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During human development, a temporary organ is formed, the placenta, which invades the uterine wall to support nutrient, oxygen, and waste exchange between the mother and fetus until birth. Most of the human placenta is formed by a syncytial villous structure lined by syncytialized trophoblasts, a specialized cell type that forms via cell-cell fusion of underlying progenitor cells. Genetic and functional studies have characterized the membrane protein fusogens Syncytin-1 and Syncytin-2, both of which are necessary and sufficient for human trophoblast cell-cell fusion. However, identification and characterization of upstream transcriptional regulators regulating their expression have been limited. Here, using CRISPR knockout in an in vitro cellular model of syncytiotrophoblast development (BeWo cells), we found that the transcription factor TFEB, mainly known as a regulator of autophagy and lysosomal biogenesis, is required for cell-cell fusion of syncytiotrophoblasts. TFEB translocates to the nucleus, exhibits increased chromatin interactions, and directly binds the Syncytin-1 and Syncytin-2 promoters to control their expression during differentiation. Although TFEB appears to play a critical role in syncytiotrophoblast differentiation, ablation of TFEB largely does not affect lysosomal gene expression or lysosomal biogenesis in differentiating BeWo cells, suggesting a previously uncharacterized role for TFEB in controlling the expression of human syncytins.

In the late stages of the HIV-1 life cycle, membrane localization and self-assembly of Gag polyproteins induce membrane deformation and budding. Release of the virion requires direct interaction between immature Gag lattice and upstream ESCRT machinery at the viral budding site, followed by assembly of downstream ESCRT-III factors, culminating in membrane scission. However, molecular details of upstream ESCRT assembly dynamics at the viral budding site remain unclear. In this work, using coarse-grained (CG) molecular dynamics (MD) simulations, we investigated the interactions between Gag, ESCRT-I, ESCRT-II, and membrane to delineate the dynamical mechanisms by which upstream ESCRTs assemble templated by late-stage immature Gag lattice. We first systematically derived "bottom-up" CG molecular models and interactions of upstream ESCRT proteins from experimental structural data and extensive all-atom MD simulations. Using these molecular models, we performed CG MD simulations of ESCRT-I oligomerization and ESCRT-I/II supercomplex formation at the neck of the budding virion. Our simulations demonstrate that ESCRT-I can effectively oligomerize to higher-order complexes templated by the immature Gag lattice both in the absence of ESCRT-II and when multiple copies of ESCRT-II are localized at the bud neck. The ESCRT-I/II supercomplexes formed in our simulations exhibit predominantly columnar structures, which has important implications for the nucleation pathway of downstream ESCRT-III polymers. Importantly, ESCRT-I/II supercomplexes bound to Gag initiate membrane neck constriction by pulling the inner edge of the bud neck closer to the ESCRT-I headpiece ring. Our findings serve to elucidate a network of interactions between upstream ESCRT machinery, immature Gag lattice, and membrane neck that regulate protein assembly dynamics at the HIV-1 budding site.

Much attention has been focused on the possibility that cytoplasmic proteins and RNA may be conveyed between cells in extracellular vesicles (EVs) and tunneling nanotube (TNT) structures. Here, we set up two quantitative delivery reporters to study cargo transfer between cells. We found that EVs are internalized by reporter cells but do not efficiently deliver functional Cas9 protein to the nucleus. In contrast, donor and acceptor cells co-cultured to permit cell contact resulted in a highly effective transfer. Among our tested donor and acceptor cell pairs, HEK293T and MDA-MB-231 recorded optimal intercellular transfer. Depolymerization of F-actin greatly decreased Cas9 transfer, whereas inhibitors of endocytosis or knockdown of genes implicated in this process had little effect on transfer. Imaging results suggest that intercellular transfer of cargos occurred through open-ended membrane tubular connections. In contrast, cultures consisting only of HEK293T cells form close-ended tubular connections ineffective in cargo transfer. Depletion of human endogenous fusogens, syncytins, especially syncytin-2 in MDA-MB-231 cells, significantly reduced Cas9 transfer. Full-length mouse syncytin, but not truncated mutants, rescued the effect of depletion of human syncytins on Cas9 transfer. Mouse syncytin overexpression in HEK293T cells partially facilitated Cas9 transfer among HEK293T cells. These findings suggest that syncytin may serve as the fusogen responsible for the formation of an open-ended connection between cells.

Despite effective antiretroviral therapies, 20–30% of persons with treated HIV infection develop a neurodegenerative syndrome termed HIV-associated neurocognitive disorder (HAND). HAND is driven by HIV expression coupled with inflammation in the brain but the mechanisms underlying neuronal damage and death are uncertain. The inflammasome-pyroptosis axis coordinates an inflammatory type of regulated lytic cell death that is underpinned by the caspase-activated pore-forming gasdermin proteins. The mechanisms driving neuronal pyroptosis were investigated herein in models of HAND, using multi-platform molecular and morphological approaches that included brain tissues from persons with HAND and simian immunodeficiency virus (SIV)-infected non-human primates as well as cultured human neurons. Neurons in the frontal cortices from persons with HAND showed increased cleaved gasdermin E (GSDME), which was associated with β-III tubulin degradation and increased HIV levels. Exposure of cultured human neurons to the HIV-encoded viral protein R (Vpr) elicited time-dependent cleavage of GSDME and Ninjurin-1 (NINJ1) induction with associated cell lysis that was inhibited by siRNA suppression of both proteins. Upstream of GSDME cleavage, Vpr exposure resulted in activation of caspases-1 and 3. Pretreatment of Vpr-exposed neurons with the caspase-1 inhibitor, VX-765, reduced cleavage of both caspase-3 and GSDME, resulting in diminished cell death. To validate these findings, we examined frontal cortical tissues from SIV-infected macaques, disclosing increased expression of GSDME and NINJ1 in cortical neurons, which was co-localized with caspase-3 detection in animals with neurological disease. Thus, HIV infection of the brain triggers the convergent activation of caspases-1 and -3, which results in GSDME-mediated neuronal pyroptosis in persons with HAND. These findings demonstrate a novel mechanism by which a viral infection causes pyroptotic death in neurons while also offering new diagnostic and therapeutic strategies for HAND and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

PURPOSE: Recent studies reported therapeutic effects of Smad7 on oral mucositis in mice without compromising radiation therapy-induced cancer cell killing in neighboring oral cancer. This study aims to assess whether a Smad7-based biologic can treat oral mucositis in a clinically relevant setting by establishing an oral mucositis model in dogs and analyzing molecular targets. METHODS AND MATERIALS: We created a truncated human Smad7 protein fused with the cell-penetrating Tat tag (Tat-PYC-Smad7). We used intensity modulated radiation therapy to induce oral mucositis in dogs and applied Tat-PYC-Smad7 to the oral mucosa in dose-finding studies after intensity modulated radiation therapy. Clinical outcomes were evaluated. Molecular targets were analyzed in biopsies and serum samples. RESULTS: Tat-PYC-Smad7 treatment significantly shortened the duration of grade 3 oral mucositis based on double-blinded Veterinary Radiation Therapy Oncology Group scores and histopathology evaluations. Topically applied Tat-PYC-Smad7 primarily penetrated epithelial cells and was undetectable in serum. NanoString nCounter Canine IO Panel identified that, compared to the vehicle samples, top molecular changes in Tat-PYC-Smad7 treated samples include reductions in inflammation and cell death and increases in cell growth and DNA repair. Consistently, immunostaining shows that Tat-PYC-Smad7 reduced DNA damage and neutrophil infiltration with attenuated TGF-β and NFκB signaling. Furthermore, IL-1β and TNF-α were lower in Tat-PYC-Smad7 treated mucosa and serum samples compared to those in vehicle controls. CONCLUSIONS: Topical Tat-PYC-Smad7 application demonstrated therapeutic effects on oral mucositis induced by intensity modulated radiation therapy in dogs. The local effects of Tat-PYC-Smad7 targeted molecules involved in oral mucositis pathogenesis as well as reduced systemic inflammatory cytokines.

Human T-lymphotropic virus 1 (HTLV-1) infection causes two serious diseases: adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy (HAM). Immunological studies have revealed that HTLV-1 Tax-specific CD8+ cytotoxic T-cells (Tax-CTLs) in asymptomatic carriers (ACs) and ATL patients play an important role in the elimination of HTLV-1-infected host cells, whereas Tax-CTLs in HAM patients trigger an excessive immune response against HTLV-1-infected host cells infiltrating the central nervous system (CNS), leading to local inflammation. Our previous evaluation of HTLV-1 Tax301-309 (SFHSLHLLF)-specific Tax-CTLs (Tax301-309-CTLs) revealed that a unique T-cell receptor (TCR) containing amino acid (AA)-sequence motif PDR, was shared among HLA-A*24:02+ ACs and ATL patients and behaved as an eliminator by strong activity against HTLV-1. However, it remains unclear whether PDR+Tax301-309-CTLs also exist in HLA-A*24:02+ HAM patients and are involved in the pathogenesis of HAM. In the present study, by high-throughput TCR repertoire analysis technology, we revealed TCR repertoires of Tax301-309-CTLs in peripheral blood (PB) of HLA-A*24:02+ HAM patients were skewed, and a unique TCR-motif PDR was conserved in HAM patients (10 of 11 cases). The remaining case dominantly expressed (-DR, P-R, and PD-), which differed by one AA from PDR. Overall, TCRs with unique AA-sequence motifs PDR, or (-DR, P-R, and PD-) accounted for a total of 0.3-98.1% of Tax301-309-CTLs repertoires of HLA-A*24:02+ HAM patients. Moreover, TCR repertoire analysis of T-cells in the cerebrospinal fluid (CSF) from four HAM patients demonstrated the possibility that PDR+Tax301-309-CTLs and (-DR, P-R, and PD-)+Tax301-309-CTLs efficiently migrated and accumulated in the CSF of HAM patients fostering increased inflammation, although we observed no clear significant correlation between the frequencies of them in PB and the levels of CSF neopterin, a known disease activity biomarker of HAM. Furthermore, to better understand the potential function of PDR+Tax301-309-CTLs, we performed immune profiling by single-cell RNA-sequencing of Tax301-309-CTLs, and the result showed that PDR+Tax301-309-CTLs up-regulated the gene expression of natural killer cell marker KLRB1 (CD161), which may be associated with T-cell activation and highly cytotoxic potential of memory T-cells. These findings indicated that unique and shared PDR+Tax301-309-CTLs have a potential role in promoting local inflammation within the CNS of HAM patients.

Epidemic Kaposi's sarcoma (KS), defined by co-infection with Human Herpes Virus 8 (HHV-8) and the Human Immunodeficiency Virus (HIV), is a major cause of mortality in sub-Saharan Africa. Antiretroviral therapy (ART) significantly reduces the risk of developing KS, and for those with KS, tumors frequently resolve with ART alone. However, for unknown reasons, a significant number of KS cases do not resolve and can progress to death. To explore how HIV responds to ART in the KS tumor microenvironment, we sequenced HIV env-nef found in DNA and RNA isolated from plasma, peripheral blood mononuclear cells, and tumor biopsies, before and after ART, in four Ugandan study participants who had unresponsive or progressive KS after 180-250 days of ART. We performed immunohistochemistry experiments to detect viral proteins in matched formalin-fixed tumor biopsies. Our sequencing results showed that HIV diversity and RNA expression in KS tumors are maintained after ART, despite undetectable plasma viral loads. The presence of spliced HIV transcripts in KS tumors after ART was consistent with a transcriptionally active viral reservoir. Immunohistochemistry staining found colocalization of HIV Nef protein and tissue-resident macrophages in the KS tumors. Overall, our results demonstrated that even after ART reduced plasma HIV viral load to undetectable levels and restored immune function, HIV in KS tumors continues to be transcriptionally and translationally active, which could influence tumor maintenance and progression.

Cataracts, a clouding of the eye lens, are a leading cause of visual impairment and are responsible for one of the most commonly performed surgical procedures worldwide. Although generally safe and effective, cataract surgery can lead to a secondary lens abnormality due to transition of lens epithelial cells to a mesenchymal phenotype (EMT) and opacification of the posterior lens capsular bag. Occurring in up to 40% of cataract cases over time, posterior capsule opacification (PCO) introduces additional treatment costs and reduced quality of life for patients. Studies have shown that PCO pathogenesis is driven in part by TGF-β, signaling through the action of the family of Smad coactivators to effect changes in gene transcription. In the present study, we evaluated the ability of Smad-7, a well characterized inhibitor of TGF-β -mediated Smad signaling, to suppress the EMT response in lens epithelial cells associated with PCO pathogenesis. Treatment of lens epithelial cells with a cell-permeable form of Smad7 variant resulted in suppressed expression of EMT markers such as alpha smooth muscle actin and fibronectin. A single application of cell-permeable Smad7 variant in the capsular bag of a mouse cataract surgery model resulted in suppression of gene transcripts encoding alpha smooth muscle actin and fibronectin. These results point to Smad7 as a promising biotherapeutic agent for prevention or substantial reduction in the incidence of PCO following cataract surgery.

The mechanism and pore architecture of the Tat complex during transport of folded substrates remain a mystery, partly due to rapid dissociation after translocation. In contrast, the proteinaceous SecY pore is a persistent structure that needs only to undergo conformational shifts between "closed" and "opened" states when translocating unfolded substrate chains. Where the proteinaceous pore model describes the SecY pore well, the toroidal pore model better accounts for the high-energy barrier that must be overcome when transporting a folded substrate through the hydrophobic bilayer in Tat transport. Membrane conductance behavior can, in principle, be used to distinguish between toroidal and proteinaceous pores, as illustrated in the examination of many antimicrobial peptides as well as mitochondrial Bax and Bid. Here, we measure the electrochromic shift (ECS) decay as a proxy for conductance in isolated thylakoids, both during protein transport and with constitutively assembled translocons. We find that membranes with the constitutively assembled Tat complex and those undergoing Tat transport display conductance characteristics similar to those of resting membranes. Membranes undergoing Sec transport and those with the substrate-engaged SecY pore result in significantly more rapid electric field decay. The responsiveness of the ECS signal in membranes with active SecY recalls the steep relationship between applied voltage and conductance in a proteinaceous pore, while the nonaccelerated electric field decay with both Tat transport and the constitutive Tat complex under the same electric field is consistent with the behavior of a toroidal pore.

The role of vaccine-induced anti-V2 Abs was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial (Anti-envelope [Env]), nine rhesus macaques were coimmunized with gp16092TH023 DNA and SIV gag and gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12, and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106-107 copies/ml developed in all nine sham controls. Overall, PVL was undetectable in one third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, Abs that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with nonneutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and noncontrollers as were Ab-dependent cellular cytotoxicity and Ab-dependent cell-mediated virus inhibition activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccine-elicited neutralizing and nonneutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for nonneutralizing V2 Abs alone in controlling PVL, as neither Ab-dependent cellular cytotoxicity, Ab-dependent cell-mediated virus inhibition, nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection.

HIV-1 Tat is essential for HIV-1 replication and appears to play an important role in the pathogenesis of HIV-associated neurological complications. Secreted from infected or transfected cells, Tat has the extraordinary ability to cross the plasma membrane. In the brain, Tat can be taken up by CNS cells via receptor-mediated endocytosis. Following endocytosis and its internalization into endolysosomes, Tat must be released in order for it to activate the HIV-1 LTR promoter and facilitate HIV-1 viral replication in the nucleus. However, the underlying mechanisms whereby Tat escapes endolysosomes remain unclear. Because Tat disrupts intracellular calcium homeostasis, we investigated the involvement of calcium in Tat endolysosome escape and subsequent LTR transactivation. We demonstrated that chelating endolysosome calcium with high-affinity rhodamine-dextran or chelating cytosolic calcium with BAPTA-AM attenuated Tat endolysosome escape and LTR transactivation. Significantly, we demonstrated that pharmacologically blocking and knocking down the endolysosome-resident two-pore channels (TPCs) attenuated Tat endolysosome escape and LTR transactivation. This calcium-mediated effect appears to be selective for TPCs because knocking down TRPML1 calcium channels was without effect. Our findings suggest that calcium released from TPCs is involved in Tat endolysosome escape and subsequent LTR transactivation. TPCs might represent a novel therapeutic target against HIV-1 infection and HIV-associated neurological complications.

Rhesus cytomegalovirus (RhCMV) strain 68-1-vectored simian immunodeficiency virus (RhCMV/SIV) vaccines are associated with complete clearance of pathogenic SIV challenge virus, non-canonical major histocompatibility complex restriction, and absent antibody responses in recipients previously infected with wild-type RhCMV. This report presents the first investigation of RhCMV/SIV vaccines in RhCMV-seronegative macaques lacking anti-vector immunity. Fifty percent of rhesus macaques (RM) vaccinated with a combined RhCMV-Gag, -Env, and -Retanef (RTN) vaccine controlled pathogenic SIV challenge despite high peak viremia. However, kinetics of viral load control by vaccinated RM were considerably delayed compared to previous reports. Impact of a TLR5 agonist (flagellin; FliC) on vaccine efficacy and immunogenicity was also examined. An altered vaccine regimen containing an SIV Gag-FliC fusion antigen instead of Gag was significantly less immunogenic and resulted in reduced protection. Notably, RhCMV-Gag and RhCMV-Env vaccines elicited anti-Gag and anti-Env antibodies in RhCMV-seronegative RM, an unexpected contrast to vaccination of RhCMV-seropositive RM. These findings confirm that RhCMV-vectored SIV vaccines significantly protect against SIV pathogenesis. However, pre-existing vector immunity and a pro-inflammatory vaccine adjuvant may influence RhCMV/SIV vaccine immunogenicity and efficacy. Future investigation of the impact of pre-existing anti-vector immune responses on protective immunity conferred by this vaccine platform is warranted.

Primate lentiviruses encode a Vif protein that counteracts the host antiviral APOBEC3 (A3) family members. The adaptation of Vif to species-specific A3 determinants is a critical event that allowed the spillover of a lentivirus from monkey reservoirs to chimpanzees and subsequently to humans, which gave rise to HIV-1 and the acquired immune deficiency syndrome (AIDS) pandemic. How Vif-A3 protein interactions are remodeled during evolution is unclear. Here, we report a 2.94 Å crystal structure of the Vif substrate receptor complex from simian immunodeficiency virus isolated from red-capped mangabey (SIVrcm). The structure of the SIVrcm Vif complex illuminates the stage of lentiviral Vif evolution that is immediately prior to entering hominid primates. Structure-function studies reveal the adaptations that allowed SIVrcm Vif to antagonize hominid A3G. These studies show a partitioning between an evolutionarily dynamic specificity determinant and a conserved protein interacting surface on Vif that enables adaptation while maintaining protein interactions required for potent A3 antagonism.

Tetherin is a host defense factor that physically prevents virion release from the plasma membrane. The Nef accessory protein of simian immunodeficiency virus (SIV) engages the clathrin adaptor AP-2 to downregulate tetherin via its DIWK motif. As human tetherin lacks DIWK, antagonism of tetherin by Nef is a barrier to simian-human transmission of non-human primate lentiviruses. To determine the molecular basis for tetherin counteraction, we reconstituted the AP-2 complex with a simian tetherin and SIV Nef and determined its structure by cryoelectron microscopy (cryo-EM). Nef refolds the first α-helix of the β2 subunit of AP-2 to a β hairpin, creating a binding site for the DIWK sequence. The tetherin binding site in Nef is distinct from those of most other Nef substrates, including MHC class I, CD3, and CD4 but overlaps with the site for the restriction factor SERINC5. This structure explains the dependence of SIVs on tetherin DIWK and consequent barrier to human transmission.

Feline leukemia virus (FeLV) is horizontally transmitted among cats and causes a variety of hematopoietic disorders. Five subgroups of FeLV, A to D and T, each with distinct receptor usages, have been described. Recently, we identified a new FeLV Env (TG35-2) gene from a pseudotyped virus that does not belong to any known subgroup. FeLV-A is the primary virus from which other subgroups have emerged via mutation or recombination of the subgroup A env gene. Retrovirus entry into cells is mediated by the interaction of envelope protein (Env) with specific cell surface receptors. Here, phenotypic screening of a human/hamster radiation hybrid panel identified SLC19A1, a feline reduced folate carrier (RFC) and potential receptor for TG35-2-phenotypic virus. RFC is a multipass transmembrane protein. Feline and human RFC cDNAs conferred susceptibility to TG35-2-pseudotyped virus when introduced into nonpermissive cells but did not render these cells permissive to other FeLV subgroups or feline endogenous retrovirus. Moreover, human cells with genomic deletion of RFC were nonpermissive for TG35-2-pseudotyped virus infection, but the introduction of feline and human cDNAs rendered them permissive. Mutation analysis of FeLV Env demonstrated that amino acid substitutions within variable region A altered the specificity of the Env-receptor interaction. We isolated and reconstructed the full-length infectious TG35-2-phenotypic provirus from a naturally FeLV-infected cat, from which the FeLV Env (TG35-2) gene was previously isolated, and compared the replication of the virus in hematopoietic cell lines with that of FeLV-A 61E by measuring the viral RNA copy numbers. These results provide a tool for further investigation of FeLV infectious disease.IMPORTANCE Feline leukemia virus (FeLV) is a member of the genus Gammaretrovirus, which causes malignant diseases in cats. The most prevalent FeLV among cats is FeLV subgroup A (FeLV-A), and specific binding of FeLV-A Env to its viral receptor, thiamine transporter feTHTR1, is the first step of infection. In infected cats, novel variants of FeLV with altered receptor specificity for viral entry have emerged by mutation or recombination of the env gene. A novel FeLV variant arose from a subtle mutation of FeLV-A Env, which altered the specific interaction of the virus with its receptor. RFC, a folate transporter, is a potential receptor for the novel FeLV variant. The perturbation of specific retrovirus-receptor interactions under selective pressure by the host results in the emergence of novel viruses.

We utilized pulsed-field gel electrophoresis (PFGE) to purify high-molecular-weight DNA from HIV-infected cells. This purification, in combination with our previously described droplet digital PCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight species of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantification of integrated HIV gag sequences in cellular DNA from cells from HIV-infected subjects after prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to the Alu-gag quantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman's rho nonparametric correlation determined PFGE ddPCR results to be positively correlated with Alu-gag qPCR results (r = 0.7052; P = 0.0273). In summary, PFGE ddPCR is a sensitive, reproducible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of integrated HIV DNA.

A 48-year-old woman was infected with a vpr-defective human immunodeficiency virus (HIV)-1 molecular clone. Seroconversion was markedly delayed, and without treatment she had durably suppressed viremia and normal T-cell levels. Neutralizing antibody and CD8+ T-cell immune responses against HIV-1 were unremarkable. Viral sequences confirmed the source but evolved defective nef, suggesting an unknown mechanistic link to vpr. There were subtle qualitative defects in T and B cells. To our knowledge, this is the only case of human infection with a characterized defective HIV-1 molecular clone, which furthermore recapitulated live-attenuated vaccination in macaque models of HIV-1 vaccine research.

Epitope escape from HIV-1-targeted CD8+ cytotoxic T lymphocyte (CTL) responses occurs rapidly after acute infection and contributes to the eventual failure of effective immune control of HIV-1 infection. Because the early CTL response is key in determining HIV-1 disease outcome, studying the process of epitope escape is crucial for understanding what leads to failure of immune control in acute HIV-1 infection and will provide important implications for HIV-1 vaccine design. HIV-1-specific CD8+ T lymphocyte responses against viral epitopes were mapped in six acutely infected individuals, and the magnitudes of these responses were measured longitudinally during acute infection. The evolution of autologous circulating viral epitopes was determined in four of these subjects. In-depth testing of CD8+ T lymphocyte responses against index and all autologous-detected variant epitopes was performed in one subject. Among the four individuals examined, 10 of a total of 35 CD8+ T cell responses within Gag, Pol, and Nef showed evidence of epitope escape. CTL responses with viral epitope variant evolution were shown in one subject, and this evolution occurred with and without measurable CTL responses against epitope variants. These results demonstrate a dynamic period of viral epitope evolution in early HIV-1 infection due to CD8+ CTL response pressure.

ObjectiveTo address the role of translationally active HIV reservoir in chronic inflammation and non-AIDS related disorders, we first need a simple and accurate assay to evaluate viral protein expression in virally suppressed subjects.DesignWe optimized an HIV Nef enzyme-linked immunosorbent assay (ELISA) and used it to quantify plasma Nef levels as an indicator of the leaky HIV reservoir in an HIV-infected cohort.MethodsThis study accessed 134 plasma samples from a well-characterized cohort study of HIV-infected and uninfected adults in San Francisco (the SCOPE cohort). We optimized an ELISA for detection of plasma Nef in HIV-negative subjects and HIV-infected non-controllers, and evaluated its utility to quantify plasma Nef levels in a cross-sectional study of ART-suppressed and elite controller HIV-infected subjects.ResultsHere, we describe the performance of an optimized HIV Nef ELISA. When we applied this assay to the study cohort we found that plasma Nef levels were correlated with plasma HIV RNA levels in untreated disease. However, we were able to detect Nef in plasma of approximately half of subjects on ART or with elite control, despite the lack of detectable plasma HIV RNA levels using standard assays. Plasma Nef levels were not consistently associated with CD4+ T-cell count, CD8+ T-cell count, self-reported nadir CD4+ T-cell count or the CD4+/CD8+ T-cell ratio in HIV-infected subjects.ConclusionSince plasma HIV RNA levels are undetectable in virally suppressed subjects, it is reasonable to assume that viral protein expression in leaky reservoir, and not plasma virions, is the source of Nef accumulating in plasma. To examine this further, improvements of the assay sensitivity, by lowering the background through improvements in the quality of Nef antibodies, and detailed characterization of the HIV reservoirs are needed.

Transposons can impact the host genome by altering gene expression and participating in chromosome rearrangements. Therefore, organisms evolved different ways to minimize the level of transposition. In Saccharomyces cerevisiae and its close relative S. paradoxus, Ty1 copy number control (CNC) is mediated by the self-encoded restriction factor p22, which is derived from the GAG capsid gene and inhibits virus-like particle (VLP) assembly and function. Based on secondary screens of Ty1 cofactors, we identified LOC1, a RNA localization/ribosome biogenesis gene that affects Ty1 mobility predominantly in strains harboring Ty1 elements. Ribosomal protein mutants rps0bΔ and rpl7aΔ displayed similar CNC-specific phenotypes as loc1Δ, suggesting that ribosome biogenesis is critical for CNC. The level of Ty1 mRNA and Ty1 internal (Ty1i) transcripts encoding p22 was altered in these mutants, and displayed a trend where the level of Ty1i RNA increased relative to full-length Ty1 mRNA. The level of p22 increased in these mutants, and the half-life of p22 also increased in a loc1Δ mutant. Transcriptomic analyses revealed small changes in the level of Ty1 transcripts or efficiency of translation initiation in a loc1Δ mutant. Importantly, a loc1Δ mutant had defects in assembly of Gag complexes and packaging Ty1 RNA. Our results indicate that defective ribosome biogenesis enhances CNC by increasing the level of p22, and raise the possibility for versatile links between VLP assembly, its cytoplasmic environment, and a novel stress response.

BackgroundHuman endogenous retroviruses (HERVs) comprise approximately 8% of the human genome and while the majority are transcriptionally silent, the most recently integrated HERV, HERV-K (HML-2), remains active. During HIV infection, HERV-K (HML-2) specific mRNA transcripts and viral proteins can be detected. In this study, we aimed to understand the antibody response against HERV-K (HML-2) Gag in the context of HIV-1 infection.ResultsWe developed an ELISA assay using either recombinant protein or 164 redundant "15mer" HERV-K (HML-2) Gag peptides to test sera for antibody reactivity. We identified a total of eight potential HERV-K (HML-2) Gag immunogenic domains: two on the matrix (peptides 16 and 31), one on p15 (peptide 85), three on the capsid (peptides 81, 97 and 117), one on the nucleocapsid (peptide 137) and one on the QP1 protein (peptide 157). Four epitopes (peptides 16, 31, 85 and 137) were highly immunogenic. No significant differences in antibody responses were found between HIV infected participants (n = 40) and uninfected donors (n = 40) for 6 out of the 8 epitopes tested. The antibody response against nucleocapsid (peptide 137) was significantly lower (p

Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for methamphetamine abuse in individuals with HIV.

Communication between intracellular organelles is essential for overall cellular function. How this communication occurs and under what circumstances alterations transpire are only the beginning to be elucidated. The pathways of calcium homeostasis, lipid transfer, mitochondrial dynamics, and mitophagy/apoptosis have been linked to the endoplasmic reticulum and tethering sites on the outer and/or inner mitochondrial membrane called mitochondria-associated endoplasmic reticulum membranes (MAM). Sensitive visualization by high-powered microscopy coupled with the advent of massive parallel sequencing has elaborated the structure, while patient’s diseases have uncovered the physiological function of these networks. Using specific patient examples from our pediatric mitochondrial center, we expand how specific genetic pathological variants in certain MAM structures induce disease. Genetic variants in MICU1, PASC-2, CYP2U1, SERAC1, and TANGO2 can induce early development abnormalities in the areas of cognition, motor, and central nervous system structures across multiple MAM pathways and implicate mitochondrial dysregulation.

The recombinant ALVAC vaccine coupled with the monomeric gp120/alum protein have decreased the risk of HIV and SIV acquisition. Ab responses to the V1/V2 regions have correlated with a decreased risk of virus acquisition in both humans and macaques. We hypothesized that the breadth and functional profile of Abs induced by an ALVAC/envelope protein regimen could be improved by substituting the monomeric gp120 boost, with the full-length single-chain (FLSC) protein. FLSC is a CD4-gp120 fusion immunogen that exposes cryptic gp120 epitopes to the immune system. We compared the immunogenicity and relative efficiency of an ALVAC-SIV vaccine boosted either with bivalent FLSC proteins or with monomeric gp120 in alum. FLSC was superior to monomeric gp120 in directing Abs to the C3 α2 helix, the V5 loop, and the V3 region that contains the putative CCR5 binding site. In addition, FLSC boosting elicited significantly higher binding Abs to V2 and increased both the Ab-dependent cellular cytotoxicity activity and the breadth of neutralizing Abs. However, the FLSC vaccine regimen demonstrated only a trend in vaccine efficacy, whereas the monomeric gp120 regimen significantly decreased the risk of SIVmac251 acquisition. In both vaccine regimens, anti-V2 Abs correlated with a decreased risk of virus acquisition but differed with regard to systemic or mucosal origin. In the FLSC regimen, serum Abs to V2 correlated, whereas in the monomeric gp120 regimen, V2 Abs in rectal secretions, the site of viral challenge, were associated with efficacy.

Neurotoxic viral protein TAT may contribute to deficits in dopaminergic and cognitive function in individuals infected with human immunodeficiency virus. Transgenic mice with brain-specific doxycycline-induced TAT expression (TAT+, TAT- control) show impaired cognition. However, previously reported TAT-induced deficits in reversal learning may be compromised by initial learning deficits. We investigated the effects of TAT expression on memory retention/recall and reversal learning, and neurotransmitter function. We also investigated if TAT-induced effects can be reversed by improving dopamine function with selegiline, a monoamine oxidase inhibitor. Mice were tested in the Barnes maze and TAT expression was induced after the task acquisition. Selegiline treatment continued throughout behavioral testing. Dopamine, serotonin and glutamate tissue levels in the prefrontal/orbitofrontal cortex, hippocampus and caudate putamen were measured using high performance liquid chromatography. Neither TAT expression nor selegiline altered memory retention. On day 2 of reversal learning testing, TAT+ mice made fewer errors and used more efficient search strategies than TAT- mice. TAT expression decreased dopamine turnover in the caudate putamen, increased serotonin turnover in the hippocampus and tended to increase the conversion of glutamate to glutamine in all regions. Selegiline decreased dopamine and serotonin metabolism in all regions and increased glutamate levels in the caudate putamen. In the absence of impaired learning, TAT expression does not impair spatial memory retention/recall, and actually facilitates reversal learning. Selegiline-induced increases in dopamine metabolism did not affect cognitive function. These findings suggest that TAT-induced alterations in glutamate signaling, but not alterations in monoamine metabolism, may underlie the facilitation of reversal learning.

Live-cell imaging has revealed unexpected features of gene expression. Here using improved single-molecule RNA microscopy, we show that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. During elongation, polymerases are spaced by few hundred nucleotides, and physical modelling suggests that DNA torsional stress may maintain polymerase spacing. We additionally observe that the HIV-1 promoter displays stochastic fluctuations on two time scales, which we refer to as multi-scale bursting. Each time scale is regulated independently: Mediator controls minute-scale fluctuation (convoys), while TBP-TATA-box interaction controls sub-hour fluctuations (long permissive/non-permissive periods). A cellular promoter also produces polymerase convoys and displays multi-scale bursting. We propose that slow, TBP-dependent fluctuations are important for phenotypic variability of single cells.

Despite tremendous progress in our understanding of human immunodeficiency virus (HIV) natural history and advances in HIV treatment, there is neither an approved vaccine nor a cure for infection. Here, we describe the development and characterization of a novel replicating vaccine vector utilizing Cytomegalovirus (CMV) and a TLR5 adjuvant. After partial truncation of the central, immunodominant hypervariable domain, flagellin (fliC) from Salmonella was cloned downstream of a codon optimized gag gene from simian immunodeficiency virus (SIV) and transiently expressed in telomerized rhesus fibroblast (TeloRF) cells in culture. Lysates generated from these transfected cells induced the tumor necrosis factor alpha (TNF-α), in a mouse macrophage cell line, in a TLR5-dependent manner. The Gag/FliC expression construct was cloned into a bacterial artificial chromosome encoding the rhesus CMV (RhCMV) genome, and infectious RhCMV was generated following transfection of TeloRF cells. This virus stably expressed an SIV Gag/FliC fusion protein through four serial passages. Lysates generated from infected cells induced TNF-α in a TLR5-dependent manner. Western blot analysis of infected cell lysates verified expression of a Gag/FliC fusion protein using a SIV p27 capsid monoclonal antibody. Lastly, rhesus macaques inoculated with this novel RhCMV virus demonstrated increased inflammatory responses at the site of inoculation seven days post-infection when compared to the parental RhCMV. These results demonstrate that an artificially constructed replicating RhCMV expressing an SIV Gag/FliC fusion protein is capable of activating TLR5 in a macrophage cell line in vitro and induction of an altered inflammatory response in vivo. Ongoing animals studies are aimed at determining vaccine efficacy, including subsequent challenge with pathogenic SIV.

BackgroundKoala retrovirus (KoRV) is an endogenous and exogenous retrovirus of koalas that may cause lymphoma. As for many other gammaretroviruses, the KoRV genome can potentially encode an alternate form of Gag protein, glyco-gag.ResultsIn this study, a convenient assay for assessing KoRV infectivity in vitro was employed: the use of DERSE cells (initially developed to search for infectious xenotropic murine leukemia-like viruses). Using infection of DERSE and other human cell lines (HEK293T), no evidence for expression of glyco-gag by KoRV was found, either in expression of glyco-gag protein or changes in infectivity when the putative glyco-gag reading frame was mutated. Since glyco-gag mediates resistance of Moloney murine leukemia virus to the restriction factor APOBEC3, the sensitivity of KoRV (wt or putatively mutant for glyco-gag) to restriction by murine (mA3) or human APOBEC3s was investigated. Both mA3 and hA3G potently inhibited KoRV infectivity. Interestingly, hA3G restriction was accompanied by extensive G → A hypermutation during reverse transcription while mA3 restriction was not. Glyco-gag status did not affect the results.ConclusionsThese results indicate that the mechanisms of APOBEC3 restriction of KoRV by hA3G and mA3 differ (deamination dependent vs. independent) and glyco-gag does not play a role in the restriction.

We describe a near infrared (NIR) light-activated gene silencing method in undifferentiated human embryonic stem cell (hESC) using a plasmonic hollow gold nanoshell (HGN) as the siRNA carrier. Our modular biotin-streptavidin coupling strategy enables positively charged TAT-peptide to coat oligonucleotides-saturated nanoparticles as a stable colloid formation. TAT-peptide coated nanoparticles with dense siRNA loading show efficient penetration into a wide variety of hESC cell lines. The siRNA is freed from the nanoparticles and delivered to the cytosol by femtosecond pulses of NIR light with potentially exquisite spatial and temporal control. The effectiveness of this approach is shown by targeting GFP and Oct4 genes in undifferentiated hESC (H9). The accelerated expression of differentiation markers for all three germ layers resulting from Oct4 knockdown confirms that this method has no detectable adverse effects that limit the range of differentiation. This biocompatible and NIR laser-activated patterning method makes possible single cell resolution of siRNA delivery for diverse studies in stem cell biology, tissue engineering and regenerative medicine.

In eukaryotes, receptor tyrosine kinases (RTKs) and trimeric G proteins are two major signaling hubs. Signal transduction via trimeric G proteins has long been believed to be triggered exclusively by G protein-coupled receptors (GPCRs). This paradigm has recently been challenged by several studies on a multimodular signal transducer, Gα-Interacting Vesicle associated protein (GIV/Girdin). We recently demonstrated that GIV's C terminus (CT) serves as a platform for dynamic association of ligand-activated RTKs with Gαi, and for noncanonical transactivation of G proteins. However, exogenous manipulation of this platform has remained beyond reach. Here we developed cell-permeable GIV-CT peptides by fusing a TAT-peptide transduction domain (TAT-PTD) to the minimal modular elements of GIV that are necessary and sufficient for activation of Gi downstream of RTKs, and used them to engineer signaling networks and alter cell behavior. In the presence of an intact GEF motif, TAT-GIV-CT peptides enhanced diverse processes in which GIV's GEF function has previously been implicated, e.g., 2D cell migration after scratch-wounding, invasion of cancer cells, and finally, myofibroblast activation and collagen production. Furthermore, topical application of TAT-GIV-CT peptides enhanced the complex, multireceptor-driven process of wound repair in mice in a GEF-dependent manner. Thus, TAT-GIV peptides provide a novel and versatile tool to manipulate Gαi activation downstream of growth factors in a diverse array of pathophysiologic conditions.

HIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL) complex as well as the non-canonical cofactor CBFβ, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac]) and non-primate (FIV, BIV, and MVV) viruses. We find that CBFβ is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA), for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity.

Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIVs reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.

Viral escape from HIV-1-specific CD8(+) T cells has been demonstrated in numerous studies previously. However, the qualitative features driving the emergence of mutations within epitopes are still unclear. In this study, we aimed to distinguish whether specific functional characteristics of HLA-B*5701-restricted CD8(+) T cells influence the emergence of mutations in high-risk progressors (HRPs) versus low-risk progressors (LRPs). Single-genome sequencing was performed to detect viral mutations (variants) within seven HLA-B*5701-restricted epitopes in Gag (n = 4) and Nef (n = 3) in six untreated HLA-B*5701 subjects followed from early infection up to 7 y. Several well-characterized effector markers (IFN-γ, IL-2, MIP-1β, TNF, CD107a, and perforin) were identified by flow cytometry following autologous (initial and emerging variant/s) epitope stimulations. This study demonstrates that specific functional attributes may facilitate the outgrowth of mutations within HLA-B*5701-restricted epitopes. A significantly lower fraction of IL-2-producing cells and a decrease in functional avidity and polyfunctional sensitivity were evident in emerging epitope variants compared with the initial autologous epitopes. Interestingly, the HRPs mainly drove these differences, whereas the LRPs maintained a directed and maintained functional response against emerging epitope variants. In addition, LRPs induced improved cell-cycle progression and perforin upregulation after autologous and emerging epitope variant stimulations in contrast to HRPs. The maintained quantitative and qualitative features of the CD8(+) T cell responses in LRPs toward emerging epitope variants provide insights into why HLA-B*5701 subjects have different risks of HIV-1 disease progression.

Disruption of the conserved motif GYxxØ in the simian immunodeficiency virus (SIV) SIVmac239 envelope (Env) cytoplasmic tail resulted in a virus (ΔGY) that exhibited a high plasma peak but uniquely failed to acutely deplete mucosal CD4(+) T cells. Here, we show that ΔGY containing a flanking S727P mutation that was acquired in ΔGY-infected macaques reacquired the ability to rapidly deplete CD4(+) T cells in lamina propria. This suggests that the GYxxØ motif and S727P each contribute to SIV's targeting to mucosal tissues.

The genomes of RNA viruses often contain RNA structures that are crucial for translation and RNA replication and may play additional, uncharacterized roles during the viral replication cycle. For the picornavirus family member poliovirus, a number of functional RNA structures have been identified, but much of its genome, especially the open reading frame, has remained uncharacterized. We have now generated a global RNA structure map of the poliovirus genome using a chemical probing approach that interrogates RNA structure with single-nucleotide resolution. In combination with orthogonal evolutionary analyses, we uncover several conserved RNA structures in the open reading frame of the viral genome. To validate the ability of our global analyses to identify functionally important RNA structures, we further characterized one of the newly identified structures, located in the region encoding the RNA-dependent RNA polymerase, 3D(pol), by site-directed mutagenesis. Our results reveal that the structure is required for viral replication and infectivity, since synonymous mutants are defective in these processes. Furthermore, these defects can be partially suppressed by mutations in the viral protein 3C(pro), which suggests the existence of a novel functional interaction between an RNA structure in the 3D(pol)-coding region and the viral protein(s) 3C(pro) and/or its precursor 3CD(pro).

APOBEC3 proteins mediate potent antiretroviral activity by hypermutating the retroviral genome during reverse transcription. To counteract APOBEC3 and gain a replicative advantage, lentiviruses such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) have evolved the Vif protein, which targets APOBEC3 proteins for proteasomal degradation. However, the proteasome plays a critical role in the generation of T cell peptide epitopes. Whether Vif-mediated destruction of APOBEC3 proteins leads to the generation and presentation of APOBEC3-derived T cell epitopes on the surfaces of lentivirus-infected cells remains unknown. Here, using peptides derived from multiple Vif-sensitive APOBEC3 proteins, we identified APOBEC3-specific T cell responses in both HIV-1-infected patients and SIV-infected rhesus macaques. These results raise the possibility that these T cell responses may be part of the larger antiretroviral immune response.

The mammalian circadian clock is composed of interlocking feedback loops. Cryptochrome is a central component in the core negative feedback loop, whereas Rev-Erbα, a member of the nuclear receptor family, is an essential component of the interlocking loop. To understand the roles of different clock genes, we conducted a genetic interaction screen by generating single- and double-mutant mice. We found that the deletion of Rev-erbα in F-box/leucine rich-repeat protein (Fbxl3)-deficient mice rescued its long-circadian period phenotype, and our results further revealed that FBXL3 regulates Rev-Erb/retinoic acid receptor-related orphan receptor-binding element (RRE)-mediated transcription by inactivating the Rev-Erbα:histone deacetylase 3 corepressor complex. By analyzing the Fbxl3 and Cryptochrome 1 double-mutant mice, we found that FBXL3 also regulates the amplitudes of E-box-driven gene expression. These two separate roles of FBXL3 in circadian feedback loops provide a mechanism that contributes to the period determination and robustness of the clock.

HIV is shed in semen but the anatomic site of virus entry into the genital secretions is unknown. We determined viral RNA (vRNA) levels and the envelope gene sequence in the SIVmac 251 viral populations in the genital tract and semen of 5 adult male rhesus monkeys (Macaca mulatta) that were infected after experimental penile SIV infection. Paired blood and semen samples were collected from 1-9 weeks after infection and the monkeys were necropsied eleven weeks after infection. The axillary lymph nodes, testes, epididymis, prostate, and seminal vesicles were collected and vRNA levels and single-genome analysis of the SIVmac251 env variants was performed. At the time of semen collection, blood vRNA levels were between 3.09 and 7.85 log10 vRNA copies/ml plasma. SIV RNA was found in the axillary lymph nodes of all five monkeys and in 3 of 5 monkeys, all tissues examined were vRNA positive. In these 3 monkeys, vRNA levels (log10 SIVgag copies/ug of total tissue RNA) in the axillary lymph node (6.48 ± 0.50) were significantly higher than in the genital tract tissues: testis (3.67 ± 2.16; p

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We recently discovered the antisense protein of human T-cell leukemia virus (HTLV) type 2 (APH-2), whose messenger RNA is encoded by the antisense strand of the HTLV-2 genome. We quantified proviral load, level of tax, and APH-2 in a series of blood samples obtained from a cohort of HTLV-2 carriers. We determined whether APH-2 promotes cell proliferation. APH-2 was detectable in most samples tested and was correlated with proviral load. APH-2 levels were not correlated with lymphocyte count in vivo, consistent with the inability of APH-2 to promote cell proliferation in vitro. APH-2 does not promote cell proliferation and does not cause lymphocytosis.

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.

Murine leukemia viruses encode a unique form of Gag polyprotein, gPr80gag or glyco-gag. Translation of this protein is initiated from full-length viral mRNA at an upstream initiation site in the same reading frame as Pr65(gag), the precursor for internal structural (Gag) proteins. Whereas gPr80gag is evolutionarily conserved among gammaretroviruses, its mechanism of action has been unclear, although it facilitates virus production at a late assembly or release step. Here, it is shown that gPr80gag facilitates release of Moloney murine leukemia virus (M-MuLV) from cells along an IFN-sensitive pathway. In particular, gPr80gag-facilitated release occurs through lipid rafts, because gPr80gag-negative M-MuLV has a lower cholesterol content, is less sensitive to inhibition of release by the cholesterol-depleting agent MbetaCD, and there is less Pr65gag associated with detergent-resistant membranes in mutant-infected cells. gPr80gag can also facilitate the release of HIV-1-based vector particles from human 293T cells.

Most human T cell leukemia virus type 1 (HTLV-1) infected subjects remain asymptomatic throughout their lives, with a few individuals developing HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukemia. Lymphocytes from about half of HTLV-1 infected subjects spontaneously proliferate in vitro, and how this phenomenon relates to symptomatic disease outcome and viral burden is poorly understood. Spontaneous proliferation was measured in lymphocyte subsets, and these findings were correlated with HTLV-1 proviral load and Tax expression in PBMCs. We found that in addition to previously described vigorous CD8+ T cell spontaneous proliferation, natural killer (NK) cells spontaneously proliferated to a similar high level, resulting in expansion of CD56-expressing NK cells. Spontaneous NK cell proliferation positively correlated with HTLV-1 proviral load but not with Tax expression or the presence of HAM/TSP. The strongest correlate with clinical outcome in this cohort was the ability of cells to express Tax, while HTLV-1 proviral load was more closely related to spontaneous NK cell proliferation. These results demonstrate that spontaneous proliferation, Tax expression, and proviral load are inter-related but not equivalent, and that spontaneous lymphocyte proliferation is not restricted to T cells, the targets of HTLV-1 infection.

Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) retroviruses infect T lymphocytes. The minus strand of the HTLV-1 genome encodes HBZ, a protein that could play a role in the development of leukemia in infected patients. Herein, we demonstrate that the complementary strand of the HTLV-2 genome also encodes a protein that we named APH-2 for "antisense protein of HTLV-2." APH-2 mRNA is spliced, polyadenylated, and initiates in the 3'-long terminal repeat at different positions. This transcript was detected in all HTLV-2-infected cell lines and short-term culture of lymphocytes obtained from HTLV-2 African patients tested and in 4 of 15 HTLV-2-infected blood donors. The APH-2 protein is 183 amino acids long, is localized in the cell nucleus, and is detected in vivo. Despite the lack of a consensus basic leucine zipper domain, APH-2 interacts with cyclic adenosine monophosphate-response element binding protein (CREB) and represses Tax2-mediated transcription in Tax2-expressing cells and in cells transfected with an HTLV-2 molecular clone. Altogether, our results demonstrate the existence of an antisense strand-encoded protein in HTLV-2, which could represent an important player in the development of disorders, such as lymphocytosis, which is frequently observed in HTLV-2 patients.

The Ty3 retrotransposon assembles into 50-nm virus-like particles that occur in large intracellular clusters in the case of wild-type (wt) Ty3. Within these particles, maturation of the Gag3 and Gag3-Pol3 polyproteins by Ty3 protease produces the structural proteins capsid (CA), spacer, and nucleocapsid. Secondary and tertiary structure predictions showed that, like retroviral CA, Ty3 CA contains a large amount of helical structure arranged in amino-terminal and carboxyl-terminal bundles. Twenty-six mutants in which alanines were substituted for native residues were used to study CA subdomain functions. Transposition was measured, and particle morphogenesis and localization were characterized by analysis of protein processing, cDNA production, genomic RNA protection, and sedimentation and by fluorescence and electron microscopy. These measures defined five groups of mutants. Proteins from each group could be sedimented in a large complex. Mutations in the amino-terminal domain reduced the formation of fluorescent Ty3 protein foci. In at least one major homology region mutant, Ty3 protein concentrated in foci but no wt clusters of particles were observed. One mutation in the carboxyl-terminal domain shifted assembly from spherical particles to long filaments. Two mutants formed foci separate from P bodies, the proposed sites of assembly, and formed defective particles. P-body association was therefore found to be not necessary for assembly but correlated with the production of functional particles. One mutation in the amino terminus blocked transposition after cDNA synthesis. Our data suggest that Ty3 proteins are concentrated first, assembly associated with P bodies occurs, and particle morphogenesis concludes with a post-reverse transcription, CA-dependent step. Particle formation was generally resistant to localized substitutions, possibly indicating that multiple domains are involved.

The ability of human immunodeficiency virus type 1 (HIV-1) to develop high levels of genetic diversity, and thereby acquire mutations to escape immune pressures, contributes to the difficulties in producing a vaccine. Possibly no single HIV-1 sequence can induce sufficiently broad immunity to protect against a wide variety of infectious strains, or block mutational escape pathways available to the virus after infection. The authors describe the generation of HIV-1 immunogens that minimizes the phylogenetic distance of viral strains throughout the known viral population (the center of tree [COT]) and then extend the COT immunogen by addition of a composite sequence that includes high-frequency variable sites preserved in their native contexts. The resulting COT(+) antigens compress the variation found in many independent HIV-1 isolates into lengths suitable for vaccine immunogens. It is possible to capture 62% of the variation found in the Nef protein and 82% of the variation in the Gag protein into immunogens of three gene lengths. The authors put forward immunogen designs that maximize representation of the diverse antigenic features present in a spectrum of HIV-1 strains. These immunogens should elicit immune responses against high-frequency viral strains as well as against most mutant forms of the virus.