MicroRNA-based Regulation of Picornavirus Tropism.

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

MicroRNA-Detargeted Mengovirus for Oncolytic Virotherapy.

UNLABELLED: Mengovirus, a member of thePicornaviridaefamily, has a broad cell tropism and can cause encephalitis and myocarditis in multiple mammalian species. Attenuation has been achieved by shortening the polycytidine tract in the 5' noncoding region (NCR). A poly(C)-truncated strain of mengovirus, vMC24, resulted in significant tumor regression in immunocompetent BALB/c mice bearing syngeneic MPC-11 plasmacytomas, but the associated toxicities were unacceptable. To enhance its safety profile, microRNA target sequences complementary to miR-124 or miR-125 (enriched in nervous tissue), miR-133 and miR-208 (enriched in cardiac tissue), or miR-142 (control; enriched in hematopoietic tissues) were inserted into the vMC24NCRs. The microRNA-detargeted viruses showed reduced replication and cell killing specifically in cells expressing the cognate microRNAs, but certain insertions additionally were associated with nonspecific suppression of viral fitnessin vivo. In vivotoxicity testing confirmed that miR-124 targets within the 5' NCR suppressed virus replication in the central nervous system while miR-133 and miR-208 targets in the 3' NCR suppressed viral replication in cardiac tissue. A dual-detargeted virus named vMC24-NC, with miR-124 targets in the 5' NCR and miR-133 plus miR-208 targets in the 3' NCR, showed the suppression of replication in both nervous and cardiac tissues but retained full oncolytic potency when administered by intratumoral (10(6)50% tissue culture infectious doses [TCID50]) or intravenous (10(7)to 10(8)TCID50) injection into BALB/c mice bearing MPC-11 plasmacytomas. Overall survival of vMC24-NC-treated tumor-bearing mice was significantly improved compared to that of nontreated mice. MicroRNA-detargeted mengoviruses offer a promising oncolytic virotherapy platform that merits further development for clinical translation. IMPORTANCE: The clinical potential of oncolytic virotherapy for cancer treatment has been well demonstrated, justifying the continued development of novel oncolytic viruses with enhanced potency. Here, we introduce mengovirus as a novel oncolytic agent. Mengovirus is appealing as an oncolytic virotherapy platform because of its small size, simple genome structure, rapid replication cycle, and broad cell/species tropism. However, mengovirus can cause encephalomyelitis and myocarditis. It can be partially attenuated by shortening the poly(C) tract in the 5' NCR but remains capable of damaging cardiac and nervous tissue. Here, we further enhanced the safety profile of a poly(C)-truncated mengovirus by incorporating muscle- and neuron-specific microRNA target sequences into the viral genome. This dual-detargeted virus has reduced pathogenesis but retained potent oncolytic activity. Our data show that microRNA targeting can be used to further increase the safety of an attenuated mengovirus, providing a basis for its development as an oncolytic platform.

Immunovirotherapy with vesicular stomatitis virus and PD-L1 blockade enhances therapeutic outcome in murine acute myeloid leukemia.

Patients with relapsed acute myeloid leukemia (AML) have limited therapeutic options. Vesicular stomatitis virus (VSV)-interferon ß (IFNß)-sodium iodide symporter (NIS) is an oncolytic VSV encoding IFNß and the NIS reporter. Syngeneic AML C1498 tumors responded to IV therapy with VSV-murine IFNß (mIFNß)-NIS in a dose-dependent manner. Imaging for NIS expression showed robust virus infection within the tumors. Virus infection did not increase programmed death ligand 1 (PD-L1) on tumor cells. Combining VSV-mIFNß-NIS with anti-PD-L1 antibody (Ab) therapy enhanced antitumor activity compared with treatment with virus alone or Ab alone; this enhancement was not significant at higher VSV-mIFNß-NIS doses. Systemic VSV therapy reduced systemic C1498-green fluorescent protein (GFP) tumor burden in the blood, bone marrow, spleen, and liver of mice with AML. Combination VSV-mIFNß-NIS and anti-PD-L1 Ab therapy significantly enhanced the survival of these mice with no evidence of toxicity, compared with isotype control, anti-PD-L1, or virus alone. There was an increase in tumor-infiltrating CD4 and CD8 cells. Single-agent VSV-mIFNß-NIS virotherapy induced both VSV-specific and GFP-specific CD8 T cells as determined by IFN-γ enzyme-linked immunospot, pentamer, and intracellular IFN-γ staining assays. Both of these responses were further enhanced by addition of anti-PD-L1 Ab. Depletion of CD8 or natural killer cells, but not CD4 cells, resulted in loss of antitumor activity in the VSV/anti-PD-L1 group. Clinical samples from chronic myelomonocytic leukemia and acute myelomonocytic leukemia appear to be especially susceptible to VSV. Overall, our studies show that oncolytic virotherapy combined with immune checkpoint blockade is a promising approach to AML therapy.

MicroRNAs and oncolytic viruses.

MicroRNAs regulate gene expression in mammalian cells and often exhibit tissue-specific expression patterns. Incorporation of microRNA target sequences can be used to control exogenous gene expression and viral tropism in specific tissues to enhance the therapeutic indices of oncolytic viruses expressing therapeutic transgenes. Continued development of this targeting strategy has resulted in the generation of unattenuated oncolytic viruses with enhanced potency, broad species-tropisms and reduced off-target toxicities in multiple-tissues simultaneously. Furthermore, oncolytic viruses have been used to enhance the delivery, duration and therapeutic efficacy of microRNA-based therapeutics designed to either restore or inhibit the function of dysregulated microRNAs in cancer cells. Recent efforts focused on combining oncolytic virotherapy and microRNA regulation have generated increasingly potent and safe cancer therapeutics.

Simian-Human immunodeficiency viruses expressing chimeric subtype B/C Vpu proteins demonstrate the importance of the amino terminal and transmembrane domains in the rate of CD4(+) T cell loss in macaques.

Previously, we reported that simian-human immunodeficiency viruses expressing either the lab adapted subtype B (SHIV(KU-1bMC33)) or subtype C (SHIV(SCVpu)) Vpu proteins of human immunodeficiency virus type 1 (HIV-1) had different rates of CD4(+) T cell loss following inoculation into macaques. In this study, we have generated SHIVs that express either the subtype B or subtype C N-terminal (NTD) and transmembrane (TMD) domains and the opposing cytoplasmic domain (SHIV(VpuBC), SHIV(VpuCB)). In culture systems, SHIV(VpuBC) replicated faster than SHIV(VpuCB) while both proteins exhibited similar ability to down-modulate CD4 surface expression. Following inoculation into macaques, SHIV(VpuBC) resulted in rapid CD4(+) T cell loss similar to the parental SHIV(KU-1bMC33), while the rate of CD4(+) T cell loss in those inoculated with SHIV(VpuCB) was intermediate of SHIV(SCVpu) and SHIV(KU-1bMC33). These results emphasize the importance of the Vpu NTD/TMD region in the rate of CD4(+) T cell loss in the pathogenic X4 SHIV/macaque model.

A new paradigm in viral resistance.

The rapid mutation of RNA viruses allows for the acquisition of resistance to drugs directly targeting viral proteins. Therefore, a novel approach to the development of antivirals centers on targeting host factors critical to viral replication. A recent report has brought to light the potential for RNA viruses to also develop resistance against compounds targeting crucial host factors, suggesting that a combination of drugs with various targets may be necessary for preventing resistance.

HIV-1 Vpu protein antagonizes innate restriction factor BST-2 via lipid-embedded helix-helix interactions.

The Vpu protein of HIV-1 antagonizes BST-2 (tetherin), a broad spectrum effector of the innate immune response to viral infection, by an intermolecular interaction that maps genetically to the α-helical transmembrane domains (TMDs) of each protein. Here we utilize NMR spectroscopy to describe key features of the helix-helix pairing that underlies this interaction. The antagonism of BST-2 involves a sequence of three alanines and a tryptophan spaced at four residue intervals within the Vpu TMD helix. Responsiveness to Vpu involves bulky hydrophobic residues in the C-terminal region of the BST-2 TMD helix that likely fit between the alanines on the interactive face of Vpu. These aspects of Vpu and BST-2 form an anti-parallel, lipid-embedded helix-helix interface. Changes in human BST-2 that mimic sequences found in nonhuman primate orthologs unresponsive to Vpu change the tilt angle of the TMD in the lipid bilayer without abrogating its intrinsic ability to interact with Vpu. These data explain the mechanism by which HIV-1 evades a key aspect of innate immunity and the species specificity of Vpu using an anti-parallel helix-helix packing model.

Differential virus restriction patterns of rhesus macaque and human APOBEC3A: implications for lentivirus evolution.

The human apolipoprotein B mRNA editing enzyme catalytic peptide-like 3 (APOBEC3; A3) family of proteins (A3A-H) are known to restrict various retroviruses and retroelements, but the full complement of rhesus macaque A3 proteins remains unclear. We report the isolation and characterization of the hA3A homologue from rhesus macaques (rhA3A) and show that the rhesus macaque and human A3 genes are orthologous. RhA3A is expressed at high levels in activated CD4+ T cells, is widely expressed in macaque tissues, and is degraded in the presence of the human immunodeficiency virus (HIV-1) and simian-human immunodeficiency virus (SHIV) genomes. Our results indicate that rhA3A is a potent inhibitor of SHIVΔvif and to a lesser extent HIV-1Δvif. Unlike hA3A, rhA3A did not inhibit adeno-associated virus 2 (AAV-2) replication and L1 retrotransposition. These data suggest an evolutionary switch in primate A3A virus specificity and provide the first evidence that a primate A3A can inhibit lentivirus replication.

Membrane raft association of the Vpu protein of human immunodeficiency virus type 1 correlates with enhanced virus release.

The Vpu protein of human immunodeficiency virus type 1 (HIV-1) is known to enhance virion release from certain cell types. To accomplish this function, Vpu interacts with the restriction factor known as bone marrow stromal cell antigen 2 (BST-2)/tetherin. In this study, we analyzed whether the Vpu protein is associated with microdomains known as lipid or membrane rafts. Our results indicate that Vpu partially partitions into detergent-resistant membrane (DRM) fractions when expressed alone or in the context of simian-human immunodeficiency virus (SHIV) infection. The ability to be partitioned into rafts was observed with both subtype B and C Vpu proteins. The use of cholesterol lowering lovastatin/M-ß-cyclodextrin and co-patching experiments confirmed that Vpu can be detected in cholesterol rich regions of membranes. Finally, we present data showing that raft association-defective transmembrane mutants of Vpu have impaired enhanced virus release function, but still maintain the ability to down-regulate CD4.

BST-2 mediated restriction of simian-human immunodeficiency virus.

Pathogenic simian-human immunodeficiency viruses (SHIV) contain HIV-1 Vpu and SIV Nef, both shown to counteract BST-2 (HM1.24; CD317; tetherin) inhibition of virus release in a species-specific manner. We show that human and pig-tailed BST-2 (ptBST-2) restrict SHIV. We found that sequential "humanization" of the transmembrane domain (TMD) of the pig-tailed BST-2 (ptBST-2) protein resulted in a fluctuation in sensitivity to HIV-1 Vpu. Our results also show that the length of the TMD in human and ptBST-2 proteins is important for BST-2 restriction and susceptibility to Vpu. Taken together, our results emphasize the importance of tertiary structure in BST-2 antagonism and suggests that the HIV-1 Vpu transmembrane domain may have additional functions in vivo unrelated to BST-2 antagonism.

Comparison of the replication and persistence of simian-human immunodeficiency viruses expressing Vif proteins with mutation of the SLQYLA or HCCH domains in macaques.

The Vif protein of primate lentiviruses interacts with APOBEC3 proteins, which results in shunting of the APOBEC3-Vif complex to the proteosome for degradation. Using the simian-human immunodeficiency virus (SHIV)/macaque model, we compared the replication and pathogenicity of SHIVs that express a Vif protein in which the entire SLQYLA (SHIV(Vif5A)) or HCCH (SHIV(VifHCCH(-))) domains were substituted with alanine residues. Each virus was inoculated into three macaques and various viral and immunological parameters followed for 6 months. All macaques maintained stable circulating CD4+ T cells, developed low viral loads, maintained the engineered mutations, yielded no histological lesions, and developed immunoprecipitating antibodies early post-inoculation. Sequence analysis of nef and vpu from three lymphoid tissues revealed a high percentage of G-to-A-substitutions. Our results show that while the presence of HCCH and SLQYLA domains are critical in vivo, there may exist APOBEC3 negative reservoirs that allow for low levels of viral replication and persistence but not disease.

The Vpu protein: new concepts in virus release and CD4 down-modulation.

Human immunodeficiency virus type 1 (HIV-1) and several simian immunodeficiency viruses (SIV) encode for a transmembrane protein known as Vpu (viral protein U). While one of the smallest of the HIV-1 proteins, it has two important functions within virus-infected cells. The first of these functions is the down-regulation of the CD4 receptor to prevent its interaction with the HIV-1 envelope glycoprotein. Vpu interacts with the CD4 receptor in the rough endoplasmic reticulum (RER), resulting in its re-translocation across the RER and subsequent degradation via the proteasomal pathway. The second major function of the Vpu protein is to facilitate release of virus from infected cells. Previous studies have shown that virus release is cell type specific, suggesting that certain cells may express a restriction factor that inhibits virus release in the absence of Vpu. Recently, bone marrow stromal antigen 2 (BST-2/HM1.24/CD317/tetherin) has been identified as this factor. This review will focus on new findings within the last four years on the role of Vpu in CD4 down-regulation and the restriction of virus release from cells. We will relate these findings to virus pathogenesis and propose questions regarding BST-2 as a restriction factor.

Identification of amino acids within the second alpha helical domain of the human immunodeficiency virus type 1 Vpu that are critical for preventing CD4 cell surface expression.

Human immunodeficiency virus type 1 (HIV-1) encodes for a Vpu protein, which interacts with CD4 resulting in its degradation. In this study, we examined the role of the 10 amino acids within the predicted second alpha-helical domain of the subtype B Vpu cytoplasmic tail in CD4 down-modulation using a VpuEGFP reporter system. Our findings indicate that the invariant leucine at position 63 and, to a lesser extent, the valine at position 68 were required for CD4 down-modulation. Mutation of analogous L63 in Vpu proteins subtypes A2, B(YU-2), C, D, and H also abolished CD4 down-modulation from the cell surface. Co-immunoprecipitation analysis revealed that L63A and V68A mutants were capable of binding CD4 and still retained the ability to interact with h-beta-TrCP1. Taken together, these results indicate that amino acid substitutions in the second alpha-helical domain that retain the predicted structure and binding to h-beta-TrCP1 can influence Vpu-mediated CD4 degradation.

Vpu antagonizes BST-2-mediated restriction of HIV-1 release via beta-TrCP and endo-lysosomal trafficking.

The interferon-induced transmembrane protein BST-2/CD317 (tetherin) restricts the release of diverse enveloped viruses from infected cells. The HIV-1 accessory protein Vpu antagonizes this restriction by an unknown mechanism that likely involves the down-regulation of BST-2 from the cell surface. Here, we show that the optimal removal of BST-2 from the plasma membrane by Vpu requires the cellular protein beta-TrCP, a substrate adaptor for a multi-subunit SCF E3 ubiquitin ligase complex and a known Vpu-interacting protein. beta-TrCP is also required for the optimal enhancement of virion-release by Vpu. Mutations in the DSGxxS beta-TrCP binding-motif of Vpu impair both the down-regulation of BST-2 and the enhancement of virion-release. Such mutations also confer dominant-negative activity, consistent with a model in which Vpu links BST-2 to beta-TrCP. Optimal down-regulation of BST-2 from the cell surface by Vpu also requires the endocytic clathrin adaptor AP-2, although the rate of endocytosis is not increased; these data suggest that Vpu induces post-endocytic membrane trafficking events whose net effect is the removal of BST-2 from the cell surface. In addition to its marked effect on cell-surface levels, Vpu modestly decreases the total cellular levels of BST-2. The decreases in cell-surface and intracellular BST-2 are inhibited by bafilomycin A1, an inhibitor of endosomal acidification; these data suggest that Vpu induces late endosomal targeting and partial degradation of BST-2 in lysosomes. The Vpu-mediated decrease in surface expression is associated with reduced co-localization of BST-2 and the virion protein Gag along the plasma membrane. Together, the data support a model in which Vpu co-opts the beta-TrCP/SCF E3 ubiquitin ligase complex to induce endosomal trafficking events that remove BST-2 from its site of action as a virion-tethering factor.

Mutations in the highly conserved SLQYLA motif of Vif in a simian-human immunodeficiency virus result in a less pathogenic virus and are associated with G-to-A mutations in the viral genome.

The simian-human immunodeficiency virus (SHIV)/macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of accessory genes in lentiviral pathogenesis. In this study, we introduced two amino acid changes in the highly conserved SLQYLA domain (to AAQYLA) of the SIV Vif protein. The resulting virus, SHIV(VifAAQYLA), was used to infect three macaques, which were followed for over six months. Plasma viral loads and circulating CD4(+) T cell levels were assessed during the course of infection. The three macaques inoculated with SHIV(VifAAQYLA) did not develop significant CD4(+) T cell loss over the course of their infection, had plasma viral RNA loads that were over 100-fold lower than macaques inoculated with parental SHIV(KU-1bMC33), and developed no histological lesions in lymphoid tissues. DNA and RT-PCR analysis revealed that only a select number of tissues were infected with this virus. Sequence analysis indicates that the site-directed changes were stable during the first three weeks after inoculation but thereafter the S147A amino acid substitution changed to a threonine in two of three macaques. The L148A substitution remained stable in the vif amplified from the PBMC of all three macaques. Sequence analysis of vif, vpu, env and nef genes revealed G-to-A mutations in the genes amplified from macaques inoculated with SHIV(VifAAQYLA), which were higher than in a macaque inoculated with parental SHIV(KU-1bMC33). We found that the majority (>85%) of the G-to-A mutations were in the context of 5'-TC (minus strand) and not 5'-CC, suggestive that one or more of the rhesus APOBEC3 proteins may be responsible for the observed mutational patterns. The data also suggest that rhesus APOBEC3G probably accounted for a minority of the mutations since its GG-to-AG mutational pattern was infrequently detected. Finally, macaques inoculated with SHIV(VifAAQYLA) developed immunoprecipitating antibody responses against the virus. The results from this study provide the first in vivo evidence of the importance of the SLQYLA domain in viral pathogenesis and show that targeted mutations in vif can lead to a persistent infection with G-to-A changes accumulating in the viral genome.

Requirements of the membrane proximal tyrosine and dileucine-based sorting signals for efficient transport of the subtype C Vpu protein to the plasma membrane and in virus release.

Previously, we showed that the Vpu protein from HIV-1 subtype C is more efficiently transported to the cell surface than the well studied subtype B Vpu (Pacyniak et al., 2005) and that a SHIV expressing the subtype C Vpu exhibited a decreased rate of CD4+ T cell loss following inoculation in macaques (Hill et al., 2008). In this study, we examined the role of overlapping tyrosine-based (YXXPhi) and dileucine-based ([D/E]XXXL[L/I]) motifs in the membrane proximal region of the subtype C Vpu (EYRKLL) in Vpu intracellular transport, CD4 surface expression and virus release from the cell surface. We constructed three site-directed mutants of the subtype C vpu and fused these genes to the gene for enhanced green fluorescent protein (EGFP). The first mutation made altered the tyrosine (EARKLL; VpuSCEGFPY35A), the second altered the dileucine motif (EYRKLG; VpuSCEGFPL39G), and the third contained both amino acid substitutions (EARKLG; VpuSCEGFPYL35,39AG) in this region of the Vpu protein. The VpuSCEGFPY35A protein was transported to the cell surface similar to the unmodified VpuSCEGFP1 while VpuSCEGFPL39G was expressed at the cell surface at significantly reduced levels. The VpuSCEGFPYL35,39AG was found to have an intermediate level of cell surface expression. All three mutant Vpu proteins were analyzed for the ability to prevent cell surface expression of CD4. We found that both single mutants did not significantly effect CD4 surface expression while the double mutant (VpuSCEGFPYL35,39AG) was significantly less efficient at preventing cell surface CD4 expression. Chimeric simian human immunodeficiency viruses were constructed with these mutations in vpu (SHIVSCVpuY35A, SHIVSCVpuL39G and SHIVSCVpuYL35,39AG). Our results indicate that SHIVSCVpuL39G replicated much more efficiently and was much more cytopathic than SHIVSCVpu. In contrast, SHIVSCVpuY35A and SHIVSCVpuYL35,39AG replicated less efficiently when compared to the parental SHIVSCVpu. Taken together, these results show for the first time that the membrane proximal tyrosine-based sorting motif in the cytoplasmic domain of Vpu is essential for efficient virus release. These results also indicate that the dileucine-based sorting motif affects the intracellular trafficking of subtype C Vpu proteins, virus replication, and release.

Modulation of the severe CD4+ T-cell loss caused by a pathogenic simian-human immunodeficiency virus by replacement of the subtype B vpu with the vpu from a subtype C HIV-1 clinical isolate.

Previously, we showed that the Vpu protein from subtype C human immunodeficiency virus type 1 (HIV-1) was efficiently targeted to the cell surface, suggesting that this protein has biological properties that differ from the well-studied subtype B Vpu protein. In this study, we have further analyzed the biological properties of the subtype C Vpu protein. Flow cytometric analysis revealed that the subtype B Vpu (strain HXB2) was more efficient at down-regulating CD4 surface expression than the Vpu proteins from four subtype C clinical isolates. We constructed a simian-human immunodeficiency virus virus, designated as SHIV(SCVpu), in which the subtype B vpu gene from the pathogenic SHIV(KU-1bMC33) was substituted with the vpu from a clinical isolate of subtype C HIV-1 (strain C.96.BW16B01). Cell culture studies revealed that SHIV(SCVpu) replicated with slightly reduced kinetics when compared with the parental SHIV(KU-1bMC33) and that the viral Env and Gag precursor proteins were synthesized and processed similarly compared to the parental SHIV(KU-1bMC33). To determine if substitution of the subtype C Vpu protein affected the pathogenesis of the virus, three pig-tailed macaques were inoculated with SHIV(SCVpu) and circulating CD4+ T-cell levels and viral loads were monitored for up to 44 weeks. Our results show that SHIV(SCVpu) caused a more gradual decline in the rate of CD4+ T cells in pig-tailed macaques compared to those inoculated with parental subtype B SHIV(KU-1bMC33). These results show for the first time that different Vpu proteins of HIV-1 can influence the rate at which CD4+ T-cell loss occurs in the SHIV/pig-tailed macaque model.

Ebola virus glycoprotein 1: identification of residues important for binding and postbinding events.

The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) are responsible for devastating hemorrhagic fever outbreaks. No therapies are available against these viruses. An understanding of filoviral glycoprotein 1 (GP1) residues involved in entry events would facilitate the development of antivirals. Towards this end, we performed alanine scanning mutagenesis on selected residues in the amino terminus of GP1. Mutant GPs were evaluated for their incorporation onto feline immunodeficiency virus (FIV) particles, transduction efficiency, receptor binding, and ability to be cleaved by cathepsins L and B. FIV virions bearing 39 out of 63 mutant glycoproteins transduced cells efficiently, whereas virions bearing the other 24 had reduced levels of transduction. Virions pseudotyped with 23 of the poorly transducing GPs were characterized for their block in entry. Ten mutant GPs were very poorly incorporated onto viral particles. Nine additional mutant GPs (G87A/F88A, K114A/K115A, K140A, G143A, P146A/C147A, F153A/H154A, F159A, F160A, and Y162A) competed poorly with wild-type GP for binding to permissive cells. Four of these nine mutants (P146A/C147A, F153A/H154A, F159A, and F160A) were also inefficiently cleaved by cathepsins. An additional four mutant GPs (K84A, R134A, D150A, and E305/E306A) that were partially defective in transduction were found to compete effectively for receptor binding and were readily cleaved by cathepsins. This finding suggested that this latter group of mutants might be defective at a postbinding, cathepsin cleavage-independent step. In total, our study confirms the role of some GP1 residues in EBOV entry that had previously been recognized and identifies for the first time other residues that are important for productive entry.

APOBEC3G expression is restricted to epithelial cells of the proximal convoluted tubules and is not expressed in the glomeruli of macaques.

The Vif protein of human immunodeficiency virus-1 (HIV-1) interacts with members of the APOBEC family of cytidine deaminases. In this study, we isolated RNA from renal cortex as well as from isolated glomeruli and tubulointerstitial fractions from two pigtailed macaques that were exsanguinated and perfused with saline. RT-PCR results indicate that APOBEC3G was detected in the tubule fractions but not in the glomerular fractions. Immunoblot analysis using lysates prepared from these same fractions and a monoclonal antibody to APOBEC3G confirmed the RT-PCR findings. To determine which cell types express APOBEC3G, immunohistochemical studies were performed using this monoclonal antibody on renal cortical sections. Our results clearly show that the glomeruli do not express APOBEC3G but that select tubules within the cortex express APOBEC3G at high levels. To further differentiate the distribution of APOBEC3G expression, serial sections were stained with the lectins Dolichos biflorus agglutinin (DBA) and Phaseolus vulgaris erythroagglutinin (PHA-E), which differentially bind to epithelial cells of the tubules and glomeruli. Our results indicate that APOBEC3G expression was restricted to PHA-E-staining tubules and not DBA-staining tubules, suggesting that APOBEC3G expression was restricted to proximal convoluted tubules. These findings suggest that infection of epithelial cells of proximal renal tubules could suppress Vif-defective HIV-1 replication, whereas infection of cells of the glomeruli, a major target of HIV-associated nephropathy, could act as a reservoir for the replication of Vif-defective HIV-1.