochondrial toxicity.

GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] 4 is a novel nucleoside phosphonate HIV-1 reverse transcriptase (RT) inhibitor with a unique resistance profile toward N(t)RTI resistance mutations. To effectively deliver 4 and its active phosphorylated metabolite 15 into target cells, a series of amidate prodrugs were designed as substrates of cathepsin A, an intracellular lysosomal carboxypeptidase highly expressed in peripheral blood mononuclear cells (PBMCs). The ethylalaninyl phosphonamidate prodrug 5 (GS-9131) demonstrated favorable cathepsin A substrate properties, in addition to favorable in vitro intestinal and hepatic stabilities. Following oral dosing (3 mg/kg) in Beagle dogs, high levels (>9.0 μM) of active metabolite 15 were observed in PBMCs, validating the prodrug design process and leading to the nomination of 5 as a clinical candidate.

The amphotropic murine leukemia virus (MuLV) can infect cells from a number of mammals, including humans, via its specific receptor. Basic knowledge of amphotropic MuLV receptor expression is likely to be useful in the development and improvement of gene therapy protocols based on amphotropic-pseudotyped vectors. To investigate the expression of the human receptor for the amphotropic MuLV (GLVR-2, newly termed Pit2), we determined its mRNA levels in several cell lines and found them to vary significantly. Induction of increased levels of mRNA after removal of phosphate from the media was observed in two osteosarcoma cell lines. The increase in GLVR-2 mRNA resulted in a concomitant rise in the levels of a 71-kDa protein specifically recognized by affinity-purified antibodies against GLVR-2. Using these antibodies, we were able to confirm the intracellular topology of the large hydrophilic domain between the proposed sixth and seventh transmembrane domains of the GLVR-2 protein. This assignment is in agreement with the fourth extracellular loop being outside the cell, consistent with the proposal that the fourth extracellular loop of GLVR-2 contains the envelope binding site.

The cellular protein BST-2/CD317/Tetherin has been shown to inhibit the release of HIV-1 and other enveloped viruses from infected cells. The HIV-1 accessory protein Vpu binds to both BST-2 and βTrCP, a substrate-recognition subunit for the SCF (Skip1-Cullin1-F-box protein) E3 ubiquitin ligase complex. This interaction leads to both the degradation of BST-2 and the enhancement of viral egress. Recently BST-2 was shown to be ubiquitinated in this process. Here we have confirmed the Vpu- and βTrCP-dependent multi/polyubiquitination of BST-2. Ubiquitinated BST-2 accumulated in cells treated with a lysosomal inhibitor but not a proteasomal inhibitor. Additionally, we observed that a BST-2 mutant deleted for its cytosolically exposed lysine residues is also ubiquitinated. Subsequent experiments suggested that Vpu promotes BST-2 ubiquitination upon amino acid residues bearing hydroxyl- but not thiol-bearing side chains. However, a BST-2 mutant bearing substitutions for its cytoplasmically exposed Ser, Thr, and Lys residues was still down-regulated, ubiquitinated, and degraded in a Vpu-dependent manner. Our results suggest that Vpu may target either the BST-2 cytoplasmic Tyr residues or the NH(2) terminus itself for ubiquitination.

Background.BST2/tetherin is an innate immune molecule with the unique ability to restrict the egress of human immunodeficiency virus (HIV) and other enveloped viruses, including Ebola virus (EBOV).Coincident with this discovery was the finding that the HIV Vpu protein down-regulates BST2 from the cell surface, thereby promoting viral release.Evidence suggests that the EBOV envelope glycoprotein (GP) also counteracts BST2, although the mechanism is unclear.Results.We find that total levels of BST2 remain unchanged in the presence of GP, whereas surface BST2 is significantly reduced.GP is known to sterically mask surface receptors via its mucin domain.Our evaluation of mutant GP molecules indicate that masking of BST2 by GP is probably responsible for the apparent surface BST2 down-regulation; however, this masking does not explain the observed virus-like particle egress enhancement.We discovered that VP40 coimmunoprecipitates and colocalizes with BST2 in the absence but not in the presence of GP.Conclusions.These results suggest that GP may overcome the BST2 restriction by blocking an interaction between VP40 and BST2.Furthermore, we have observed that GP may enhance BST2 incorporation into virus-like particles.Understanding this novel EBOV immune evasion strategy will provide valuable insights into the pathogenicity of this deadly pathogen.

The development of gene transfer systems for the efficient transduction of human primary cells including lymphocytes and CD34+ cells is a significant step in the advancement of gene therapy and cell marking protocols. Efficient gene transfer systems also represent useful tools for basic research. Here we show that human primary lymphocytes and CD34+ cells can be efficiently transduced using a VSV-G pseudotyped HIV-1-based gene transfer system. The enhanced green fluorescent protein (EGFP) was chosen as the marker transgene, because it can be easily visualized and quantitated using fluorescence microscopy and flow cytometry, thus eliminating the need for selection or PCR to score transduction. Vectors produced with this system did not generate replication-competent retroviruses (RCRs) and efficiently transduced human cell lines (40-90%), PBMCs (60%), mobilized CD34+ cells (39%), and CD34+ cells from umbilical cord blood (60%) as measured by flow cytometry. Cells treated with AZT prior to infection did not express EGFP, ruling out passive protein or plasmid DNA transfer. This was further confirmed in methylcellulose cultures, where expression in myeloid and erythroid colonies was maintained for at least 3 weeks. In addition, this HIV-based vector was able to efficiently transduce freshly isolated, not-prestimulated CD34+ cells (70% EGFP positive) in serum-free medium. Under these same conditions, a Moloney murine leukemia virus-based vector failed to transduce not-prestimulated CD34+ cells. These characteristics make this gene transfer system an excellent choice for both basic science and possible gene therapy applications.

Abstract The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34+ cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin− bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

Kaposi's sarcoma (KS) is a complex cancer characterized by angioproliferative multifocal tumors of the skin, mucosa and viscera. KS lesions are comprised of both distinctive spindle cells of endothelial origin and a variable inflammatory infiltrate. There are four different epidemiological forms of KS: classic (sporadic), African (endemic), AIDS-associated (epidemic), and immunosupression-associated (iatrogenic). Although these various forms of KS have different environmental and immunological components, the development of each depends upon infection with Kaposi's sarcoma herpesvirus/human herpesvirus-8 (KSHV/HHV8). KSHV encodes an arsenal of gene products that induce cellular proliferation, transformation, cell signaling, cytokine production, immune evasion, antiapoptosis and angiogenesis. Yet, KSHV alone is insufficient to give rise to KS. The exact origin of the tumor cell (spindle cell), which is generally agreed to be a type of endothelial cell, remains elusive. Current evidence supports their derivation from lymphatic endothelium. However, both lymphatic and vascular endothelial cell types can be infected by KSHV in vitro, and recent studies suggest that this virus may reprogram the target cell, thus masking the cell's true origin. It is also possible that the original target cell is an uncommitted progenitor. In addition to the potentially neoplastic spindle cells, the KS lesion also contains dendritic cells, macrophages, plasma cells and lymphocytes. The presence of this admixed immune infiltrate has led to the suggestion that KS may result from reactive hyperproliferation induced by chronic inflammation, and that it is therefore not a true neoplasm. This review details the data that support KS as a model of both oncogenesis and chronic inflammation.

Immortalization of primary cells is a multistep process. The adenovirus E1A 12S gene product is a member of the class of oncoproteins that have the ability to establish primary cells as cell lines in culture. It is encoded by two exons. Extensive mutational analysis demonstrates that four regions of the E1A 12S gene, encoded by both exons, are necessary for immortalization of primary epithelial cells. Expression of two regions is necessary to activate quiescent cells into the cell cycle but is unable to extend the life span of these cells in culture and thus cannot immortalize them. These regions are encoded by the first exon. A third first-exon region, for which no function has yet been identified, is also required. These three regions are also required for 12S to cooperate with an activated ras gene to bring about tumorigenic transformation. The fourth region is required to maintain the cells in a proliferative mode, extend their life span in culture, and induce an autocrine growth factor. These functions are encoded by the second exon. The cells immortalized by wild-type 12S and immortalization-competent mutants retain their epithelial morphology and expression of keratin and vimentin intermediate filament proteins.

A series of nucleoside phosphonate reverse transcriptase (RT) inhibitors have been synthesized and their anti-HIV activity and resistance profiles evaluated. The most potent analog [5-(6-amino-purin-9-yl)-2,5-dihydro-furan-2-yloxymethyl]-phosphonic acid (d4AP) demonstrated a HIV EC50 = 2.1 μM, and the most favorable resistance profile against HIV-1 variants with K65R, M184V or multiple thymidine analog mutations in RT.

Phosphonomethoxy nucleoside analogs of the thymine containing nucleoside reverse transcriptase inhibitors (NRTIs), 3′-azido-2′,3′-dideoxythymidine (AZT), 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), and 2′,3′-dideoxythymidine (ddT), were synthesized. The anti-HIV activity against wild-type and several major nucleoside-resistant strains of HIV-1 was evaluated together with the inhibition of wild-type HIV reverse transcriptase (RT). Phosphonomethoxy analog of d4T, 8 (d4TP), demonstrated antiviral activity with an EC50 value of 26 μM, whereas, phosphonomethoxy analogs of ddT, 7 (ddTP), and AZT, 6 (AZTP), were both inactive at concentrations up to 200 μM. Bis-isopropyloxymethylcarbonyl (bisPOC) prodrugs improved the anti-HIV activity of 7 and 8 by >150-fold and 29-fold, respectively, allowing for antiviral resistance to be determined. The K65R RT mutant virus was more resistant to the bisPOC prodrugs of 7 and 8 than bisPOC PMPA (tenofovir DF) 1. However, bisPOC prodrug of 7 demonstrated superior resistance toward the RT virus containing multiple thymidine analog mutations (6TAMs) indicating that new phosphonate nucleoside analogs may be suitable for targeting clinically relevant nucleoside resistant HIV-1 strains.

Modified purine analogs of GS-9148 [5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl]-phosphonic acid (2'-Fd4AP) were synthesized and their anti-HIV potency evaluated. The antiviral activity of guanosine analog (2'-Fd4GP) was comparable that of to 2'-Fd4AP in MT-2 cells, but selectivity was reduced.

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E–restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E–restricted CD8 + T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E–restricted CD8 + T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E + prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8 + T cell–based immunotherapies.

The inefficient transduction of human hematopoietic stem cells (HSC) with amphotropic retroviral vectors has been an obstacle to gene therapy for hematopoietic diseases. We have previously reported low levels of amphotropic retrovirus receptor (Pit-2) mRNA and higher levels of gibbon ape leukemia virus (GALV) or 10A1 retrovirus receptor (Pit-1) mRNA in mouse and human HSC. The vesicular stomatitis virus (VSV-G) uses an abundant membrane phospholipid as a receptor. We hypothesized that transduction of HSC requires relatively high levels of retrovirus receptor molecules. Because mouse HSC can be efficiently transduced by ecotropic virus through the abundant ecotropic receptor, the mouse is an ideal model to compare receptor levels and transduction. We have developed a cotransduction assay where ecotropic retrovirus transduction is a positive internal control for downstream steps in retrovirus transduction. A comparison of mouse HSC transduction with amphotropic, 10A1, and VSV-G envelopes showed that the level of amphotropic and 10A1 receptor mRNA in HSC correlated with the frequency of transduction. Transduction with VSV-G vectors was similar to that with 10A1 vectors. We conclude that the level of retrovirus receptor on HSC is critical for HSC transduction and that GALV or VSV-G vectors would be better for human HSC transduction. The inefficient transduction of human hematopoietic stem cells (HSC) with amphotropic retroviral vectors has been an obstacle to gene therapy for hematopoietic diseases. We have previously reported low levels of amphotropic retrovirus receptor (Pit-2) mRNA and higher levels of gibbon ape leukemia virus (GALV) or 10A1 retrovirus receptor (Pit-1) mRNA in mouse and human HSC. The vesicular stomatitis virus (VSV-G) uses an abundant membrane phospholipid as a receptor. We hypothesized that transduction of HSC requires relatively high levels of retrovirus receptor molecules. Because mouse HSC can be efficiently transduced by ecotropic virus through the abundant ecotropic receptor, the mouse is an ideal model to compare receptor levels and transduction. We have developed a cotransduction assay where ecotropic retrovirus transduction is a positive internal control for downstream steps in retrovirus transduction. A comparison of mouse HSC transduction with amphotropic, 10A1, and VSV-G envelopes showed that the level of amphotropic and 10A1 receptor mRNA in HSC correlated with the frequency of transduction. Transduction with VSV-G vectors was similar to that with 10A1 vectors. We conclude that the level of retrovirus receptor on HSC is critical for HSC transduction and that GALV or VSV-G vectors would be better for human HSC transduction.

The methods available to efficiently transduce human CD34+ hematopoietic stem cells (HSCs) derived from mobilized peripheral blood, such that they fully retain their engraftment potential and maintain high levels of transgene expression in vivo, have been unsatisfactory. The current murine retrovirus-based gene transfer systems require dividing cells for efficient transduction, and therefore the target HSCs must be activated ex vivo by cytokines to cycle, which may limit their engrafting ability. Lentivirus-based gene transfer systems do not require cell division and, thus, may allow for efficient gene transfer to human HSCs in the absence of any ex vivo cytokine stimulation. We constructed human immunodeficiency virus (HIV)-based vectors and compared them in vitro and in vivo with MuLV-based vectors in their ability to transduce unstimulated human CD34+ HSCs isolated from mobilized peripheral blood. Both sets of vectors contained the marker gene that expresses the enhanced green fluorescent protein (EGFP) for evaluating transduction efficiency and were pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or the amphotropic murine leukemia virus envelope (A-MULV Env). The VSV-G-pseudotyped HIV-based vectors containing an internal mouse phosphoglycerate kinase promoter (PGK) were able to transduce up to 48% of the unstimulated CD34+ cells as measured by EGFP expression. When these cells were injected into the human fetal thymus implants of irradiated SCID-hu Thy/Liv mice, up to 18% expressed EGFP after 8 weeks in vivo. In contrast, the MULV-based vectors were effective at transducing HSCs only in the presence of cytokines. Our results demonstrate that the improved HIV-based gene transfer system can effectively transduce unstimulated human CD34+ HSCs, which can then differentiate into thymocytes and provide long-term transgene expression in vivo.

Cyclic phosphonomethoxy pyrimidine nucleosides that are bioisosteres of the monophosphate metabolites of HIV reverse transcriptase (RT) inhibitors AZT, d4T, and ddC have been synthesized. The RT inhibitory activities of the phosphonates were reduced for both dideoxy (dd) and dideoxydidehydro (d4) analogs compared to the nucleosides. Bis-isopropyloxymethylcarbonyl (BisPOC) prodrugs were prepared on selected compounds and provided > 150-fold improvements in antiviral activity.

Expression of adenovirus type 5 E1A 12S is sufficient to immortalize primary baby rat kidney cells, but another viral or cellular oncogene, such as E1B or T24ras, is necessary for complete transformation. The regions of 12S sufficient for T24ras cotransformation have been well characterized and are located in the first exon. The second exon is dispensable for ras cotransformation, although it contains a region which appears to modulate the transforming phenotype. The same 12S first exon regions important in ras transformation are also necessary for E1B transformation. Analysis of an extensive series of second exon deletion and amino acid point mutations demonstrated that mutations affecting either the efficient nuclear localization and/or the immortalizing ability of the 12S protein also prevented cooperation with E1B. In general, the entire C-terminal half of 12S, including the nuclear localization signal, was necessary for efficient cotransformation with E1B. In addition to the differences between T24ras and E1B regarding 12S regions necessary for cotransformation, the characteristics of E1B-cotransformed foci differed from those of T24ras. The E1B foci took longer to appear and had a much slower growth rate. No hypertransformed foci were produced with E1B cotransfections, and established E1A-E1B lines exhibited minimal growth in soft agar compared with that of E1A-T24ras lines.

We have undertaken a genetic strategy to map Vpu regions necessary for BST-2 antagonism and viral egress. This approach is based on our identification of an egress-defective Vpu variant encoded by an HIV-1 subtype C strain. We constructed a series of chimeric Vpu molecules made from the Vpu C variant and Vpu B from a standard laboratory strain. The TM domain from the inactive Vpu C, which contains multiple non-conserved residues, was responsible for a significant decrease in egress activity and BST-2 downregulation, confirming the functional importance of the Vpu TM domain. However, for complete inactivation, both the N-terminus and TM domain from the inactive Vpu C molecule were required, suggesting a new role for the Vpu N-terminus. In addition, determinants in the C-terminus of Vpu B that may be involved in efficient TGN accumulation were also necessary for enhanced viral egress but are missing or non-functional in Vpu C.

DNA sequences corresponding to a novel herpesvirus (human herpesvirus 8 [HHV8]) are associated with Kaposi's sarcoma (KS), Castleman's disease, and body cavity-based lymphomas (BCBL). Studies of a BCBL-derived cell line suggest a direct correlation between seropositivity against antigens specifically present in such lines and the development of KS. We have generated recombinant proteins corresponding to open reading frame (ORF) 26 of HHV8 and have produced affinity-purified antibodies. Using these antibodies, we studied the expression of HHV8 ORF26 in a BCBL-derived cell line and found that it encodes a cytoplasmic protein whose expression is induced 16-fold by treatment with phorbol ester or sodium butyrate. This protein induction correlates with a significant induction of viral RNA transcripts. Interestingly, under our experimental conditions minimal increases in viral DNA were observed. No antibodies to the ORF26 protein of HHV8 were found in the sera from two human immunodeficiency virus-positive patients with KS as determined by immunoprecipitation analysis. However, antibodies in the sera from the two KS patients immunoprecipitated a 34-kDa protein found in extracts from induced BCBL1 cells that was not recognized by the control sera.

Respiratory syncytial virus has been an ongoing health problem for 50 years. Hospitalization rates due to virus-induced respiratory illness continue to be substantial for infants, small children, the elderly and the immunocompromised. The only currently available treatments are a broad-spectrum antiviral and two immunoprophylactic antibodies, all of which are reserved for high-risk patients. The combination of this limited therapeutic repertoire and the lack of a vaccine clearly demonstrates the need to continue the search for more efficacious and safe agents against respiratory syncytial virus. The following is a review on the current progress of that search.

The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34+ cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin− bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

In the fields of virology and innate immunity, BST-2/tetherin is well known for its ability to block the egress of enveloped viruses from infected cells. This appears to be accomplished by ‘tethering’ virions to the cell surface, thereby limiting virion release. In the past year, several groups have discovered that BST-2/tetherin can activate NF-κB, a transcriptional activator that leads to the rapid expression of both proinflammatory cytokines and proteins involved in cell survival. While this new BST-2 function has been interpreted as a possible viral-sensing mechanism, there may also be broader implications for HIV gene regulation. This article reviews the evidence for BST-2-dependent NF-κB activation, and explores the significance of these exciting new results.

Human CD34(+) cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous hematopoietic disorders. However, the efficient introduction of exogenous genes into this small, quiescent population of cells continues to present a significant challenge. To circumvent the need for high initial transduction efficiency of human hematopoietic cells, we investigated a dominant selection strategy using a variant of the DHFR gene (DHFR(L22Y)). For this purpose, we constructed a lentivirus-based bicistronic vector expressing EGFP and DHFR(L22Y). Here we demonstrate efficient in vitro selection and enrichment of lentivirus vector-transduced human CD34(+) hematopoietic cells from fetal liver, umbilical cord blood, bone marrow, and peripheral blood after cytokine mobilization. Growth of transduced human CD34(+) cells in semisolid culture under selective pressure resulted in enrichment of transduced progenitor cells to 99.5% (n = 14). Selection for DHFR(L22Y)(+) cells after expansion of transduced progenitors in liquid culture resulted in a 7- to 13-fold increase in the percentage of marked cells. Thus we have shown that transduced human hematopoietic cells may be effectively enriched in vitro by dominant selection, suggesting that development of such strategies holds promise for future in vivo application.

Abstract Systemic injection of hematoporphyrin derivative (HpD) in combination with visible light (red or blue‐green) delivered by laser was used to treat a patient with psoriasis. The psoriatic lesions responded vigorously to laser treatments, forming eschars by 1 week post irradiation. In contrast, only minimal erythema was observed in the noninvolved, clinically normal appearing skin. Two approaches for localized HpD administration were investigated in the guinea‐pig and minipig models as a means of achieving local photodynamic effects. Intracutaneous injection of HpD produced localized cutaneous photosensitization with either UVA or red light. Azone increased percutaneous penetration of HpD in human skin in vitro. Topical application of HpD and irradiation with UVA produced localized cutaneous photosensitivity and inhibition of epidermal DNA synthesis.

<b>Aims:</b> Recent results generated in a mouse model suggest that tumour angiogenesis/vasculogenesis can be initiated and maintained by bone marrow derived endothelial progenitor cells. This present study investigated the distribution and frequency of CD133 positive endothelial progenitor cells in patients with non-small cell lung cancer (NSCLC) (tumour tissue and tumour free lung regions) and healthy controls using fresh frozen specimens. The novel marker CD133 identifies human haemopoetic precursor cells, in addition to human endothelial progenitor cells. <b>Methods:</b> Seventy nine lung cancer specimens and 66 adjacent histologically tumour free tissues of the same patient cohort were analysed; 11 postmortem specimens from control patients who did not suffer from malignant disease served as controls. Cryostat sections were stained for CD133, CD31, vascular endothelial growth factor receptor 2 (VEGFR-2; KDR), p53, and the proliferation marker Ki-67, and the correlations were analysed. <b>Results:</b> Forty three of 63 evaluable tumour specimens had increased numbers of CD133 positive cells and in some cases capillary forming CD133 positive structures were detectable. In addition, 30 of 63 specimens had raised expression of KDR and 29 of 63 had increased MVD. Increased CD133 expression marginally correlated with raised KDR expression but not with p53 and Ki-67. <b>Conclusion:</b> A significant increase in CD133 positive cells was documented in patients with NSCLC, suggesting an involvement of endothelial progenitor cells in tumour vasculogenesis and tumour growth in these patients.

Kaposi's sarcoma (KS) herpesvirus (KSHV) is the etiological agent of several immunodeficiency-linked cancers, including KS. Our previous work showed that the proto-oncogene c-kit is upregulated in KSHV-infected endothelial cells (ECs), as well as in KS lesions. We show here that KSHV-dependent induction of both c-kit mRNA and protein requires the establishment of a latent infection and that this upregulation occurs in primary DMVECs as well as in immortalized DMVECs (eDMVECs). Interestingly, we find that while the lymphatic EC (LEC) subpopulation exhibits KSHV-induced c-Kit upregulation, the blood EC (BEC) subpopulation does not. Despite this upregulation of c-Kit, receptor activation and phosphorylation of downstream effectors such as MAP Kinase Erk 1/2 and GSK-3 still requires the addition of exogenous c-Kit ligand, stem cell factor (SCF). These data indicate that KSHV does not induce constitutive c-Kit signaling, but instead upregulates c-Kit receptor levels, thus allowing infected ECs to respond to endogenous and exogenous SCF. Nonetheless, inhibition of either c-Kit activation or its downstream effectors reverses the characteristic spindle phenotype of infected eDMVECs. Together, these results contribute to our overall understanding of the role that the c-kit proto-oncogene plays in KS pathogenesis.

The Ad5 E1A 12S gene encodes an oncoprotein with the ability to immortalize and cooperate with other viral or cellular oncoproteins to transform primary epithelial cells. The immortalizing function is dependent on the protein's efficient localization to the nucleus. A five amino acid nuclear localization signal (NLS), Lys-Arg-Pro-Arg-Pro, has been identified at the extreme COOH-terminus. This signal is necessary but not sufficient for efficient nuclear localization. A mutational analysis has been undertaken to further characterize the 12S NLS. The individual amino acids of the signal appear to have varying functional relevance. The lysine residue (a.a. 239) and the first arginine residue (a.a. 240) are the most critical. Changing the second arginine (a.a. 242) to threonine or either proline (a.a. 241 or 243) to alanine marginally diminishes signal function. Replacing the 12S NLS with the SV40 large T antigen (LT) NLS does not measurably affect the protein's nuclear localization. Sequences directly upstream of the NLS have a significant role in the proper localization of the 12S protein as illustrated by inefficiently localized mutants that have deletions of these sequences. Analyses of these mutants using a monoclonal antibody that recognizes the COOH-terminal four amino acids of the NLS have revealed that their signals are probably masked. To further investigate the importance of protein context in signal function, several NLS insertion mutants were constructed. Two regions in the first exon with predicted high surface probabilities and no known functions were chosen as sites for NLS insertions. Neither a wild-type 12S- nor a SV40 LT-NLS was functional in any of the new locations, indicating that for 12S, positioning of the NLS in the protein is critical.

Immortalization of primary cells is an early and important event in multistep tumorigenesis and is itself a multistep process. Adenovirus E1A 12S encodes an oncoprotein that can rescue cells from senescence and overcome apoptosis, leading to their immortalization. Five regions of 12S, located in both exons, are required for immortalization. Two regions in the first exon are necessary to activate the cell cycle, increase the number of population doublings, and overcome the M1 stage of mortality. However, extension of life span requires overcoming crisis or M2, which can be accomplished by the expression of the second exon. Several cellular proteins associate with the peptide encoded by the first exon of 12S including pRB, p107, p130, and p300. The importance of pRB-E1A and p300-E1A complexes in transformation is well established; however, their roles in 12S-mediated immortalization remain undefined. Results obtained from the present study using a panel of second exon immortalization-defective mutants demonstrate that formation of pRB-E1A and p300-E1A complexes is insufficient for immortalization of primary cells. We further demonstrate that the expression levels of another tumor suppressor protein, p53, also do not correlate with the inability of the mutants to immortalize. Thus, mutations in the second exon of 12S do not affect the early steps in the immortalization pathway. The second exon mutants are defective in performing a late function in immortalization, involving the reactivation of the cell cycle, indicating that it is a crucial event in immortalization.

CXCR7 was recently characterized as an alternative receptor for the chemokine CXCL12/SDF-1, previously thought to bind and signal exclusively through CXCR4. We recently identified CXCR7 as a key cellular factor in the endothelial cell (EC) dysfunction associated with KSHV infection. CXCL12 signaling is critically associated with tumor growth, angiogenesis and metastasis in several diverse tumors and is one of the most studied chemokine/chemokine receptor interactions in cancer systems. The tumorigenic activity of the CXCL12 signaling axis offers an attractive target for therapeutic intervention in multiple cancers including Kaposi’ sS arcoma (KS). However, most of the research to date was based on the assumption that CXCR4 was the sole CXCL12 receptor, and thus focused on the development of CXCR4-targeted treatments. CXCR4 participates in important homeostatic functions including hematopoiesis and mucosal immunity, while CXCR7 is rarely expressed in normal adult cells. As a result, CXCR7 may be a more specific chemotherapeutic target for tumor cells and tumor-associated vasculature with fewer adverse effects than treatments targeting CXCR4. CXCR7 is poorly studied throughout the cancer literature and although CXCR7 expression has been found in tumor-associated vasculature, no studies comprehensively examine the biology of CXCR7 in EC and its implications for tumor biology. We seek to define th er ole of CXCR7-mediated CXCL12 signaling in EC biology, and in the context of KSHV infection, in order to determine potential contributions of CXCR7 signaling to KSHV-mediated EC transformation and KS tumorigenesis. We demonstrate that CXCR7 is strongly expressed on LANA+ spindle cells in KS biopsy tissue at all stages of tumor progression. We further demonstrate that CXCR7 induction by KSHV in vitro is specific to lymphatic EC lineages and occurs coincident with the acquisition of spindle morphology. Detailed examination of CXCR7 functions in EC biology reveals multiple roles for CXCR7 that could impact KS tumorigenesis, including effects on cellular proliferation, junctional integrity, cell survival and metastatic capacity. Specifically, we determine that CXCR7 expression results in a loss of PECAM/CD31 expression, perturbing the formation and maintenance of EC monolayers. Moreover, CXCR7+ EC display significant SDF-1 dependent hypermotility, as measured via Electrical Cell-Substrate Impedence Sensing (ECIS). We also demonstrate that SDF-1 signaling through CXCR7 expression is enhanced in EC undergoing anchorage-deprivation, affecting EC cell survival and invasion into SDF-1 rich niches. Taken together, these results demonstrate that CXCR7 is a novel KSHV-induced oncogene with the capacity to influence multiple aspects of KS pathogenesis including tumor growth, seeding and metastasis.

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Abstract The transmembrane ubiquitin ligase K5/MIR2 of Kaposi’s sarcoma herpesvirus (KSHV) mediates internalization and lysosomal degradation of glycoproteins involved in antigen presentation and co-stimulation. In endothelial cells (ECs), K5 additionally reduced expression of CD31/PECAM, an adhesion molecule regulating cell-cell interactions of ECs, platelets, monocytes and T cells. K5 also reduced EC migration, a CD31-dependent process. Unlike other K5 substrates, both newly synthesized and pre-existing CD31 molecules were targeted by K5. K5 was transported to the cell surface and ubiquitinated pre-existing CD31 resulting in endocytosis and lysosomal degradation. In the endoplasmic reticulum, newly synthesized CD31 was degraded by proteasomes which required binding of phosphofurin acidic cluster sorting protein-2 (PACS-2) to acidic residues in the carboxyterminal tail of K5. Thus, CD31, a novel target of K5, is efficiently removed from ECs by a dual degradation mechanism that is regulated by the subcellular sorting of the ubiquitin ligase. K5-mediated degradation of CD31 is likely to affect EC function in KS tumors.

The primary roles attributed to the HIV-1 Vpu protein are the targeted downregulation of the viral receptor CD4 and the enhancement of virion release. With regards to CD4 degradation, Vpu has been shown to act as an adaptor linking CD4 with the ubiquitin/proteasome machinery via interaction with the F-box protein βTrCP. This interaction was previously thought to be dispensable for Vpu's virion release function. In an attempt to identify other cellular βTrCP-dependent Vpu targets, we performed quantitative proteomics on the plasma membrane fraction of Hela cells expressing either a wildtype Vpu, or a Vpu double point mutant (S52N/S56N) that no longer binds to βTrCP. We identified one cellular protein, BST-2 (CD317), that was significantly downregulated by wildtype Vpu, but not the mutant. Recently, BST-2 has been identified as the IFN-inducible cellular factor Tetherin, which limits HIV virion release in the absence of Vpu. We address here the mechanism of Vpu-mediated BST-2 downregulation. To verify the biological relevance of this phenotype, we show that in T-cells infected with HIV-1, BST-2 downregulation does indeed occur and is Vpu dependent. Interestingly, HIV-2, which does not encode Vpu, did not downregulate BST-2. Because our screen was designed to identify those proteins downregulated by Vpu in a βTrCP-dependent manner, we next sought to determine if Vpu targets BST-2 for degradation. We show via immunofluorescence and co-immunoprecipitations that both wildtype and mutant Vpu are able to interact with BST-2, but only wildtype Vpu results in BST-2 degradation. Further support for βTrCP's role was provided by showing that the dominant negative mutant ΔF-βTrCP, which can no longer interact with the SCF ubiquitin ligase complex, blocks Vpu's ability to downregulate BST-2. In addition, treatment of cells with either the proteasome inhibitor MG132 or the lysosome acidification inhibitor concanamycin A reduces BST-2 downregulation by wildtype Vpu. Taken together, these data support the hypothesis that, similar to its role in CD4 degradation, Vpu acts as an adaptor molecule linking BST-2 to the cellular ubiquitination machinery via βTrCP. This interaction then leads to the degradation of BST-2, which prevents viral tethering and therefore results in enhanced virion release.

Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Abstract Pre-clinical models in non-human primates demonstrate that cytomegalovirus (CMV)-vectored vaccines are unique in their ability to elicit and indefinitely maintain high frequencies of polyfunctional effector memory T cells to heterologous pathogen antigens, including in animals that are already chronically CMV infected. By introducing defined genetic modifications into the CMV backbone it is possible to program CD8+ T cell responses that are either directed to MHC-I, MHC-II or the non-classical MHC-I molecule MHC-E. In progress clinical studies are evaluating CMV-based vaccine immunity in humans as potential vaccines against HIV. To see if the pre-clinical findings could be extended to cancer antigens we inserted known tumor associated antigens (TAA) or viral TAA into genetically modified rhesus CMV (RhCMV) and characterized the T cell response in rhesus macaques. We found T cell responses to all TAA that were comparable to pathogen antigen-specific responses in frequency, duration, phenotype, epitope density and MHC-restriction. Since many of these TAA are expressed in healthy tissue, this suggests that CMV-vectored cancer vaccines are well-suited to overcome immunological tolerance. As such, CMV-based vectors expressing TAAs could be a powerful new tool for cancer immunotherapy. We show that TAA-specific, MHC-E restricted CD8+ T cells from RhCMV/TAA-immunized RM are stimulated by TAA-expressing cancer tissues and cell lines, indicating that cancer cells can present TAA-derived peptides via MHC-E. Since MHC-E is often upregulated in cancer cells to engage the inhibitory NKG2A receptor on tumor-infiltrating T and NK cells, these results suggest that MHC-E could be used as a novel target for T cell-based immunotherapies.

The chapter traces the development of Zionism among Leeds Jewry, taking inspiration from the work of Theodore Herzl. In many ways, Zionism acted as a unifying influence in what was often a fragmented community, particularly since support did not depend on the degree of religious orthodoxy. Zionism in Leeds received a great stimulus from the arrival of Selig Brodetsky, who became the main organiser and leader. The city also was inspired by the visits of Chaim Weizmann.

The chapter traces the development of Zionism among Leeds Jewry, taking inspiration from the work of Theodore Herzl. In many ways, Zionism acted as a unifying influence in what was often a fragmented community, particularly since support did not depend on the degree of religious orthodoxy. Zionism in Leeds received a great stimulus from the arrival of Selig Brodetsky, who became the main organiser and leader. The city also was inspired by the visits of Chaim Weizmann.