Mimicking SIV chimpanzee viral evolution toward HIV-1 during cross-species transmission

HIV-1 evolved from SIV during cross-species transmission events, though viral genetic changes are not well understood. Here, we studied the evolution of SIVcpzLB715 into HIV-1 Group M using humanized mice. High viral loads, rapid CD4⁺ T-cell decline, and non-synonymous substitutions were identified throughout the viral genome suggesting viral adaptation.     

Evolution of SIVsm in humanized mice towards HIV-2

Through the accumulation of adaptive mutations, HIV-2 originated from SIVsm. To identify these evolutionary changes, a humanized mouse model recapitulated the process that likely enabled this cross-species transmission event. Various adaptive mutations arose, as well as increased virulence and CD4⁺ T-cell decline as the virus was passaged in humanized mice.     

HIV-1 tracing method of systemic viremia in vivo using an artificially mutated virus pool

The appearance of human immunodeficiency virus type 1 (HIV-1) plasma viremia is associated with progression to symptomatic disease and CD4⁺ T cell depletion. To locate the source of systemic viremia, this study employed a novel method to trace HIV-1 infection in vivo. We created JRCSFξnef, a pool of infectious HIV-1 (strain JR-CSF) with highly mutated nef gene regions by random mutagenesis PCR and infected this mutated virus pool into both Jurkat-CCR5 cells and hematopoietic stem cell-transplanted humanized mice. Infection resulted in systemic plasma viremia in humanized mice and viral RNA sequencing helped us to identify multiple lymphoid organs such as spleen, lymph nodes, and bone marrow but not peripheral blood cells as the source of systemic viremia. Our data suggest that this method could be useful for the tracing of viral trafficking in vivo.     

Development of a chronically catheterized maternal-fetal macaque model to study in utero mother-to-fetus HIV transmission: A preliminary report

Abstract: The lack of a representative animal model that permits frequent in utero fetal blood sampling is a major limiting factor for the study of maternal-fetal HIV transmission. Therefore, we have developed a maternal-fetal virus infection model using chronically catheterized macaques to simultaneously study the time-course of viral infection in the mother and the response of the fetus to maternal HIV infection. Pregnant macaques were infected with 10³ infectious units of HIV-2₂₈₇; every 3 days blood samples from both the mother and the fetus as well as amniotic fluid samples were collected. We found a varying degree of peak and time-to-peak virus load, virus-infected PBMCs, and free virus (determined by QC-RNA-PCR method) in maternal blood. Two of the three mothers with more than 10⁸ copies of viral RNA/ml of plasma at peak viremia transmitted the virus to their fetuses at about 14 days post-infection. As observed with HIV-2₂₈₇ infected mothers, virus-infected fetuses also produced a rapid rate of CD4⁺ cell decline in utero.     

Structure of antibody F425-B4e8 in complex with a V3 peptide reveals a new binding mode for HIV-1 neutralization

F425-B4e8 (B4e8) is a monoclonal antibody isolated from a human immunodeficiency virus type 1 (HIV-1)-infected individual that recognizes the V3 variable loop on the gp120 subunit of the viral envelope spike. B4e8 neutralizes a subset of HIV-1 primary isolates from subtypes B, C and D, which places this antibody among the very few human anti-V3 antibodies with notable cross-neutralizing activity. Here, the crystal structure of the B4e8 Fab′ fragment in complex with a 24-mer V3 peptide (RP142) at 2.8 Å resolution is described. The complex structure reveals that the antibody recognizes a novel V3 loop conformation, featuring a five-residue α-turn around the conserved GPGRA apex of the β-hairpin loop. In agreement with previous mutagenesis analyses, the Fab′ interacts primarily with V3 through side-chain contacts with just two residues, Ile^P309 and Arg^P315, while the remaining contacts are to the main chain. The structure helps explain how B4e8 can tolerate a certain degree of sequence variation within V3 and, hence, is able to neutralize an appreciable number of different HIV-1 isolates.     

Adaptation of HIV-1 to its human host

Human immunodeficiency virus type 1 (HIV-1) originated from three independent cross-species transmissions of simian immunodeficiency virus (SIVcpzPtt) infecting chimpanzees (Pan troglodytes troglodytes) in west central Africa, giving rise to pandemic (group M) and non-pandemic (groups N and O) clades of HIV-1. To identify host-specific adaptations in HIV-1 we compared the inferred ancestral sequences of HIV-1 groups M, N and O to 12 full length genome sequences of SIVcpzPtt and four of the outlying but closely related SIVcpzPts (from P. t. schweinfurthii). This analysis revealed a single site that was completely conserved among SIVcpzPtt strains but different (due to the same change) in all three groups of HIV-1. This site, Gag-30, lies within p17, the gag-encoded matrix protein. It is Met in SIVcpzPtt, underwent a conservative replacement by Leu in one lineage of SIVcpzPts but changed radically to Arg on all three lineages leading to HIV-1. During subsequent diversification this site has been conserved as a basic residue (Arg or Lys) in most lineages of HIV-1. Retrospective analysis revealed that Gag-30 had reverted to Met in a previous experiment in which HIV-1 was passaged through chimpanzees. To examine whether this substitution conferred a species specific growth advantage, we used site-directed mutagenesis to generate variants of these chimpanzee-adapted HIV-1 strains with Lys at Gag-30, and tested their replication in both human and chimpanzee CD4+ T lymphocytes. Remarkably, viruses encoding Met replicated to higher titers than viruses encoding Lys in chimpanzee T cells, but the opposite was found in human T cells. Taken together, these observations provide compelling evidence for host-specific adaptation during the emergence of HIV-1 and identify the viral matrix protein as a modulator of viral fitness following transmission to the new human host.     

Modeling sequence evolution in acute HIV-1 infection

We describe a mathematical model and Monte Carlo (MC) simulation of viral evolution during acute infection. We consider both synchronous and asynchronous processes of viral infection of new target cells. The model enables an assessment of the expected sequence diversity in new HIV-1 infections originating from a single transmitted viral strain, estimation of the most recent common ancestor (MRCA) of the transmitted viral lineage, and estimation of the time to coalesce back to the MRCA. We also calculate the probability of the MRCA being the transmitted virus or an evolved variant. Excluding insertions and deletions, we assume HIV-1 evolves by base substitution without selection pressure during the earliest phase of HIV-1 infection prior to the immune response. Unlike phylogenetic methods that follow a lineage backwards to coalescence, we compare the observed data to a model of the diversification of a viral population forward in time. To illustrate the application of these methods, we provide detailed comparisons of the model and simulations results to 306 envelope sequences obtained from eight newly infected subjects at a single time point. The data from patients were in good agreement with model predictions, and hence compatible with a single-strain infection evolving under no selection pressure. The diversity of the samples from the other two patients was too great to be explained by the model, suggesting multiple HIV-1-strains were transmitted. The model can also be applied to longitudinal patient data to estimate within-host viral evolutionary parameters.     

Intra-amniotic inoculation of pigtailed macaque (Macaca nemestrina) fetuses with SIV and HIV-1

Six pregnant pigtailed macaques (Macaca nemestrina) were inoculated intra-amniotically (i.a.) with SIV_Mne. All became viremic and seroconverted; three viable offspring were SIV-positive and at autopsy showed disseminated viral infection; one of three abortuses had SIV-infected thymic macrophages. Three of five pregnant macaques inoculated i.v. and/or i.a. with HIV-1_LAI became virus-positive, and four seroconverted, suggesting fetal-maternal transmission. One abortus had HIV-1-antigen in lymph nodes and brain; one infant, culture-positive at birth, died at age 11 days of disseminated HIV-1 infection.     

Determination of a T cell receptor of potent CD8+ T cells against simian immunodeficiency virus infection in Burmese rhesus macaques

Cumulative studies on human immunodeficiency virus (HIV)-infected individuals have shown association of major histocompatibility complex class I (MHC-I) polymorphisms with lower viral load and delayed AIDS progression, suggesting that HIV replication can be controlled by potent CD8⁺ T-cell responses. We have previously established an AIDS model of simian immunodeficiency virus (SIV) infection in Burmese rhesus macaques and found a potent CD8⁺ T cell targeting the Mamu-A1*065:01-restricted Gag_241-249 epitope, which is located in a region corresponding to the HIV Gag_240-249 TW10 epitope restricted by a protective MHC-I allele, HLA-B*57. In the present study, we determined a T cell receptor (TCR) of this Gag_241-249 epitope-specific CD8⁺ T cell. cDNA clones encoding TCR-α and TCR-β chains were obtained from a Gag_241-249-specific CD8⁺ T-cell clone. Coexpression of these TCR-α and TCR-β cDNAs resulted in reconstitution of a functional TCR specifically detected by Gag_241-249 epitope-Mamu-A1*065:01 tetramer. Two of three previously-reported CD8⁺ T-cell escape mutations reduced binding affinity of Gag_241-249 peptide to Mamu-A1*065:01 but the remaining one not. This is consistent with the data obtained by molecular modeling of the epitope-MHC-I complex and TCR. These results would contribute to understanding how viral CD8⁺ T-cell escape mutations are selected under structural constraint of viral proteins.     

Persistent hepatitis C virus infections and hepatopathological manifestations in immune-competent humanized mice

The majority of hepatitis C virus (HCV) infection develops chronic infection, which causes steatosis, cirrhosis and hepatocellular carcinoma. However, understanding HCV chronicity and pathogenesis is hampered by its narrow host range, mostly restricted to human and chimpanzee. Recent endeavour to infect a variety of humanized mice has not been able to achieve persistent HCV infection unless the essential innate immune responsive genes are knocked out. Nevertheless, such immune-compromised humanized mice still lacked HCV infection-induced hepatopathogenesis. Here we report that transgenic mice in ICR background harboring both human CD81 and occludin genes (C/O^Tg) are permissive to HCV infection at a chronicity rate comparable to humans. In this mouse model, HCV accomplishes its replication cycle, leading to sustained viremia and infectivity for more than 12 months post infection with expected fibrotic and cirrhotic progression. Host factors favorable for HCV replication, and inadequate innate immune-response may contribute to the persistence. Lastly, NS3/4 protease inhibitor telaprevir can effectively inhibit de novo RNA synthesis and acute HCV infection of C/O^Tg mice. Thus, chronic HCV infection with complete replication cycle and hepatopathologic manifestations is recapitulated, for the first time, in immune-competent mice. This model will open a new venue to study the mechanisms of chronic hepatitis C and develop better treatments.     

Efficient gene transfer mediated by HIV-1-based defective lentivector and inhibition of HIV-1 replication

Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5–1.2 × 10⁷IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols. Foundation items: National Institute of Health (S11 NS43499); RCMI (G12RR/AI03061, USA.)     

Enigma of HIV-1 latent infection in astrocytes: an in-vitro study using protein kinase C agonist as a latency reversing agent

Purging HIV-1 to cure the infection in patients undergoing suppressive antiretroviral therapy requires targeting all possible viral reservoirs. Other than the memory CD4⁺ T cells, several other HIV-1 reservoirs have been identified. HIV-1 infection in the brain as a reservoir is well documented, but not fully characterized. There, microglia, perivascular macrophages, and astrocytes can be infected by HIV-1. HIV-1 infection in astrocytes has been described as a nonproductive and primarily a latent infection. Using primary human astrocytes, we investigated latent HIV-1 infection and tested phorbol 12-myristate 13-acetate (PMA), a protein kinase C agonist, as an HIV-1-latency- reversing agent in infected astrocytes. Chloroquine (CQ) was used to facilitate initial HIV-1 escape from endosomes in astrocytes. CQ significantly increased HIV-1 infection. But treatment with PMA or viral Tat protein was similar to untreated HIV-1-infected astrocytes. Long-term follow-up of VSV-envelope-pseudotyped HIV-1 infected astrocytes showed persistent infection for 110 days, indicating the active state of the virus.     

Studies on toll-like receptor stimuli responsiveness in HIV-1 and HIV-2 infections

Background: HIV-1 and HIV-2 are two related viruses with distinct clinical outcomes, where HIV-1 is more pathogenic and transmissible than HIV-2. The pathogenesis of both infections is influenced by the dysregulation and deterioration of the adaptive immune system. However, their effects on the responsiveness of innate immunity are less well known. Here, we report on toll-like receptor (TLR) stimuli responsiveness in HIV-1 or HIV-2 infections. Methods: Whole blood from 235 individuals living in Guinea-Bissau who were uninfected, infected with HIV-1, infected with HIV-2, and/or infected with HTLV-I, was stimulated with TLR7/8 and TLR9 agonists, R-848 and unmethylated CpG DNA. After TLR7/8 and TLR9 stimuli, the expression levels of IL-12 and IFN-α were related to gender, age, infection status, CD4⁺ T cell counts, and plasma viral load. Results: Defective TLR9 responsiveness was observed in the advanced disease stage, along with CD4⁺ T cell loss in both HIV-1 and HIV-2 infections. Moreover, TLR7/8 responsiveness was reduced in HIV-1 infected individuals compared with uninfected controls. Conclusions: Innate immunity responsiveness can be monitored by whole blood stimulation. Both advanced HIV-1 and HIV-2 infections may cause innate immunity dysregulation.     

A role for Rev in the association of HIV-1 gag mRNA with cytoskeletal β-actin and viral protein expression

Human immunodeficiency virus type-1 (HIV-1) Rev acts by inducing the specific nucleocytoplasmic transport of a class of incompletely spliced RNAs that encodes the viral structural proteins. The transfection of HeLA cells with a rev-defective HIV-1 expression plasmid, however, resulted in the export of overexpressed, intron-containing species of viral RNAs, possibly through a default process of nuclear retention. Thus, this system enabled us to directly compare Rev⁺ and Rev⁻ cells as to the usage of RRE-containing mRNAs by the cellular translational machinery. Biochemical examination of the transfected cells revealed that although significant levels of gag and env mRNAs were detected in both the presence and absence of Rev, efficient production of viral proteins was strictly dependent on the presence of Rev. A fluoroscence in situ hybridisation assay confirmed these findings and provided further evidence that even in the presence of Rev, not all of the viral mRNA was equally translated. At the early phase of RNA export in Rev⁺ cells, gag mRNA was observed throughout both the cytoplasm and nucleoplasm as uniform fine stippling. In addition, the mRNA formed clusters mainly in the perinuclear region, which were not observed in Rev⁻ cells. In the presence of Rev, expression of the gag protein was limited to these perinuclear sites where the mRNA accumulated. Subsequent staining of the cytoskeletal proteins demonstrated that in Rev⁺ cells gag mRNA is colocalized with β-actin in the sites where the RNA formed clusters. In the absence of Rev, in contrast, the gag mRNA failed to associate with the cytoskeletal proteins. These results suggest that in addition to promoting the emergence of intron-containing RNA from the nucleus, Rev plays an important role in the compartmentation of translation by directing RRE-containing mRNAs to the β-actin to form the perinuclear clusters at which the synthesis of viral structural proteins begins.     

HIV-1 evolution: frustrating therapies,but disclosing molecular mechanisms

Replication of HIV-1 under selective pressure frequently results in the evolution of virus variants that replicate more efficiently under the applied conditions. For example, in patients on antiretroviral therapy, such evolution can result in variants that are resistant to the HIV-1 inhibitors, thus frustrating the therapy. On the other hand, virus evolution can help us to understand the molecular mechanisms that underlie HIV-1 replication. For example, evolution of a defective virus mutant can result in variants that overcome the introduced defect by restoration of the original sequence or by the introduction of additional mutations in the viral genome. Analysis of the evolution pathway can reveal the requirements of the element under study and help to understand its function. Analysis of the escape routes may generate new insight in the viral life cycle and result in the identification of unexpected biological mechanisms. We have developed in vitro HIV-1 evolution into a systematic research tool that allows the study of different aspects of the viral replication cycle. We will briefly review this method of forced virus evolution and provide several examples that illustrate the power of this approach.     

Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection

HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4⁺ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1.