Molecular interactions of cytomegalovirus and the human and simian immunodeficiency viruses

In HuT 78 cells chronically infected with SIV, super-infection with rhesus cytomegalovirus (rhCMV) stimulated an increase in SIV replication. Utilizing transient expression assays with the SIV long terminal repeat (LTR) driving expression of the chloramphenicol acetyltransferase (CAT) reporter gene, the increase in SIV replication, by coinfection with CMV, was due to transactivation of the SIV LTR by the immediate early gene products (IE) of rhesus CMV. Similarly, IE of human CMV stimulated expression from both the SIV and HIV LTRs.     

Vif counteracts a cyclophilin A-imposed inhibition of simian immunodeficiency viruses in human cells

Vif is a primate lentiviral accessory protein that is crucial for viral infectivity. Vif counteracts the antiviral activity of host deaminases such as APOBEC3G and APOBEC3F. We now report a novel function of African green monkey simian immunodeficiency virus (SIVagm) Vif that promotes replication of SIVagm in human cells lacking detectable deaminase activity. We found that cyclophilin A (CypA) was excluded from wild-type SIV particles but was efficiently packaged into vif-deficient SIVagm virions. The presence of CypA in vif-defective SIVagm was correlated with reduced viral replication. Infection of CypA knockout Jurkat cells or treatment of Jurkat cells with cyclosporine A eliminated the Vif-sensitive inhibition and resulted in replication profiles that were similar for wild-type and vif-deficient SIVagm. Importantly, the inhibitory effect of CypA was restricted to virus-producing cells and was TRIM5alpha independent. The abilities of SIVagm Vif to inhibit encapsidation of CypA and to increase viral infectivity were shared by rhesus macaque SIV Vif and thus seem to be general properties of SIV Vif proteins. Exclusion of CypA from SIVagm particles was not associated with intracellular degradation, suggesting a mode of Vif action distinct from that proposed for APOBEC3G. This is the first report of a novel vif-sensitive antiviral activity of human CypA that may limit zoonotic transmission of SIV and the first demonstration of CypA encapsidation into a virus other than human immunodeficiency virus type 1.     

A novel approach for producing lentiviruses that are limited to a single cycle of infection

We have devised a novel approach for producing simian immunodeficiency virus (SIV) strains and, potentially, human immunodeficiency virus type 1 (HIV-1) strains that are limited to a single cycle of infection. Unlike previous lentiviral vectors, our single-cycle SIV is capable of expressing eight of the nine viral gene products and infected cells release immature virus particles that are unable to complete subsequent rounds of infection. Single-cycle SIV (scSIV) was produced by using a two-plasmid system specifically designed to minimize the possibility of generating replication-competent virus by recombination or nucleotide reversion. One plasmid carried a full-length SIV genome with three nucleotide substitutions in the gag-pol frameshift site to inactivate Pol expression. To ensure inactivation of Pol and to prevent the recovery of wild-type virus by nucleotide reversion, deletions were also introduced into the viral pol gene. In order to provide Gag-Pol in trans, a Gag-Pol-complementing plasmid that included a single nucleotide insertion to permanently place gag and pol in the same reading frame was constructed. We also mutated the frameshift site of this Gag-Pol expression construct so that any recombinants between the two plasmids would remain defective for replication. Cotransfection of both plasmids into 293T cells resulted in the release of Gag-Pol-complemented virus that was capable of one round of infection and one round of viral gene expression but was unable to propagate a spreading infection. The infectivity of scSIV was limited by the amount of Gag-Pol provided in trans and was dependent on the incorporation of a functional integrase. Single-cycle SIV produced by this approach will be useful for addressing questions relating to viral dynamics and viral pathogenesis and for evaluation as an experimental AIDS vaccine in rhesus macaques.     

Simian immunodeficiency virus negative factor suppresses the level of viral mRNA in COS cells.

The nef gene is conserved among all human and simian lentiviruses. However, the amino acid similarity between simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 NEF is only 38%. To assess the role of SIV NEF on virus replication and compare its activity with that of its human immunodeficiency virus type 1 counterpart, we examined the activity of an intact nef gene from proviral clone pSIV 102, an isolate from SIV-MAC-251-infected cells. Proviral clone pSIV BA was constructed by introducing a premature termination codon at codon 40 of the nef gene without altering the predicted amino acid sequence of the overlapping env gene. These two clones were transfected into CD4- COS cells, and virus replication was monitored by p27 enzyme-linked immunosorbent assay kits. In seven independent experiments, clone pSIV BA afforded two- to sixfold greater levels of viral antigen compared with those in clone pSIV 102 and two- to sixfold-increased levels of viral mRNAs as indicated with Northern (RNA) blot and S1 nuclease protection analyses. Nuclear run-on assays demonstrated a two- to threefold increased rate of RNA synthesis with nuclei isolated from cells transfected with pSIV BA compared with that from cells transfected with pSIV 102. In contrast, there was no apparent destabilization of SIV mRNAs by NEF, as measured in dactinomycin-treated cells. This study demonstrates that SIV NEF is a negative regulator of virus replication and acts by suppressing the level of mRNA synthesis and accumulation in COS cells.     

The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable.

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.     

A Stretch of Unpaired Purines in the Leader Region of Simian Immunodeficiency Virus (SIV) Genomic RNA is Critical for its Packaging into Virions

Simian immunodeficiency virus (SIV) is an important lentivirus used as a non-human primate model to study HIV replication, and pathogenesis of human AIDS, as well as a potential vector for human gene therapy. This study investigated the role of single-stranded purines (ssPurines) as potential genomic RNA (gRNA) packaging determinants in SIV replication. Similar ssPurines have been implicated as important motifs for gRNA packaging in many retroviruses like, HIV-1, MPMV, and MMTV by serving as Gag binding sites during virion assembly. In examining the secondary structure of the SIV 5′ leader region, as recently deduced using SHAPE methodology, we identified four specific stretches of ssPurines (I-IV) in the region that harbors major packaging determinants of SIV. The significance of these ssPurine motifs were investigated by mutational analysis coupled with a biologically relevant single round of replication assay. These analyses revealed that while ssPurine II was essential, the others (ssPurines I, III, & IV) did not significantly contribute to SIV gRNA packaging. Any mutation in the ssPurine II, such as its deletion or substitution, or other mutations that caused base pairing of ssPurine II loop resulted in near abrogation of RNA packaging, further substantiating the crucial role of ssPurine II and its looped conformation in SIV gRNA packaging. Structure prediction analysis of these mutants further corroborated the biological results and further revealed that the unpaired nature of ssPurine II is critical for its function during SIV RNA packaging perhaps by enabling it to function as a specific binding site for SIV Gag.     

Genital tract sequestration of SIV following acute infection

We characterized the evolution of simian immunodeficiency virus (SIV) in the male genital tract by examining blood- and semen-associated virus from experimentally and sham vaccinated rhesus monkeys during primary infection. At the time of peak virus replication, SIV sequences were intermixed between the blood and semen supporting a scenario of high-level virus "spillover" into the male genital tract. However, at the time of virus set point, compartmentalization was apparent in 4 of 7 evaluated monkeys, likely as a consequence of restricted virus gene flow between anatomic compartments after the resolution of primary viremia. These findings suggest that SIV replication in the male genital tract evolves to compartmentalization after peak viremia resolves.     

Enhanced replication of simian immunodeficiency virus adjacent to catecholaminergic varicosities in primate lymph nodes

Clinical and in vitro studies have shown that activity of the autonomic nervous system (ANS) can stimulate lentivirus replication. To define the potential anatomical basis for this effect, we analyzed the spatial relationship between catecholaminergic neural fibers and sites of simian immunodeficiency virus (SIV) replication in lymph nodes from rhesus macaques experimentally infected with SIVmac251. Viral replication was mapped by in situ hybridization for SIV env, gag, and nef RNA, and catecholaminergic varicosities from the ANS were mapped by sucrose phosphate glyoxylic acid chemofluorescence. Spatial statistical analyses showed that the likelihood of active SIV replication increased by 3.9-fold in the vicinity of catecholaminergic varicosities (P < 0.0001). The densities of both ANS innervation and SIV replication differed across cortical, paracortical, and medullary regions of the lymph node, but analyses of each region separately continued to show increased replication of SIV adjacent to catecholaminergic varicosities. Ancillary analyses ruled out the possibility that SIV-induced alterations in lymph node architecture might create a spurious spatial association. These data support human clinical studies and in vitro molecular analyses showing that catecholamine neurotransmitters from the ANS can increase lentiviral replication by identifying a specific anatomic context for interactions between ANS neural fibers and replication of SIV in lymphoid tissue.     

The simian immunodeficiency virus (SIV) rev gene regulates env expression

The rev gene product is a trans-acting nuclear regulatory protein that is essential for AIDS virus replication. To define the effect of the SIV rev gene on its gp160 expression, two SV40 constructs have been made: pBC17 is a rev+env+ construct, and pBD21 is a rev-env+ construct. After transfecting the constructs into COS-1 cells, RNA, protein, syncytium formation, and monoclonal antibody blocking assays were performed. The results indicated that the SIV rev gene positively influenced the level of full-length env mRNA and was required for expression of SIV gp160.     

Temporal and anatomic relationship between virus replication and cytokine gene expression after vaginal simian immunodeficiency virus infection

The current knowledge about early innate immune responses at mucosal sites of human immunodeficiency virus (HIV) entry is limited but likely to be important in the design of effective HIV vaccines against heterosexual transmission. This study examined the temporal and anatomic relationship between virus replication, lymphocyte depletion, and cytokine gene expression levels in mucosal and lymphoid tissues in a vaginal-transmission model of HIV in rhesus macaques. The results of the study show that the kinetics of cytokine gene expression levels in the acute phase of infection are positively correlated with virus replication in a tissue. Thus, cytokine responses after vaginal simian immunodeficiency virus (SIV) inoculation are earliest and strongest in mucosal tissues of the genital tract and lowest in systemic lymphoid tissues. Importantly, the early cytokine response was dominated by the induction of proinflammatory cytokines, while the induction of cytokines with antiviral activity, alpha/beta interferon, occurred too late to prevent virus replication and dissemination. Thus, the early cytokine response favors immune activation, resulting in the recruitment of potential target cells for SIV. Further, unique cytokine gene expression patterns were observed in distinct anatomic locations with a rapid and persistent inflammatory response in the gut that is consistent with the gut being the major site of early CD4 T-cell depletion in SIV infection.     

TRIM5α does not affect simian immunodeficiency virus SIV(mac251) replication in vaccinated or unvaccinated Indian rhesus macaques following intrarectal challenge exposure

TRIM5α is a natural resistance factor that binds retroviral capsid proteins and restricts virus replication. The B30.2/SPRY domain of TRIM5α is polymorphic in rhesus macaques, and some alleles are associated with reduced simian immunodeficiency virus (SIV) SIV(mac251) and SIV(smE543) replication in vivo. We determined the distribution of TRIM5α alleles by PCR and sequence analysis of the B30.2/SPRY domain in a cohort of 82 macaques. Thirty-nine of these macaques were mock vaccinated, 43 were vaccinated with either DNA-SIV/ALVAC-SIV/gp120, ALVAC-SIV/gp120, or gp120 alone, and all were exposed intrarectally to SIV(mac251) at one of three doses. We assessed whether the TRIM5α genotype of the macaques affected the replication of challenge virus by studying the number of SIV variants transmitted, the number of exposures required, the SIV(mac251) viral level in plasma and tissue, and the CD4(+) T-cell counts. Our results demonstrated that TRIM5α alleles, previously identified as restrictive for SIV(mac251) replication in vivo following intravenous exposure, did not affect SIV(mac251) replication following mucosal exposure, regardless of prior vaccination, challenge dose, or the presence of the protective major histocompatibility complex alleles (MamuA01(+), MamuB08(+), or MamuB017(+)). The TRIM5α genotype had no apparent effect on the number of transmitted variants or the number of challenge exposures necessary to infect the animals. DNA sequencing of the SIV(mac251) Gag gene of the two stocks used in our study revealed SIV(mac239)-like sequences that are predicted to be resistant to TRIM5α restriction. Thus, the TRIM5α genotype does not confound results of mucosal infection of rhesus macaques with SIV(mac251).     

CD4-independent entry and replication of simian immunodeficiency virus in primary rhesus macaque astrocytes are regulated by the transmembrane protein

Previous studies have demonstrated that the genetic determinants of simian immunodeficiency virus (SIV) neurovirulence map to the env and nef genes. Recent studies from our laboratory demonstrated that SIV replication in primary rhesus macaque astrocyte cultures is dependent upon the nef gene. Here, we demonstrate that macrophage tropism is not sufficient for replication in astrocytes and that specific amino acids in the transmembrane (TM) portion of Env are also important for optimal SIV replication in astrocytes. Specifically, a Gly at amino acid position 751 and truncation of the cytoplasmic tail of TM are required for efficient replication in these cells. Studies using soluble CD4 demonstrated that these changes within the TM protein regulate CD4-independent, CCR5-dependent entry of virus into astrocytes. In addition, we observed that two distinct CD4-independent, neuroinvasive strains of SIV/DeltaB670 also replicated efficiently in astrocytes, further supporting the role of CD4 independence as an important determinant of SIV infection of astrocytes in vitro and in vivo.     

Transduction of CD34+ hematopoietic progenitor cells with an antitat gene protects T-cell and macrophage progeny from AIDS virus infection.

Transduction of hematopoietic stem cells with genes that inhibit human immunodeficiency virus (HIV) replication has the potential to reconstitute immune function in individuals with AIDS. We evaluated the ability of an autoregulated gene, antitat, to inhibit replication of simian immunodeficiency virus (SIV) and HIV type 1 (HIV-1) in hematopoietic cells derived from transduced progenitor cells. The antitat gene expresses an antiviral RNA encoding polymeric Tat activation response elements in combination with an antisense tat moiety under the control of the HIV-1 long terminal repeat. CD34+ hematopoietic progenitor cells were transduced with a retroviral vector containing the antitat gene and then cultured under conditions that support in vitro differentiation of T cells or macrophage-like cells. Rhesus macaque CD4+ T cells and macrophage-like cells derived from CD34+ bone marrow cells transduced with the antitat gene were highly resistant to challenge with SIV, reflecting a 2- to 3-log reduction in peak SIV replication compared with controls. Similarly, human CD4+ T cells derived from CD34+ cord blood cells transduced with antitat were also resistant to infection with HIV-1. No evidence for toxicity of the antitat gene was observed in any of five different lineages derived from transduced hematopoietic cells. These results demonstrate that a candidate therapeutic gene introduced into hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication following T-cell differentiation and support the potential use of the antitat gene for stem cell gene therapy.     

Molecular and biological characterization of a neurovirulent molecular clone of simian immunodeficiency virus.

To identify the molecular determinants of neurovirulence, we constructed an infectious simian immunodeficiency virus (SIV) molecular clone, SIV/17E-Fr, that contained the 3' end of a neurovirulent strain of SIV, SIV/17E-Br, derived by in vivo virus passage. SIV/17E-Fr is macrophage tropic in vitro and neurovirulent in macaques. In contrast, a molecular clone, SIV/17E-Cl, that contains the SU and a portion of the TM sequences of SIV/17E-Br is macrophage tropic but not neurovirulent. To identify the amino acids that accounted for the replication differences between SIV/17E-Fr and SIV/17E-Cl in primary macaque cells in vitro, additional infectious molecular clones were constructed. Analysis of these recombinant viruses revealed that changes in the TM portion of the envelope protein were required for the highest level of replication in primary macaque macrophages and brain cells derived from the microvessel endothelium. In addition, a full-length Nef protein is necessary for optimum virus replication in both of these cell types. Finally, viruses expressing a full-length Nef protein in conjunction with the changes in the TM had the highest specific infectivity in a sMAGI assay. Thus, changes in the TM and nef genes between SIV/17E-Cl and SIV/17E-Fr account for replication differences in vitro and correlate with replication in the central nervous system in vivo.     

Inhibition of Simian Immunodeficiency Virus (SIV) Replication by CD8+ T Lymphocytes from Macaques Immunized with Live Attenuated SIV

Characterization of immune responses induced by live attenuated simian immunodeficiency virus (SIV) strains may yield clues to the nature of protective immunity induced by this vaccine approach. We investigated the ability of CD8⁺ T lymphocytes from rhesus macaques immunized with the live, attenuated SIV strain SIVmac239Δnef or SIVmac239Δ3 to inhibit SIV replication. CD8⁺ T lymphocytes from immunized animals were able to potently suppress SIV replication in autologous SIV-infected CD4⁺ T cells. Suppression of SIV replication by unstimulated CD8⁺ T cells required direct contact and was major histocompatibility complex (MHC) restricted. However, CD3-stimulated CD8⁺ T cells produced soluble factors that inhibited SIV replication in an MHC-unrestricted fashion as much as 30-fold. Supernatants from stimulated CD8⁺ T cells were also able to inhibit replication of both CCR5- and CXCR4-dependent human immunodeficiency virus type 1 (HIV-1) strains. Stimulation of CD8⁺ cells with cognate cytotoxic T-lymphocyte epitopes also induced secretion of soluble factors able to inhibit SIV replication. Production of RANTES, macrophage inhibitory protein 1α (MIP-1α), or MIP-1β from stimulated CD8⁺ T cells of vaccinated animals was almost 10-fold higher than that from stimulated CD8⁺ T cells of control animals. However, addition of antibodies that neutralize these β-chemokines, either alone or in combination, only partly blocked inhibition of SIV and HIV replication by soluble factors produced by stimulated CD8⁺ T cells. Our results indicate that inhibition of SIV replication by CD8⁺ T cells from animals immunized with live attenuated SIV strains involves both MHC-restricted and -unrestricted mechanisms and that MHC-unrestricted inhibition of SIV replication is due principally to soluble factors other than RANTES, MIP-1α, and MIP-1β.     

Primate and feline lentivirus vector RNA packaging and propagation by heterologous lentivirus virions

Development of safe and effective gene transfer systems is critical to the success of gene therapy protocols for human diseases. Currently, several primate lentivirus-based gene transfer systems, such as those based on human and simian immunodeficiency viruses (HIV/SIV), are being tested; however, their use in humans raises safety concerns, such as the generation of replication-competent viruses through recombination with related endogenous retroviruses or retrovirus-like elements. Due to the greater phylogenetic distance from primate lentiviruses, feline immunodeficiency virus (FIV) is becoming the lentivirus of choice for human gene transfer systems. However, the safety of FIV-based vector systems has not been tested experimentally. Since lentiviruses such as HIV-1 and SIV have been shown to cross-package their RNA genomes, we tested the ability of FIV RNA to get cross-packaged into primate lentivirus particles such as HIV-1 and SIV, as well as a nonlentiviral retrovirus such as Mason-Pfizer monkey virus (MPMV), and vice versa. Our results reveal that FIV RNA can be cross-packaged by primate lentivirus particles such as HIV-1 and SIV and vice versa; however, a nonlentivirus particle such as MPMV is unable to package FIV RNA. Interestingly, FIV particles can package MPMV RNA but cannot propagate the vector RNA further for other steps of the retrovirus life cycle. These findings reveal that diverse retroviruses are functionally more similar than originally thought and suggest that upon coinfection of the same host, cross- or copackaging may allow distinct retroviruses to generate chimeric variants with unknown pathogenic potential.     

Viral escape from dominant simian immunodeficiency virus epitope-specific cytotoxic T lymphocytes in DNA-vaccinated rhesus monkeys

Virus-specific cytotoxic T lymphocytes (CTL) are critical for control of human immunodeficiency virus type 1 replication. However, viral escape from CTL recognition can undermine this immune control. Here we demonstrate the high frequency and pattern of viral escape from dominant epitope-specific CTL in SIV gag DNA-vaccinated rhesus monkeys following a heterologous simian immunodeficiency virus (SIV) challenge. DNA-vaccinated monkeys exhibited initial effective control of the SIV challenge, but this early control was lost by serial breakthroughs of viral replication over a 3-year follow-up period. Increases in plasma viral RNA correlated temporally with declines of dominant SIV epitope-specific CD8(+) T-lymphocyte responses and the emergence of viral mutations that escaped recognition by dominant epitope-specific CTL. Viral escape from CTL occurred in a total of seven of nine vaccinated and control monkeys, including three animals that initially controlled viral replication to undetectable levels of plasma viral RNA. These data suggest that CTL exert selective pressure on viral replication and that viral escape from CTL may be a limitation of CTL-based AIDS vaccine strategies.