Functional comparison of transactivation by simian immunodeficiency virus from rhesus macaques and human immunodeficiency virus type 1.

Simian immunodeficiency virus from rhesus macaques (SIVmac), like human immunodeficiency virus type 1 (HIV-1), encodes a transactivator (tat) which stimulates long terminal repeat (LTR)-directed gene expression. We performed cotransfection assays of SIVmac and HIV-1 tat constructs with LTR-CAT reporter plasmids. The primary effect of transactivation for both SIVmac and HIV-1 is an increase in LTR-directed mRNA accumulation. The SIVmac tat gene product partially transactivates an HIV-1 LTR, whereas the HIV-1 tat gene product fully transactivates an SIVmac LTR. Significant transactivation is achieved by the product of coding exon 1 of the HIV-1 tat gene; however, inclusion of coding exon 2 results in a further increase in mRNA accumulation. In contrast, coding exon 2 of the SIVmac tat gene is required for significant transactivation. These results imply that the tat proteins of SIVmac and HIV-1 are functionally similar but not interchangeable. In addition, an in vitro-generated mutation in SIVmac tat disrupts splicing at the normal splice acceptor site at the beginning of coding exon 2 and activates a site approximately 15 nucleotides downstream. The product of this splice variant stimulates LTR-directed gene expression. This alternative splice acceptor site is also used by a biologically active provirus with an efficiency of approximately 5% compared with the upstream site. These data suggest that a novel tat protein is encoded during the course of viral infection.     

Role for human immunodeficiency virus type 1 membrane cholesterol in viral internalization

The membrane of human immunodeficiency virus type 1 (HIV-1) virions contains high levels of cholesterol and sphingomyelin, an enrichment that is explained by the preferential budding of the virus through raft microdomains of the plasma membrane. Upon depletion of cholesterol from HIV-1 virions with methyl-beta-cyclodextrin, infectivity was almost completely abolished. In contrast, this treatment had only a mild effect on the infectiousness of particles pseudotyped with the G envelope of vesicular stomatitis virus. The cholesterol-chelating compound nystatin had a similar effect. Cholesterol-depleted HIV-1 virions exhibited wild-type patterns of viral proteins and contained normal levels of cyclophilin A and glycosylphosphatidylinositol-anchored proteins. Nevertheless, and although they could still bind target cells, these virions were markedly defective for internalization. These results indicate that the cholesterol present in the HIV-1 membrane plays a prominent role in the fusion process that is key to viral entry and suggest that drugs capable of disturbing the lipid composition of virions could serve as a basis for the development of microbicides.     

Vif substitution enables persistent infection of pig-tailed macaques by human immunodeficiency virus type 1

Among Old World monkeys, pig-tailed macaques (Pt) are uniquely susceptible to human immunodeficiency virus type 1 (HIV-1), although the infection does not persist. We demonstrate that the susceptibility of Pt T cells to HIV-1 infection is due to the absence of postentry inhibition by a TRIM5 isoform. Notably, substitution of the viral infectivity factor protein, Vif, with that from pathogenic SIVmne enabled replication of HIV-1 in Pt T cells in vitro. When inoculated into juvenile pig-tailed macaques, the Pt-tropic HIV-1 persistently replicated for more than 1.5 to 2 years, producing low but measurable plasma viral loads and persistent proviral DNA in peripheral blood mononuclear cells. It also elicited strong antibody responses. However, there was no decline in CD4(+) T cells or evidence of disease. Surprisingly, the Pt-tropic HIV-1 was rapidly controlled when inoculated into newborn Pt macaques, although it transiently rebounded after 6 months. We identified two notable differences between the Pt-tropic HIV-1 and SIVmne. First, SIV Vif does not associate with Pt-tropic HIV-1 viral particles. Second, while Pt-tropic HIV-1 degrades both Pt APOBEC3G and APOBEC3F, it prevents their inclusion in virions to a lesser extent than pathogenic SIVmne. Thus, while SIV Vif is necessary for persistent infection by Pt-tropic HIV-1, improved expression and inhibition of APOBEC3 proteins may be required for robust viral replication in vivo. Additional adaptation of the virus may also be necessary to enhance viral replication. Nevertheless, our data suggest the potential for the pig-tailed macaque to be developed as an animal model of HIV-1 infection and disease.     

Cellular and viral specificities of human immunodeficiency virus type 1 vif protein

The vif gene of human immunodeficiency virus type 1 (HIV-1) greatly enhances the infectivity of HIV-1 virions that are released from cells classified as nonpermissive (e.g., lymphocytes, macrophages, and H9 leukemic T cells) but is irrelevant in permissive cells (e.g., HeLa or COS cells). Recently, it was reported that vif expression in nonpermissive cells dramatically increases infectivity not only of HIV-1 but also of other enveloped viruses, including murine leukemia viruses (MLVs). This was surprising in part because MLVs and other murine retroviruses lack vif genes yet replicate efficiently in T lymphocytes. To investigate these issues, we first developed improved methods for producing substantial quantities of HIV-1 virions with vif deletions from healthy H9 cells. These virions had approximately the same amounts of major core proteins and envelope glycoproteins as the control wild-type virions but were only approximately 1% as infectious. We then produced H9 cells that contained wild-type or vif deletion HIV-gpt proviruses, which lack a functional env gene. After superinfection with either xenotropic or amphotropic MLVs, these cells released HIV-gpt virions pseudotyped with an MLV envelope plus replication-competent MLV. Interestingly, the pseudotyped HIV-gpt (vif deletion) virions were noninfectious, whereas the MLV virions simultaneously released from the same H9 cells were fully infectious. These results strongly suggest that the Vif protein functions in a manner that is both cell specific and at least substantially specific for HIV-1 and related lentiviruses. In addition, these results confirm that vif deletion HIV-1 virions from nonpermissive cells are blocked at a postpenetration stage of the infection pathway.     

Immunogenicity of multiple gene and clade human immunodeficiency virus type 1 DNA vaccines

The ability to elicit an immune response to a spectrum of human immunodeficiency virus type 1 (HIV-1) gene products from divergent strains is a desirable feature of an AIDS vaccine. In this study, we examined combinations of plasmids expressing multiple HIV-1 genes from different clades for their ability to elicit humoral and cellular immune responses in mice. Immunization with a modified Env, gp145DeltaCFI, in combination with a Gag-Pol-Nef fusion protein plasmid elicited similar CD4(+) and CD8(+) cellular responses to immunization with either vector alone. Further, when mice were immunized with a mixture of Env from three clades, A, B, and C, together with Gag-Pol-Nef, the overall potency and balance of CD4(+)- and CD8(+)-T-cell responses to all viral antigens were similar, with only minor differences noted. In addition, plasmid mixtures elicited antibody responses comparable to those from individual inoculations. These findings suggest that a multigene and multiclade vaccine, including components from A, B, and C Env and Gag-Pol-Nef, can broaden antiviral immune responses without immune interference. Such combinations of immunogens may help to address concerns about viral genetic diversity for a prospective HIV-1 vaccine.     

Regulation of human immunodeficiency virus type 1 Env-mediated membrane fusion by viral protease activity

We and others have presented evidence for a direct interaction between the matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein and the cytoplasmic tail of the transmembrane envelope (Env) glycoprotein gp41. In addition, it has been postulated that the MA domain of Gag undergoes a conformational change following Gag processing, and the cytoplasmic tail of gp41 has been shown to modulate Env-mediated membrane fusion activity. Together, these results raise the possibility that the interaction between the gp41 cytoplasmic tail and MA is regulated by protease (PR)-mediated Gag processing, perhaps affecting Env function. To examine whether Gag processing affects Env-mediated fusion, we compared the ability of wild-type (WT) HIV-1 Env and a mutant lacking the gp41 cytoplasmic tail to induce fusion in the context of an active (PR(+)) or inactive (PR(-)) viral PR. We observed that PR(-) virions bearing WT Env displayed defects in cell-cell fusion. Impaired fusion did not appear to be due to differences in the levels of virion-associated Env, in CD4-dependent binding to target cells, or in the formation of the CD4-induced gp41 six-helix bundle. Interestingly, truncation of the gp41 cytoplasmic tail reversed the fusion defect. These results suggest that interactions between unprocessed Gag and the gp41 cytoplasmic tail suppress fusion.     

Immune escape by human immunodeficiency virus type 1 from neutralizing antibodies: evidence for multiple pathways.

Sera from many HIV-1-infected individuals contain broadly reactive, specific neutralizing antibodies. Despite their broad reactivity, variant viruses, resistant to neutralization, can be selected in vitro in the presence of such antisera. We have previously shown that neutralization resistance of an escape mutant with an amino acid substitution in the transmembrane protein (A582T) occurs because of alteration of a conformational epitope that is recognized by neutralizing antibodies directed against the CD4 binding site. In this report we demonstrate that immune escape via a single-amino-acid substitution (A281V) within a conserved region of the envelope glycoprotein gp120 confers neutralization resistance against a broadly reactive neutralizing antiserum from a seropositive individual. We show this alteration affects V3 and additional regions unrelated to V3 or the CD4 binding site. Together with previous studies on escape mutants selected in vitro, our findings suggest that immune-selective pressure can arise by multiple pathways.     

Comparison of human immunodeficiency virus type 1 viral loads in Kenyan women,men, and infants during primary and early infection

Steady-state levels of human immunodeficiency virus type 1 (HIV-1) RNA in plasma reached at approximately 4 months postinfection are highly predictive of disease progression. Several studies have investigated viral levels in adults or infants during primary and early infection. However, no studies have directly compared these groups. We compared differences in peak and set point plasma HIV-1 RNA viral loads among antiretrovirus-naive Kenyan infants and adults for whom the timing of infection was well defined. Peak and set point viral loads were significantly higher in infants than in adults. We did not observe any gender-specific differences in viral set point in either adults or infants. However, infants who acquired HIV-1 in the first 2 months of life, either in utero, intrapartum, or through early breast milk transmission, had significantly higher set point HIV-1 RNA levels than infants who were infected after 2 months of age through late breast milk transmission or adults who were infected through heterosexual transmission.     

Molecular evolution analysis of the human immunodeficiency virus type 1 envelope in simian/human immunodeficiency virus-infected macaques: implications for challenge dose selection

Since the demonstration that almost 80% of human immunodeficiency virus type 1 (HIV-1) infections result from the transmission of a single variant from the donor, biological features similar to those of HIV mucosal transmission have been reported for macaques inoculated with simian immunodeficiency virus (SIV). Here we describe the early diversification events and the impact of challenge doses on viral kinetics and on the number of variants transmitted in macaques infected with the chimeric simian/human immunodeficiency virus SHIV(sf162p4). We show that there is a correlation between the dose administered and the number of variants transmitted and that certain inoculum variants are preferentially transmitted. This could provide insight into the viral determinants of transmission and could aid in vaccine development. Challenge through the mucosal route with high doses results in the transmission of multiple variants in all the animals. Such an unrealistic scenario could underestimate potential intervention measures. We thus propose the use of molecular evolution analysis to aid in the determination of challenge doses that better mimic the transmission dynamics seen in natural HIV-1 infection.     

Role of the human immunodeficiency virus type 1 envelope gene in viral fitness

A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r = 0.93; P = 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.     

In vivo evidence for instability of episomal human immunodeficiency virus type 1 cDNA

Current regimens for the management of human immunodeficiency virus type 1 (HIV-1) infection suppress plasma viremia to below detectable levels for prolonged intervals. Nevertheless, there is a rapid resumption in plasma viremia if therapy is interrupted. Attempts to characterize the extent of viral replication under conditions of potent suppression and undetectable plasma viremia have been hampered by a lack of convenient assays that can distinguish latent from ongoing viral replication. Using episomal viral cDNA as a surrogate for ongoing replication, we previously presented evidence that viral replication persists in the majority of infected individuals with a sustained aviremic status. The labile nature of viral episomes and hence their validity as surrogate markers of ongoing replication in individuals with long-term-suppressed HIV-1 infection have been analyzed in short-term in vitro experiments with conflicting results. Since these in vitro experiments do not shed light on the long-term in vivo dynamics of episomal cDNA or recapitulate the natural targets of infection in vivo, we have analyzed the dynamics of episomal cDNA turnover in vivo by following the emergence of an M184V polymorphism in plasma viral RNA, in episomal cDNA, and in proviral DNA in patients on suboptimal therapies. We demonstrate that during acquisition of drug resistance, wild-type episomal cDNAs are replaced by M184V-harboring episomes. Importantly, a complete replacement of wild-type episomes with M184V-containing episomes occurred while proviruses remained wild type. This indicates that episomal cDNAs are turned over by degradation rather than through death or tissue redistribution of the infected cell itself. Therefore, evolution of episomal viral cDNAs is a valid surrogate of ongoing viral replication in HIV-1-infected individuals.     

Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: evidence for viral compartmentalization and selection during sexual transmission.

To explore the mechanism of sexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared HIV-1 gp120 sequences in longitudinal samples from five acute seroconvertors with those from their corresponding sexual partners (transmitters). We used a quantitative homoduplex tracking assay to compare the overall genetic composition of HIV-1 quasispecies in each transmission pair and to track the transmitted viruses during the acute and asymptomatic stages of HIV-1 infection. In the chronically infected transmitters, HIV-1 variants in genital secretions differed from those in blood and variants in cells differed from those in cell-free plasma, indicating remarkable sequence heterogeneity in these subjects as well as compartmentalization of the virus in different bodily sites. Conversely, two of five seroconvertors had only a few related variants and three of five harbored only one viral population, indicating that in these subjects the transmitted viruses were typically homogeneous. Transmitted viruses were evident in the donor's seminal plasma (one of five cases) and even more so in their seminal cells (three of five cases), suggesting that both cell-associated and cell-free viruses can be transmitted. In every pair studied, the transmitted variant(s) represents only a minor population in the semen of the corresponding transmitter, thereby providing evidence that HIV-1 selection indeed occurs during sexual transmission.     

Modulation by morphine of viral set point in rhesus macaques infected with simian immunodeficiency virus and simian-human immunodeficiency virus

Six rhesus macaques were adapted to morphine dependence by injecting three doses of morphine (5 mg/kg of body weight) for a total of 20 weeks. These animals along with six control macaques were infected intravenously with mixture of simian-human immunodeficiency virus KU-1B (SHIV(KU-1B)), SHIV(89.6P), and simian immunodeficiency virus 17E-Fr. Levels of circulating CD4(+) T cells and viral loads in the plasma and the cerebrospinal fluid were monitored in these macaques for a period of 12 weeks. Both morphine and control groups showed precipitous loss of CD4(+) T cells. However this loss was more prominent in the morphine group at week 2 (P = 0.04). Again both morphine and control groups showed comparable peak plasma viral load at week 2, but the viral set points were higher in the morphine group than that in the control group. Likewise, the extent of virus replication in the cerebral compartment was more pronounced in the morphine group. These results provide a definitive evidence for a positive correlation between morphine and levels of viral replication.     

Autologous neutralizing humoral immunity and evolution of the viral envelope in the course of subtype B human immunodeficiency virus type 1 infection

Most human immunodeficiency virus type 1 (HIV-1)-infected individuals develop an HIV-specific neutralizing antibody (NAb) response that selects for escape variants of the virus. Here, we studied autologous NAb responses in five typical CCR5-using progressors in relation to viral NAb escape and molecular changes in the viral envelope (Env) in the period from seroconversion until after AIDS diagnosis. In sera from three patients, high-titer neutralizing activity was observed against the earliest autologous virus variants, followed by declining humoral immune responses against subsequent viral escape variants. Autologous neutralizing activity was undetectable in sera from two patients. Patients with high-titer neutralizing activity in serum showed the strongest positive selection pressure on Env early in infection. In the initial phase of infection, gp160 length and the number of potential N-linked glycosylation sites (PNGS) increased in viruses from all patients. Over the course of infection, positive selection pressure declined as the NAb response subsided, coinciding with reversions of changes in gp160 length and the number of PNGS. A number of identical amino acid changes were observed over the course of infection in the viral quasispecies of different patients. Our results indicate that although neutralizing autologous humoral immunity may have a limited effect on the disease course, it is an important selection pressure in virus evolution early in infection, while declining HIV-specific humoral immunity in later stages may coincide with reversion of NAb-driven changes in Env.