Retrovirology - Latest Articles

Response of a simian immunodeficiency virus (SIVmac251) to raltegravir: a basis for a new treatment for simian AIDS and an animal model for studying lentiviral persistence during antiretroviral therapy

Mark Lewis — 2010-03-16T00:00:00Z

Background: In this study we successfully created a new approach to ART in SIVmac251 infected nonhuman primates. This drug regimen is entirely based on drugs affecting the pre-integration stages of replication and consists of only two nucleotidic/nucleosidic reverse transcriptase inhibitors (Nt/NRTIs) and raltegravir, a promising new drug belonging to the integrase strand transfer inhibitor (INSTI) class. Results: In acutely infected human lymphoid CD4+ T-cell lines MT-4 and CEMx174, SIVmac251 replication was efficiently inhibited by raltegravir, which showed an EC90 in the low nanomolar range. This result was confirmed in primary macaque PBMCs and enriched CD4+ T cell fractions. In vivo monotherapy with raltegravir for only ten days resulted in reproducible decreases in viral load in two different groups of animals. When emtricitabine (FTC) and tenofovir disoproxil fumarate (PMPA) were added to treatment, undetectable viral load was reached in two weeks, and a parallel increase in CD4 counts was observed. In contrast, the levels of proviral DNA did not change significantly during the treatment period, thus showing persistence of this lentiviral reservoir during therapy. Conclusions: In line with the high conservation of the three main amino acids Y143, Q148 and N155 (responsible for raltegravir binding) and molecular docking simulations showing similar binding modes of raltegravir at the SIVmac251 and HIV-1 IN active sites, raltegravir is capable of inhibiting SIVmac251 replication both in tissue culture and in vivo. This finding may help to develop effective ART regimens for the simian AIDS model entirely based on drugs adopted for treatment in humans. This ART-treated AIDS nonhuman primate model could be employed to find possible strategies for virus eradication from the body.

GPG-NH2 acts via the metabolite alphaHGA to target HIV-1 Env to the ER-associated protein degradation pathway

Alenka Jejcic — 2010-03-15T00:00:00Z

Background: The synthetic peptide glycyl-prolyl-glycine amide (GPG-NH2) was previously shown to abolish the ability of HIV-1 particles to fuse with the target cells, by reducing the content of the viral envelope glycoprotein (Env) in progeny HIV-1 particles. The loss of Env was found to result from GPG-NH2 targeting the Env precursor protein gp160 to the ER-associated protein degradation (ERAD) pathway during its maturation. However, the anti-viral effect of GPG-NH2 has been shown to be mediated by its metabolite alpha-hydroxy-glycine amide (alphaHGA), which is produced in the presence of fetal bovine serum, but not human serum. In accordance, we wanted to investigate whether the targeting of gp160 to the ERAD pathway by GPG-NH2 was attributed to its metabolite alphaHGA. Results: In the presence of fetal bovine serum, GPG-NH2, its intermediary metabolite glycine amide (G-NH2), and final metabolite alphaHGA all induced the degradation of gp160 through the ERAD pathway. However, when fetal bovine serum was replaced with human serum only alphaHGA showed an effect on gp160, and this activity was further shown to be completely independent of serum. This indicated that GPG-NH2 acts as a pro-drug, which was supported by the observation that it had to be added earlier to the cell cultures than alphaHGA to induce the degradation of gp160. Furthermore, the substantial reduction of Env incorporation into HIV-1 particles that occurs during GPG-NH2 treatment was also achieved by treating HIV-1 infected cells with alphaHGA. Conclusions: The previously observed specificity of GPG-NH2 towards gp160 in HIV-1 infected cells, resulting in the production of Env (gp120/gp41) deficient fusion incompetent HIV-1 particles, was most probably due to the action of the GPG-NH2 metabolite alphaHGA.

Mutagenesis analysis of the zinc-finger antiviral protein

Xinlu Wang — 2010-03-13T00:00:00Z

Background: The zinc-finger antiviral protein (ZAP) specifically inhibits the replication of certain viruses, including murine leukemia virus (MLV), by preventing the accumulation of viral mRNA in the cytoplasm. ZAP directly binds to the viral mRNA through the zinc-finger motifs and recruits the RNA exosome to degrade the target RNA. RNA helicase p72 is required for the optimal function of ZAP. In an attempt to understand the structure-function relationship of ZAP, we performed alanine scanning analysis. Results: A series of ZAP mutants was generated, in which three consecutive amino acids were replaced with three alanines. The mutants were analyzed for their antiviral activities against pseudotyped MLV vector. Out of the nineteen mutants analyzed, seven displayed significantly lower antiviral activities. Two mutations were in the very N-terminal domain, and five mutations were within or around the first and second zinc-finger motifs. These mutants were further analyzed for their abilities to bind to the target RNA, the exosome, and the RNA helicase p72. Mutants Nm3 and Nm63 lost the ability to bind to RNA. Mutants Nm 63 and Nm93 displayed compromised interaction with p72, while the binding of Nm133 to p72 was very modest. The interactions of all the mutants with the exosome were comparable to wild type ZAP. Conclusions: The integrity of the very N-terminal domain and the first and second zinc-finger motifs appear to be required for ZAP's antiviral activity. Analyses of the mutants for their abilities to interact with the target RNA and RNA helicase p72 confirmed our previous results. The mutants that bind normally to the target RNA, the exosome, and the RNA helicase p72 may be useful tools for further understanding the mechanism underlying ZAP's antiviral activity.

GCN5-dependent acetylation of HIV-1 integrase enhances viral integration

Mariaelena Terreni — 2010-03-12T00:00:00Z

Background: An essential event during the replication cycle of HIV-1 is the integration of the reverse transcribed viral DNA into the host cellular genome. Our former report revealed that HIV-1 integrase (IN), the enzyme that catalyzes the integration reaction, is positively regulated by acetylation mediated by the histone acetyltransferase (HAT) p300. Results: In this study we demonstrate that another cellular HAT, GCN5, acetylates IN leading to enhanced 3'-end processing and strand transfer activities. GCN5 participates in the integration step of HIV-1 replication cycle as demonstrated by the reduced infectivity, due to inefficient provirus formation, in GCN5 knockdown cells. Within the C-terminal domain of IN, four lysines (K258, K264, K266, and K273) are targeted by GCN5 acetylation, three of which (K264, K266, and K273) are also modified by p300. Replication analysis of HIV-1 clones carrying substitutions at the IN lysines acetylated by both GCN5 and p300, or exclusively by GCN5, demonstrated that these residues are required for efficient viral integration. In addition, a comparative analysis of the replication efficiencies of the IN triple- and quadruple-mutant viruses revealed that even though the lysines targeted by both GCN5 and p300 are required for efficient virus integration, the residue exclusively modified by GCN5 (K258) does not affect this process. Conclusions: The results presented here further demonstrate the relevance of IN post-translational modification by acetylation, which results from the catalytic activities of multiple HATs during the viral replication cycle. Finally, this study contributes to clarifying the recent debate raised on the role of IN acetylated lysines during HIV-1 infection.

Tax gene expression and cell cycling but not cell death are selected during HTLV-1 infection in vivo

Linda Zane — 2010-03-11T00:00:00Z

Background: Adult T cell leukemia results from the malignant transformation of a CD4+ lymphoid clone carrying an integrated HTLV-1 provirus that has undergone several oncogenic events over a 30-60 year period of persistent clonal expansion. Both CD4+ and CD8+ lymphocytes are infected in vivo; their expansion relies on CD4+ cell cycling and on the prevention of CD8+ cell death. Cloned infected CD4+ but not CD8+ T cells from patients without malignancy also add up nuclear and mitotic defects typical of genetic instability related to the expression of the virus-encoded oncogene tax. HTLV-1 expression is cancer-prone in vitro, but in vivo numerous selection forces act to maintain T cell homeostasis and are possibly involved in clonal selection. Results: Here we demonstrate that the HTLV-1 associated CD4+ preleukemic phenotype and the specific patterns of CD4+ and CD8+ clonal expansion are in vivo selected processes. By comparing the effects of recent (1 month) experimental infections performed in vitro and those observed in cloned T cells from patients infected for > 6-26 years, we found that in chronically HTLV-1 infected individuals, HTLV-1 positive clones are selected for tax expression. In vivo, infected CD4+ cells are positively selected for cell cycling whereas infected CD8+ cells and uninfected CD4+ cells are negatively selected for the same processes. In contrast, the known HTLV-1-dependent prevention of CD8+ T cell death pertains to both in vivo and in vitro infected cells. Conclusions: Therefore, virus-cell interactions alone are not sufficient to initiate early leukemogenesis in vivo.

Detection of a gammaretrovirus, XMRV, in the human population: Open questions and implications for xenotransplantation

Joachim Denner — 2010-03-10T00:00:00Z

XMRV (xenotropic murine leukaemia virus-related virus) is a gammaretrovirus that has been detected in human patients with prostate carcinoma, chronic fatigue syndrome (CFS) and also in a small percentage of clinically healthy individuals. It is not yet clear whether the distribution of this virus is primarily limited to the USA or whether it is causally associated with human disease. If future investigations confirm a broad distribution of XMRV and its association with disease, this would have an impact on xenotransplantation of porcine tissues and organs. Xenotransplantation is currently being developed to compensate for the increasing shortage of human material for the treatment of tissue and organ failure but could result in the transmission of porcine pathogens. Maintenance of pathogen-free donor animals will dramatically reduce this risk, but some of the porcine endogenous retroviruses (PERVs) found in the genome of all pigs, can produce infectious virus and infect cultured human cells. PERVs are closely related to XMRV so it is critical to develop tests that discriminate between them. Since recombination can occur between viruses, and recombinants can exhibit synergism, recipients should be tested for XMRV before xenotransplantation.

High systemic levels of interleukin-10, interleukin-22 and C-reactive protein in Indian patients are associated with low in vitro replication of HIV-1 subtype C viruses

Juan Arias — 2010-03-09T00:00:00Z

Background: HIV-1 subtype C (HIV-1C) accounts for almost 50% of all HIV-1 infections worldwide and predominates in countries with the highest case-loads globally. Functional studies suggest that HIV-1C is unique in its biological properties, and there are contradicting reports about its replicative characteristics. The present study was conducted to evaluate whether the host cytokine environment modulates the in vitro replication capacity of HIV-1C viruses. Methods: A small subset of HIV-1C isolates showing efficient replication in peripheral blood mononuclear cells (PBMC) is described, and the association of in vitro replication capacity with disease progression markers and the host cytokine response was evaluated. Viruses were isolated from patient samples, and the corresponding in vitro growth kinetics were determined by monitoring for p24 production. Genotype, phenotype and co-receptor usage were determined for all isolates, while clinical category, CD4 cell counts and viral loads were recorded for all patients. Plasmatic concentrations of cytokines and, acute-phase response, and microbial translocation markers were determined; and the effect of cytokine treatment on in vitro replication rates was also measured. Results: We identified a small number of viral isolates showing high in vitro replication capacity in healthy-donor PBMC. HIV-1C usage of CXCR4 co-receptor was rare; therefore, it did not account for the differences in replication potential observed. There was also no correlation between the in vitro replication capacity of HIV-1C isolates and patients' disease status. Efficient virus growth was significantly associated with low interleukin-10 (IL-10), interleukin-22 (IL-22), and C-reactive protein (CRP) levels in plasma (p < .0001). In vitro, pretreatment of virus cultures with IL-10 and CRP resulted in a significant reduction of virus production, whereas IL-22, which lacks action on immune cells appears to mediate its anti-HIV effect through interaction with both IL-10 and CRP, and its own protective effect on mucosal membranes. Conclusions: These results indicate that high systemic levels of IL-10, CRP and IL-22 in HIV-1C-infected Indian patients are associated with low viral replication in vitro, and that the former two have direct inhibitory effects whereas the latter acts through downstream mechanisms that remain uncertain.

Dominance of highly divergent feline leukemia virus A progeny variants in a cat with recurrent viremia and fatal lymphoma

A Helfer-Hungerbuehler — 2010-02-19T00:00:00Z

Background: In a cat that had ostensibly recovered from feline leukemia virus (FeLV) infection, we observed the reappearance of the virus and the development of fatal lymphoma 8.5 years after the initial experimental exposure to FeLV-A/Glasgow-1. The goals of the present study were to investigate this FeLV reoccurrence and molecularly characterize the progeny viruses. Results: The FeLV reoccurrence was detected by the presence of FeLV antigen and RNA in the blood and saliva. The cat was feline immunodeficiency virus positive and showed CD4+ T-cell depletion, severe leukopenia, anemia and a multicentric monoclonal B-cell lymphoma. FeLV-A, but not -B or -C, was detectable. Sequencing of the envelope gene revealed three FeLV variants that were highly divergent from the virus that was originally inoculated (89-91% identity to FeLV-A/Glasgow-1). In the long terminal repeat 31 point mutations, some previously described in cats with lymphomas, were detected. The FeLV variant tissue provirus and viral RNA loads were significantly higher than the FeLV-A/Glasgow-1 loads. Moreover, the variant loads were significantly higher in lymphoma positive compared to lymphoma negative tissues. An increase in the variant provirus blood load was observed at the time of FeLV reoccurrence. Conclusions: Our results demonstrate that ostensibly recovered FeLV provirus-positive cats may act as a source of infection following FeLV reactivation. The virus variants that had largely replaced the inoculation strain had unusually heavily mutated envelopes. The mutations may have led to increased viral fitness and/or changed the mutagenic characteristics of the virus.

Anti-tetherin activities in Vpu-expressing primate lentiviruses

Su Jung Yang — 2010-02-18T00:00:00Z

Background: The anti-viral activity of the cellular restriction factor, BST-2/tetherin, was first observed as an ability to block the release of Vpu-minus HIV-1 from the surface of infected cells. However, tetherin restriction is also counteracted by primate lentiviruses that do not express a Vpu protein, where anti-tetherin functions are provided by either the Env protein (HIV-2, SIVtan) or the Nef protein (SIVsm/mac and SIVagm). Within the primate lentiviruses, Vpu is also present in the genomes of SIVcpz and certain SIVsyk viruses. We asked whether, in these viruses, anti-tetherin activity was always a property of Vpu, or if it had selectively evolved in HIV-1 to perform this function. Results: We found that despite the close relatedness of HIV-1 and SIVcpz, the chimpanzee viruses use Nef instead of Vpu to counteract tetherin. Furthermore, SIVcpz Nef proteins had activity against chimpanzee but not human tetherin. This specificity mapped to a short sequence that is present in the cytoplasmic tail of primate but not human tetherins, and this also accounts for the specificity of SIVsm/mac Nef for primate but not human tetherins. In contrast, Vpu proteins from four diverse members of the SIVsyk lineage all displayed an anti-tetherin activity that was active against macaque tetherin. Interestingly, Vpu from a SIVgsn isolate was also found to have activity against human tetherin. Conclusions: Primate lentiviruses show a high degree of flexibility in their use of anti-tetherin factors, indicating a strong selective pressure to counteract tetherin restriction. The identification of an activity against human tetherin in SIVgsn Vpu suggests that the presence of Vpu in the ancestral SIVmus/mon/gsn virus believed to have contributed the 3' half of the HIV-1 genome may have played a role in the evolution of viruses that could counteract human tetherin and infect humans.

In situ detection of Gag-specific CD8+ cells in the GI tract of SIV infected Rhesus macaques

Annelie Tjernlund — 2010-02-16T00:00:00Z

Background: SIV and HIV predominantly replicate in lymphoid tissue, but the study of virus specific CD8+ T cells in intact lymphoid tissue is difficult, as traditional in situ tetramer staining requires fresh tissue. Results: In this report, we demonstrate a novel technique using Qdot 655-conjugated peptide-MHC multimers to directly visualize SIV specific cells in cryopreserved tissue biopsies from chronically SIVmac239 infected Rhesus macaques. Qdot 655 multimers showed similar sensitivity and specificity to APC-conjugated tetramers by flow cytometry analysis, but yielded ten-fold higher signal intensity when imaged by fluorescence microscopy. Using this technique, we detected CD8+ T cells which recognize an immunodominant epitope (Gag CM9) in the spleen, lymph nodes, ileum and colon. In all these tissues, the Gag CM9 positive cells were mainly located in the extra follicular T cell zone. In the ileum and colon, we found Gag CM9 positive cells concentrated in Peyer's patches and solitary lymphoid follicles, a pattern of localization not previously described. Conclusions: The use of Qdot multimers provide an anatomic and quantitative evaluation of SIV specific CD8+ T cell responses in SIV pathogenesis, and may prove useful to studies of SIV specific CD8+ T cell responses elicited by vaccines and other immunotherapies in the non-human primate model.