Rhesus Macaque Simian Immunodeficiency Virus Research Articles

Transformation of brain myeloid cell populations by SIV in rhesus macaques revealed by multiomics.

The primary immune constituents in the brain, microglia and macrophages, are the target for HIV in people and the simian immunodeficiency virus (SIV) in nonhuman primates. This infection can lead to neurological dysfunction, known as HIV-associated neurocognitive disorder (HAND). Given the gaps in our knowledge on how these cells respond in vivo to CNS infection, we performed single-cell multiomic sequencing, including gene expression and ATAC-seq, on myeloid cells from the brains of rhesus macaques with SIV-induced encephalitis (SIVE) as well as uninfected controls. We found that the myeloid cell populations were significantly changed by SIVE. In SIVE microglia-like cells express high levels of chemoattractants capable of recruiting highly activated CAM-like cells to the site of infection/inflammation. A unique population of microglia-like cells was found in which the chromatin accessibility of genes diverged from their RNA expression. Additionally, we observed a dramatic shift of upstream gene regulators and their targets in brain myeloid cells during SIVE. In summary, this study further uncovers the transcriptome, gene regulatory events and potential roles of different brain myeloid phenotypes in SIVE.

PD-1 blockade following ART interruption enhances control of pathogenic SIV in rhesus macaques

Programmed death-1 (PD-1) blockade during chronic Simian immunodeficiency virus (SIV) infection results in restoration of CD8 T-cell function and enhances viral control. Here, we tested the therapeutic benefits of PD-1 blockade administered soon after anti-retrovial therapy (ART) interruption (ATI) by treating SIV-infected and ART-suppressed macaques with either an anti-PD-1 antibody (n = 7) or saline (n = 4) at 4 wk after ATI. Following ATI, the plasma viremia increased rapidly in all animals, and the frequency of SIV-specific CD8 T cells also increased in some animals. PD-1 blockade post ATI resulted in higher proliferation of total memory CD8 and CD4 T cells and natural killer cells. PD-1 blockade also resulted in higher proliferation of SIV-specific CD8 T cells and promoted their differentiation toward better functional quality. Importantly, four out of the seven anti-PD-1 antibody-treated animals showed a rapid decline in plasma viremia by 100- to 2300-fold and this was observed only in animals that showed measurable SIV-specific CD8 T cells post PD-1 blockade. These results demonstrate that PD-1 blockade following ATI can significantly improve the function of anti-viral CD8 T cells and enhance viral control and strongly suggests its potential synergy with other immunotherapies that induce functional CD8 T-cell response under ART. These results have important implications for HIV cure research.

Mutation in the Disordered Linker Region of Capsid Disrupts Viral Kinetics of a Neuropathogenic SIV in Rhesus Macaques.

ABSTRACTTRIM5α polymorphism in rhesus macaques (RM) limits the genetic pool of animals in which we can perform simian immunodeficiency virus (SIV) studies without first screening animals for permissive TRIM5α genotypes. We have previously shown that polymorphisms in the TRIM5α B30.2/SPRY domain impact the level of SIVsmm viremia in RM and that amino acid substitutions (P37S/R98S) in the capsid N-terminal domain (CA-NTD) enables the virus to overcome restriction in RMs with the restrictive homozygous TRIM5αTFP/TFP genotype. Since this genotype also negatively impacted the development of central nervous system (CNS) lesions in animals infected with the parental source of CL757, we sought to generate a TRIM5αTFP/TFP-resistant clone, SIV-804E-CL757-P37S/R98S (CL757-SS), using a similar strategy. Unexpectedly, viral replication of CL757-SS was impaired in RMs with either the permissive TRIM5αTFP/Q or the restrictive TRIM5αTFP/TFP genotype. Analysis of the virus which emerged in the latter animals led to the discovery of a preexisting mutation relative to other SIVs. This P146T substitution in a conserved disordered linker region in the C-terminal domain of capsid (CA-CTD) has been shown to inhibit proper formation of HIV-1 capsid particles. Restoration of this residue to proline in the context of the TRIM5α-SS escape mutations not only restored viral replication, but also enhanced the infectivity of our previously reported neurotropic clone, even in RMs with permissive TRIM5α genotypes.IMPORTANCE SIV infection of rhesus macaques has become a valuable model for the development of AIDS vaccines and antiretroviral therapies. Polymorphisms in the rhesus macaque TRIM5α gene can affect SIV replication, making it necessary to genetically screen macaques for TRIM5α alleles that are permissive for SIV replication. This limits the pool of animals that can be used in a study, thereby making the acquisition of animals needed to fulfill study parameters difficult. We have constructed a viral clone that induces neuroAIDS in rhesus macaques regardless of their TRIM5α genotype, while also highlighting the important role the disordered linker domain plays in viral infectivity.

Protection against SIV in Rhesus Macaques Using Albumin and CD4-Based Vector-Mediated Gene Transfer

Antibody-like molecules were evaluated with potent simian immunodeficiency virus (SIV) neutralizing properties (immunoadhesins) that were delivered by a recombinant adeno-associated virus (rAAV) vector in the SIV-infected rhesus macaque model. When injected intramuscularly into the host, the vector directs in vivo production of the transgenes with antibody-like binding properties that lead to serum neutralizing activity against SIV. To extend the half-life of the immunoadhesins, rhesus cluster of differentiation 4 (CD4) and a single-chain antibody (4L6) were fused with albumin molecules, and these constructs were tested in our model of SIV infection. Antibody-based immunoadhesins provided high serum neutralizing titers against the original SIV strain. CD4-based immunoadhesins provided a wider spectrum of neutralization against different SIV strains in comparison to antibody-based therapeutics and had the potential to protect against high viral challenging doses. Although the albumin-antibody fusion immunoadhesin provided strong and prolonged protection of the immunized animals against SIV challenge, the albumin-CD4 fusion altered the specificity and decreased the overall protection effectiveness of the immunoadhesin in comparison to the antibody-based molecules. Albumin-based immunoadhesins increase in vivo longevity of the immune protection; however, they present challenges likely linked to the induction of anti-immunoadhesin antibodies.

SIV-Mediated Synaptic Dysfunction Is Associated with an Increase in Synapsin Site 1 Phosphorylation and Impaired PP2A Activity.

Although the reduction of viral loads in people with HIV undergoing combination antiretroviral therapy has mitigated AIDS-related symptoms, the prevalence of neurological impairments has remained unchanged. HIV-associated CNS dysfunction includes impairments in memory, attention, memory processing, and retrieval. Here, we show a significant site-specific increase in the phosphorylation of Syn I serine 9, site 1, in the frontal cortex lysates and synaptosome preparations of male rhesus macaques infected with simian immunodeficiency virus (SIV) but not in uninfected or SIV-infected antiretroviral therapy animals. Furthermore, we found that a lower protein phosphatase 2A (PP2A) activity, a phosphatase responsible for Syn I (S9) dephosphorylation, is primarily associated with the higher S9 phosphorylation in the frontal cortex of SIV-infected macaques. Comparison of brain sections confirmed higher Syn I (S9) in the frontal cortex and greater coexpression of Syn I and PP2A A subunit, which was observed as perinuclear aggregates in the somata of the frontal cortex of SIV-infected macaques. Synaptosomes from SIV-infected animals were physiologically tested using a synaptic vesicle endocytosis assay and FM4-64 dye showing a significantly higher baseline depolarization levels in synaptosomes of SIV+-infected than uninfected control or antiretroviral therapy animals. A PP2A-activating FDA-approved drug, FTY720, decreased the higher synaptosome depolarization in SIV-infected animals. Our results suggest that an impaired distribution and lower activity of serine/threonine phosphatases in the context of HIV infection may cause an indirect effect on the phosphorylation levels of essential proteins involving in synaptic transmission, supporting the occurrence of specific impairments in the synaptic activity during SIV infection.SIGNIFICANCE STATEMENT Even with antiretroviral therapy, neurocognitive deficits, including impairments in attention, memory processing, and retrieval, are still major concerns in people living with HIV. Here, we used the rhesus macaque simian immunodeficiency virus model with and without antiretroviral therapy to study the dynamics of phosphorylation of key amino acid residues of synapsin I, which critically impacts synaptic vesicle function. We found a significant increase in synapsin I phosphorylation at serine 9, which was driven by dysfunction of serine/threonine protein phosphatase 2A in the nerve terminals. Our results suggest that an impaired distribution and lower activity of serine/threonine phosphatases in the context of HIV infection may cause an indirect effect on the phosphorylation levels of essential proteins involved in synaptic transmission.

Analysis of Complement-Mediated Lysis of Simian Immunodeficiency Virus (SIV) and SIV-Infected Cells Reveals Sex Differences in Vaccine-Induced Immune Responses in Rhesus Macaques

An effective human immunodeficiency virus (HIV) vaccine has yet to be developed, and defining immune correlates of protection against HIV infection is of paramount importance to inform future vaccine design. The complement system is a component of innate immunity that can directly lyse pathogens and shape adaptive immunity. To determine if complement lysis of simian immunodeficiency virus (SIV) and/or SIV-infected cells represents a protective immune correlate against SIV infection, sera from previously vaccinated and challenged rhesus macaques were analyzed for the induction of antibody-dependent complement-mediated lysis (ADCML). Importantly, the vaccine regimen, consisting of a replication-competent adenovirus type 5 host-range mutant SIV recombinant prime followed by a monomeric gp120 or oligomeric gp140 boost, resulted in overall delayed SIV acquisition only in females. Here, sera from all vaccinated animals induced ADCML of SIV and SIV-infected cells efficiently, regardless of sex. A modest correlation of SIV lysis with a reduced infection rate in males but not females, together with a reduced peak viremia in all animals boosted with gp140, suggested a potential for influencing protective efficacy. Gag-specific IgG and gp120-specific IgG and IgM correlated with SIV lysis in females, while Env-specific IgM correlated with SIV-infected cell lysis in males, indicating sex differences in vaccine-induced antibody characteristics and function. In fact, gp120/gp140-specific antibody functional correlates between antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis, and ADCML as well as the gp120-specific IgG glycan profiles and the corresponding ADCML correlations varied depending on the sex of the vaccinees. Overall, these data suggest that sex influences vaccine-induced antibody function, which should be considered in the design of globally effective HIV vaccines in the future.IMPORTANCE An HIV vaccine would thwart the spread of HIV infection and save millions of lives. Unfortunately, the immune responses conferring universal protection from HIV infection are poorly defined. The innate immune system, including the complement system, is an evolutionarily conserved, basic means of protection from infection. Complement can prevent infection by directly lysing incoming pathogens. We found that vaccination against SIV in rhesus macaques induces antibodies that are capable of directing complement lysis of SIV and SIV-infected cells in both sexes. We also found sex differences in vaccine-induced antibody species and their functions. Overall, our data suggest that sex affects vaccine-induced antibody characteristics and function and that males and females might require different immune responses to protect against HIV infection. This information could be used to generate highly effective HIV vaccines for both sexes in the future.

Effects of alcohol consumption on antigen-specific cellular and humoral immune responses to SIV in rhesus macaques.

Simian immunodeficiency virus (SIV) infection in macaques chronically receiving ethanol results in significantly higher plasma viral loads and more rapid progression to end-stage disease. We thus hypothesized that the increased plasma viral load in ethanol-treated, SIV-infected macaques would negatively correlate with antigen-specific immune responses. Rhesus macaques were administered ethanol or sucrose (n = 12 per group) by indwelling gastric catheters for 3 months and then intravenously infected with SIVMAC251. Peripheral blood T- and B-cell immunophenotyping and quantification were performed. Plasma was examined for viremia, levels of SIVEnv-specific binding, and neutralizing antibodies. Virus-specific interferon γ and tumor necrosis factor α cytokine responses to SIV-Nef, Gag, or Env peptide pools were measured in peripheral blood CD8 T cells. Macaques receiving ethanol had both higher plasma viremia and virus-specific cellular immune responses compared with the sucrose-treated group. The emergence of virus-specific cytokine responses temporally correlated with the decline in mean plasma viral load after 14 days postinfection in all SIV-infected animals. However, neither the breadth and specificity nor the magnitude of virus-specific CD8 T-cell responses correlated with early postpeak reductions in plasma viral loads. In fact, increased cytokine responses against Gag, gp120, and gp41 positively correlated with plasma viremia. Levels of SIV envelope-specific immunoglobulin G and neutralizing antibodies were similar over the disease course in both groups of macaques. Persistently higher antigen-specific cytokine responses in animals receiving ethanol are likely an effect of the higher viral loads and antigen persistence, rather than a cause of the increased viremia.

Evidence for an Increased Risk of Transmission of Simian Immunodeficiency Virus and Malaria in a Rhesus Macaque Coinfection Model

In sub-Saharan Africa, HIV-1 infection frequently occurs in the context of other coinfecting pathogens, most importantly, Mycobacterium tuberculosis and malaria parasites. The consequences are often devastating, resulting in enhanced morbidity and mortality. Due to the large number of confounding factors influencing pathogenesis in coinfected people, we sought to develop a nonhuman primate model of simian immunodeficiency virus (SIV)-malaria coinfection. In sub-Saharan Africa, Plasmodium falciparum is the most common malaria parasite and is responsible for most malaria-induced deaths. The simian malaria parasite Plasmodium fragile can induce clinical symptoms, including cerebral malaria in rhesus macaques, that resemble those of P. falciparum infection in humans. Thus, based on the well-characterized rhesus macaque model of SIV infection, this study reports the development of a novel rhesus macaque SIV-P. fragile coinfection model to study human HIV-P. falciparum coinfection. Using this model, we show that coinfection is associated with an increased, although transient, risk of both HIV and malaria transmission. Specifically, SIV-P. fragile coinfected macaques experienced an increase in SIV viremia that was temporarily associated with an increase in potential SIV target cells and systemic immune activation during acute parasitemia. Conversely, primary parasitemia in SIV-P. fragile coinfected animals resulted in higher gametocytemia that subsequently translated into higher oocyst development in mosquitoes. To our knowledge, this is the first animal model able to recapitulate the increased transmission risk of both HIV and malaria in coinfected humans. Therefore, this model could serve as an essential tool to elucidate distinct immunological, virological, and/or parasitological parameters underlying disease exacerbation in HIV-malaria coinfected people.

Recombinant varicella vaccines induce neutralizing antibodies and cellular immune responses to SIV and reduce viral loads in immunized rhesus macaques

The development of an effective AIDS vaccine remains one of the highest priorities in HIV research. The live, attenuated varicella-zoster virus (VZV) Oka vaccine, safe and effective for prevention of chickenpox and zoster, also has potential as a recombinant vaccine against other pathogens, including human immunodeficiency virus (HIV). The simian varicella model, utilizing simian varicella virus (SVV), offers an approach to evaluate recombinant varicella vaccine candidates. Recombinant SVV (rSVV) vaccine viruses expressing simian immunodeficiency virus (SIV) env and gag antigens were constructed. The hypothesis tested was that a live, attenuated rSVV-SIV vaccine will induce immune responses against SIV in the rhesus macaques and provide protection against SIV challenge. The results demonstrated that rSVV-SIV vaccination induced low levels of neutralizing antibodies and cellular immune responses to SIV in immunized rhesus macaques and significantly reduced viral loads following intravenous challenge with pathogenic SIVmac251-CX-1.

Limited CD4+ T cell proliferation leads to preservation of CD4+ T cell counts in SIV-infected sooty mangabeys

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections result in chronic virus replication and progressive depletion of CD4+ T cells, leading to immunodeficiency and death. In contrast, 'natural hosts' of SIV experience persistent infection with high virus replication but no severe CD4+ T cell depletion, and remain AIDS-free. One important difference between pathogenic and non-pathogenic infections is the level of activation and proliferation of CD4+ T cells. We analysed the relationship between CD4+ T cell number and proliferation in HIV, pathogenic SIV in macaques, and non-pathogenic SIV in sooty mangabeys (SMs) and mandrills. We found that CD4+ T cell proliferation was negatively correlated with CD4+ T cell number, suggesting that animals respond to the loss of CD4+ T cells by increasing the proliferation of remaining cells. However, the level of proliferation seen in pathogenic infections (SIV in rhesus macaques and HIV) was much greater than in non-pathogenic infections (SMs and mandrills). We then used a modelling approach to understand how the host proliferative response to CD4+ T cell depletion may impact the outcome of infection. This modelling demonstrates that the rapid proliferation of CD4+ T cells in humans and macaques associated with low CD4+ T cell levels can act to 'fuel the fire' of infection by providing more proliferating cells for infection. Natural host species, on the other hand, have limited proliferation of CD4+ T cells at low CD4+ T cell levels, which allows them to restrict the number of proliferating cells susceptible to infection.

Inhibition of HIV-1 Replication by Amphotericin B Methyl Ester: SELECTION FOR RESISTANT VARIANTS

Membrane cholesterol plays an important role in human immunodeficiency virus type 1 (HIV-1) particle production and infectivity. Here, we have investigated the target and mechanism of action of a cholesterol-binding compound, the polyene antifungal antibiotic amphotericin B methyl ester (AME). We found that AME potently inhibited the replication of a highly divergent panel of HIV-1 isolates in various T-cell lines and primary cells irrespective of clade or target cell tropism. The defects in HIV-1 replication caused by AME were due to profoundly impaired viral infectivity as well as a defect in viral particle production. To elucidate further the mechanism of action of AME, we selected for and characterized AME-resistant HIV-1 variants. Mutations responsible for AME resistance mapped to a highly conserved and functionally important endocytosis motif in the cytoplasmic tail of the transmembrane glycoprotein gp41. Interestingly, truncation of the gp41 cytoplasmic tail in the context of either HIV-1 or rhesus macaque simian immunodeficiency virus also conferred resistance to AME. The infectivity of HIV-1 virions bearing murine leukemia virus or vesicular stomatitis virus glycoproteins was unaffected by AME. Our data define the target and mechanism of action of AME and provide support for the concept that cholesterol-binding compounds should be pursued as antiretroviral drugs to disrupt HIV-1 replication.

CpG-C ISS-ODN activation of blood-derived B cells from healthy and chronic immunodeficiency virus-infected macaques

Cytosine-phosphate-guanine class C (CpG-C) immunostimulatory sequence oligodeoxynucleotides (ISS-ODNs) activate human B cells and dendritic cells (DCs), properties that suggest potential use as a novel adjuvant to enhance vaccine efficacy. After demonstrating that the CpG-C ISS-ODN C274 activates macaque DCs, we examined in vitro activation of macaque B cells by C274 as a prelude to evaluation of this molecule as an adjuvant in the testing of candidate human immunodeficiency virus vaccines in the rhesus macaque-simian immunodeficiency virus (SIV) model. C274 induced macaque CD20(+) B cells to proliferate more strongly than CD40 ligand or CpG-B ISS-ODN. C274 enhanced B cell survival; increased viability was most evident after 3-7 days of culture. Increased expression of CD40, CD80, and CD86 by B cells was apparent within 24 h of exposure to C274 and persisted for up to 1 week. C274-stimulated, B cell-enriched and peripheral blood mononuclear cell suspensions from naïve and immunodeficiency virus-infected monkeys secreted several cytokines [e.g., interleukin (IL)-3, IL-6, IL-12, interferon-alpha] and chemokines [e.g., monocyte chemoattractant protein-1/CC chemokine ligand 2 (CCL2), macrophage-inflammatory protein-1alpha/CCL3, IL-8/CXC chemokine ligand 8]. In comparison, exposure of macaque B cells to SIV had minimal impact on surface phenotype, despite inducing cytokine and chemokine production in cells from infected and uninfected animals. These observations emphasize the need to identify strategies to optimally boost immune function, as immunodeficiency viruses themselves only partially activate B cells and DCs. The ability of C274 to stimulate B cells and DCs in healthy and infected monkeys suggests its possible use as a broad-acting adjuvant to be applied in the rhesus macaque model for the development of preventative and therapeutic vaccines.

760. Overcoming Rhesus Macaque Endogenous Restriction Factors during HIV-1 Vector Transduction

Top of pageAbstract The ability to deliver genes into hematopoietic stem cells via HIV-1 derived vectors holds promise to investigate the pathobiology and therapeutic applications for infectious diseases. In vivo HIV-1 infection models are limited. The NOD/SCID-hu mouse has been an excellent model to study thymic function and HIV-1 thymic depletion. However, to better understand HIV-1 pathogenesis in humans, non-human primate models must be used. The rhesus macaque simian immunodeficiency virus (SIV) model recapitulates aspects of the pathology seen in human HIV-1 infection. Our laboratory has designed and previously described a CCR5 intrabody HIV-1 vector that efficiently prevents R5-tropic HIV-1 entry and provides transduced CD4+ T cells with a selective advantage during a dendritic cell-mediated infection. Unfortunately, transduction with a HIV-1 derived vector undergoes a post-entry restriction when introduced into rhesus macaque cells. The target for rhesus macaque endogenous restriction factors lies within the HIV-1 capsid protein, and may be due to rhesus macaque Cyclophilin A (CypA) incorporation and TRIM-5|[alpha]| capsid interactions. To increase transduction efficacy in both non-human primate and human hematopoietic cells we have assessed a naturally occurring HIV-1 capsid motif (V86P/H87Q/I91V/M96I), which evades both CypA- dependence in human cells and TRIM-5|[alpha]|-CypA restriction in owl monkey kidney cells during HIV-1 infection (Chatterji, et al. JBC, 2005). The V86P/H87Q/I91V/M96I capsid mutation within the CypA binding loop also rendered VSV-G pseudotyped HIV-1 vectors independent of CypA incorporation during human and non-human primate cell transduction. Interestingly, HIV-1 vectors with the V86P/H87Q/I91V/M96I capsid mutation were able to efficiently transduce non-human primate Vero and sMAGI cell lines and yielded a 1.4-fold increase in transduction efficiency in primary human CD4+ T cells. However, the V86P/H87Q/I91V/M96I HIV-1 vector did not efficiently transduce primary rhesus macaque CD4+ T cells. We are now in the process of evaluating novel HIV-1 capsid mutations that lie within and around the CypA binding loop in our HIV-1 vector system, such as H87Q and Q50Y/T54Q. Some of these capsid mutations in HIV-1 are required to efficiently infect primary rhesus macaque macrophages. We will present our findings on HIV-1 capsid mutations that allow HIV-1 vector entry into primary rhesus macaque CD4+ T cells.

Simian immunodeficiency virus envelope compartmentalizes in brain regions independent of neuropathology

Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) gp160s obtained from the brain are often genetically distinct from those isolated from other organs, suggesting the presence of brain-specific selective pressures or founder effects that result in the compartmentalization of viral quasi-species. Whereas HIV has also been found to compartmentalize within different regions of the brain, the extent of brain-regional compartmentalization of SIV in rhesus macaques has not been characterized. Furthermore, much is still unknown about whether phenotypic differences exist in envelopes from different brain regions. To address these questions, env DNA sequences were amplified from four SIVmac239-infected macaques and subjected to phylogenetic and phenetic analysis. The authors demonstrated that sequences from different areas of the brain form distinct clades, and that the long-term progressing macaques demonstrated a greater degree of regional compartmentalization compared to the rapidly progressing macaques. In addition, regional compartmentalization occurred regardless of the presence of giant-cell encephalitis. Nucleotide substitution rates at synonymous and nonsynonymous sites (ds:dn rates) indicated that positive selection varied among envelopes from different brain regions. In one macaque, envelopes from some but not all brain regions acquired changes in a conserved CD4-binding motif GGGDPE at amino acids 382 to 387. Furthermore, gp160s with the mutation G383E were able to mediate cell-to-cell fusion in a CD4-independent manner and were more susceptible to fusion inhibition by pooled plasma from infected macaques. Reversion of this mutation by site-directed mutagenesis resulted in reduction of CD4-independence and resistance to fusion inhibition in cell fusion assays. These studies demonstrate that SIV evolution within the brain results in a heterogeneous viral population with different phenotypes among different regions.

A single amino acid substitution in human APOBEC3G antiretroviral enzyme confers resistance to HIV-1 virion infectivity factor-induced depletion.

HIV-1 and other retroviruses occasionally undergo hypermutation, characterized by a high rate of G-to-A substitution. Recently, the human apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G), first identified as CEM15, was shown to be packaged into retroviral virions and to deaminate deoxycytidine to deoxyuridine in newly synthesized viral minus-strand DNA, thereby inducing G-to-A hypermutation. This innate mechanism of resistance to retroviral infection is counteracted by the HIV-1 viral infectivity factor (Vif), which protects the virus by preventing the incorporation of APOBEC3G into virions by rapidly inducing its ubiquitination and proteasomal degradation. To gain insights into the mechanism by which Vif protects HIV-1 from APOBEC3G, we substituted several amino acids in human APOBEC3G with equivalent residues in simian APOBEC3Gs that are resistant to HIV-1 Vif and determined the effects of the mutations on HIV-1 replication in the presence and absence of Vif. We found that a single amino acid substitution mutant of human APOBEC3G (D128K) can interact with HIV-1 Vif but is not depleted from cells; thus, it inhibits HIV-1 replication in an HIV-1 Vif-resistant manner. Interestingly, rhesus macaque simian immunodeficiency virus 239 or HIV-2 Vif coexpression depleted the intracellular steady state levels of the D128K mutant and abrogated its antiviral activity, indicating that it can be a substrate for the proteasomal pathway. The HIV-1 Vif-resistant mutant APOBEC3G could provide a gene therapy approach to combat HIV-1 infection.

Viral and immunological factors associated with breast milk transmission of SIV in rhesus macaques

BackgroundThe viral and host factors involved in transmission of HIV through breastfeeding are largely unknown, and intervention strategies are urgently needed to protect at-risk populations. To evaluate the viral and immunological factors directly related to milk transmission of virus, we have evaluated the disease course of Simian Immunodeficiency Virus (SIV) in lactating rhesus macaques (Macaca mulatta) as a model of natural breast milk transmission of HIV.ResultsFourteen lactating macaques were infected intravenously with SIV/DeltaB670, a pathogenic isolate of SIV and were pair-housed with their suckling infants throughout the disease course. Transmission was observed in 10 mother-infant pairs over a one-year period. Two mothers transmitted virus during the period of initial viremia 14–21 days post inoculation (p.i.) and were classified as early transmitters. Peak viral loads in milk and plasma of early transmitters were similar to other animals, however the early transmitters subsequently displayed a rapid progressor phenotype and failed to control virus expression as well as other animals at 56 days p.i. Eight mothers were classified as late transmitters, with infant infection detected at time points in the chronic stage of the maternal SIV disease course (81 to 360 days). Plasma viral loads, CD4+ T cell counts and SIV-specific antibody titers were similar in late transmitters and non-transmitters. Late breast milk transmission, however, was correlated with higher average milk viral loads and more persistent viral expression in milk 12 to 46 weeks p.i. as compared to non-transmitters. Four mothers failed to transmit virus, despite disease progression and continuous lactation.ConclusionThese studies validate the SIV-infected rhesus macaque as a model for breast milk transmission of HIV. As observed in studies of HIV-infected women, transmission occurred at time points throughout the period of lactation. Transmission during the chronic stage of SIV-infection correlated with a threshold level of virus expression as well as more persistent shedding in milk. This model will be a valuable resource for deciphering viral and host factors responsible for transmission of HIV through breastfeeding.

Route of simian immunodeficiency virus inoculation determines the complexity but not the identity of viral variant populations that infect rhesus macaques.

A better understanding of the host and viral factors associated with human immunodeficiency virus (HIV) transmission is essential to developing effective strategies to curb the global HIV epidemic. Here we used the rhesus macaque-simian immunodeficiency virus (SIV) animal model of HIV infection to study the range of viral genotypes that are transmitted by different routes of inoculation and by different types of viral inocula. Analysis of transmitted variants was undertaken in outbred rhesus macaques inoculated intravenously (IV) or intravaginally (IVAG) with a genetically heterogeneous SIVmac251 stock derived from a well-characterized rhesus macaque viral isolate. In addition, we performed serial IV and IVAG passage experiments using plasma from SIV-infected macaques as the inoculum. We analyzed the V1-V2 region of the SIV envelope gene from virion-associated RNA in plasma from infected animals by the heteroduplex mobility assay (HMA) and by DNA sequence analysis. We found that a more diverse population of SIV genetic variants was present in the earliest virus-positive plasma samples from all five IV SIVmac251-inoculated monkeys and from two of five IVAG SIVmac251-inoculated monkeys. In contrast, we found a relatively homogeneous population of SIV envelope variants in three of five monkeys inoculated IVAG with SIVmac251 stock and in two monkeys infected after IVAG inoculation with plasma from an SIV-infected animal. In some IVAG-inoculated animals, the transmitted SIV variant was the most common variant in the inoculum. However, a specific viral variant in the SIVmac251 stock was not consistently transmitted by IVAG inoculation. Thus, it is likely that host factors or stochastic processes determine the specific viral variants that infect an animal after IVAG SIV exposure. In addition, our results clearly demonstrate that the route of inoculation is associated with the extent and breadth of the genetic complexity of the viral variant population in the earliest stages of systemic infection.

Immune response of rhesus macaques to recombinant simian immunodeficiency virus gp130 does not protect from challenge infection

Simian immunodeficiency virus (SIV) infection of rhesus macaques is a model for human immunodeficiency virus (HIV) infection in humans. Inactivated and modified live whole-virus vaccines have provided limited protective immunity against SIV in rhesus macaques. Because of safety concerns in the use of inactivated and live whole-virus vaccines, we evaluated the protective immunity of vaccinia virus recombinants expressing the surface glycoprotein (gp130) of SIVmac and subunit preparations of gp130 expressed in mammalian cells (CHO). Three groups of animals were immunized with recombinant SIV gp130. The first group received SIV gp130 purified from genetically engineered CHO cells (cSIVgp130), the second group was vaccinated with recombinant vaccinia virus expressing SIVmac gp130 (vSIVgp130), and the third group was first primed with vSIVgp130 and then given a booster immunization with cSIVgp130. Although anti-gp130 binding antibodies were elicited in all three groups, neutralizing antibodies were transient or undetectable. None of the immunized animals resisted intravenous challenge with a low dose of cell-free virus. However, the group primed with vSIVgp130 and then boosted with cSIVgp130 had the lowest antigen load (p27) compared with the other groups. The results of these studies suggest that immunization of humans with HIV type 1 surface glycoprotein may not provide protective immunity against virus infection.

Role of TAR RNA splicing in translational regulation of simian immunodeficiency virus from rhesus macaques.

The untranslated leader sequences of rhesus macaque simian immunodeficiency virus mRNAs form a stable secondary structure, TAR. This structure can be modified by RNA splicing. In this study, the role of TAR splicing in virus replication was investigated. The proportion of viral RNAs containing a spliced TAR structure is high early after infection and decreases at later times. Moreover, proviruses containing mutations which prevent TAR splicing are significantly delayed in replication. These mutant viruses require approximately 20 days to achieve half-maximal virus production, in contrast to wild-type viruses, which require approximately 8 days. We attribute this delay to the inefficient translation of unspliced-TAR-containing mRNAs. The molecular basis for this translational effect was examined in in vitro assays. We found that spliced-TAR-containing mRNAs were translated up to 8.5 times more efficiently than were similar mRNAs containing an unspliced TAR leader. Furthermore, these spliced-TAR-containing mRNAs were more efficiently associated with ribosomes. We postulate that the level of TAR splicing provides a balance for the optimal expression of both viral proteins and genomic RNA and therefore ultimately controls the production of infectious virions.

Identification of viral determinants of macrophage tropism for simian immunodeficiency virus SIVmac

Simian immunodeficiency virus (SIV), a lymphocytopathic lentivirus, induces an AIDS-like disease in rhesus macaques (Macaca mulatta). A pathogenic molecular clone of rhesus macaque SIV (SIVmac), SIVmac-239, replicates and induces cytopathology in T lymphocytes but is restricted for replication in macrophages. In contrast, a nonpathogenic molecular clone of SIVmac, SIVmac-1A11, replicates and induces syncytia (multinucleated giant cells) in cultures of both T lymphocytes and macrophages. SIVmac-1A11 does not cause disease in macaques. To map the viral determinants of macrophage tropism, reciprocal recombinant genomes were constructed between molecular clones of SIVmac-239 and SIVmac-1A11. Infectious recombinant viruses were rescued by transfection of cloned viral genomes into permissive lymphoid cells. Analysis of one pair of reciprocal recombinants revealed that an internal 6.2-kb DNA fragment of SIVmac-1A11 was necessary and sufficient for both syncytium formation and efficient replication in macrophages. This region includes the coding sequences for a portion of the gag gene, all of the pol, vif, vpr, and vpx genes, the first coding exons of tat and rev, and the external env glycoprotein gp130. Thus, the transmembrane glycoprotein of env, the nef gene, the second coding exons of tat and rev, and the long terminal repeats are not essential for in vitro macrophage tropism. Analysis of additional recombinants revealed that syncytium formation, but not virus production, was controlled by a 1.4-kb viral DNA fragment in SIVmac-1A11 encoding only the external env glycoprotein gp130. Thus, gp130 env of SIVmac-1A11 is necessary for entry of virus into macrophages but is not sufficient for a complete viral replication cycle in this cell type. We therefore conclude that gp130 env and one or more genetic elements (exclusive of the long terminal repeats, transmembrane glycoprotein of env, and second coding exons of tat and rev, and nef) are essential for a complete replication cycle of SIVmac in rhesus macaque macrophages.