The human immunodeficiency virus type 2 vpr gene is essential for productive infection of human macrophages.

The human immunodeficiency virus (HIV) genetic determinant(s) responsible for tropism in human T cells or macrophages are not well defined. We studied the role of the HIV type 2 (HIV-2) nef and vpr genes in viral tropism. HIV-2 mutants, lacking either vpr or nef genes, or both vpr and nef, were obtained by site-specific mutagenesis of a biologically active HIV-2 proviral clone (HIV-2sbl/isy), which is infectious in both human T cells and macrophages. Viral progeny carrying mutations of nef, vpr, or of both nef and vpr genes replicated more efficiently than the parental virus in primary human peripheral blood cells and in the human Hut 78 T-cell line. In contrast, the HIV-2 nef- mutant infected human macrophages as efficiently as the parental virus, whereas viruses lacking the vpr gene either alone or in conjunction with the lack of the nef gene did not replicate in macrophages. Thus, some lack of nef in HIV-2 enhances viral replication in T cells and does not interfere with viral replication in primary macrophages, whereas vpr is essential for replication of HIV-2 in human macrophages. Because the parental HIV-2sbl/isy cloned virus also infects rhesus macaques, the use in animal studies of these HIV-2 mutants with differences in cell tropism and rates of replication will be highly useful in understanding the mechanism of viral infectivity and possibly pathogenicity in vivo.     

HIV with multiple gene deletions as a live attenuated vaccine for AIDS.

Most viral vaccines currently in use in humans are live attenuated strains of virus that lack pathogenic potential. In general, such live attenuated vaccines induce the strongest longest-lasting immunity. Live attenuated strains of human immunodeficiency virus type 1 (HIV-1) have not been previously considered as vaccines for acquired immunodeficiency syndrome (AIDS) because of an inability to envision how their safety could be adequately assured. This report describes a means for making live, nonpathogenic strains of SIVmac and HIV-1 that cannot revert to a virulent form and a stepwise scheme for demonstrating their safety. Replication-competent, multiply deleted derivatives that are currently being tested are missing combinations of auxiliary genes (nef, vpr, vif, vpx, vpu) and certain control elements in the negative regulatory element (NRE) of the long terminal repeat (LTR). Since these genomic regions are in large part conserved among the SIVs and HIVs, they are likely to be important for the virus life cycle in vivo. Consistent with this line of reasoning, a replication-competent nef deletion mutant of SIVmac apparently has lost most or all of its pathogenic potential, yet it still induces strong immune responses. Multiply deleted derivatives of SIVmac and HIV-1 will have to be extensively tested in animal models prior to moving a promising HIV-1 candidate to initial trials in high-risk human volunteers. Definitive evidence for safety and general acceptance for this approach can only evolve gradually over a prolonged period of time.     

Allelic variation in the effects of the nef gene on replication of human immunodeficiency virus type 1.

The effects of the viral gene nef on human immunodeficiency virus type 1 (HIV-1) replication in culture were investigated using nef alleles of the HIV-1 IIIB and ELI strains. The results demonstrate significant allelic variation in the effect of nef on virus replication in both an established human CD4+ T-cell line and primary human lymphocytes. In the context of the HXB2 virus, the ELI nef allele but not the IIIB nef allele permits initiation of efficient low-multiplicity infection in primary peripheral blood mononuclear cells, including unfractionated peripheral blood lymphocytes, T cells, and monocyte/macrophages. Within the same genetic context, the IIIB nef allele slightly retards replication of the virus in a T-cell line, whereas the ELI nef allele accelerates replication of the virus. Sequences in the IIIB and ELI genomes outside of nef also moderate the effects of nef on HIV-1 replication. nef did not appear to determine the host-cell preference of the virus. These studies may help to reconcile apparently conflicting reports on the role of nef in HIV-1 replication and suggest that HIV-1 nef may play an important role in viral pathogenesis.     

The Vif and Vpr accessory proteins independently cause HIV-1-induced T cell cytopathicity and cell cycle arrest

HIV type I (HIV-1) can cause G(2) cell cycle arrest and death of CD4(+) T lymphocytes in vitro and inexorable depletion of these cells in vivo. However, the molecular mechanism of viral cytopathicity has not been satisfactorily elucidated. Previously, we showed that HIV-1 kills T cells by a necrotic form of cell death that requires high level expression of an integrated provirus but not the env or nef genes. To determine which viral protein(s) are required for cell death, we systematically mutated, alone and in combination, the ORFs of the NL4-3 strain of HIV-1. We found that the elimination of the viral functions encoded by gag-pol and vpu, tat, and rev did not mitigate cytopathicity. However, elimination of the vif and vpr accessory genes together, but not individually, renders the virus incapable of causing cell death and G(2) cell cycle blockade. We thus identify vif and vpr as necessary for T cell cytopathic effects induced by HIV-1. These findings may provide an important insight into the molecular mechanism of viral pathogenesis in AIDS.     

Human immunodeficiency virus vpr gene encodes a virion-associated protein

The vpr gene of human immunodeficiency virus type 1 (HIV-1) is one of the seven accessory genes that are believed to have roles in the virus replication cycle. We report here the detection of a 13 kD vpr protein in sucrose gradient-purified HIV-1. This protein was not detected in cells infected with a virus having a truncated vpr gene that lacks the potential to encode for 26 C-terminal amino acid residues. These findings raise the possibility that virion-associated vpr proteins may be involved in the early life cycle of HIV-1 replication and suggest that the C-terminal region of the vpr gene is essential for its expression.     

Serum reactivity to HIV-1 accessory gene products distinguishes East African from West African HIV strains as infecting agent

The existence of dual infections with human immunodeficiency virus (HIV)-1 and 2 in West African countries has been controversial, although the current consensus is that dual infection is not the cause of the extensive cross-reactivity observed between these 2 viruses. To evaluate the role of antibody reactivity to the HIV-1 accessory gene products in type-specific HIV serology, proteins encoded for nef, tat, rev, vpr, and vpu were developed and used as an antigen. 5 of the 7 exclusively HIV-2 reactive sera were not reactive to the HIV-1 accessory gene products. Moreover, the 2 sera that showed reactivity to the HIV-1 envelope were the only ones reactive to HIV-1 accessory gene products. These findings indicate that type 2 viruses may be as diverse as type 1 viruses. A subsequent analysis of sera from 24 West Africans revealed reactivity with a simian immunodeficiency virus (SIV) peptide but not with an HIV-1 peptide previously shown to be discriminatory in a direct binding assay between HIV-1 and HIV-2. Compared to 29 control sera from East Africans, the West Africa sera had significantly lower reactivity to antibodies specific to nef, tat, and rev; there was not reactivity to vpr and vpu. 38% of the West African sera compared with 93% of the East African sera showed reactivity to HIV-1 accessory gene products. It is concluded that, while reactivity to the HIV-1 accessory gene products vpr and vpu indicate HIV-1 infection, reactivity to the other accessory gene products cannot be used to identify virus type given the documented cross-reactivity to HIV-1 accessory gene products of antibodies elicited by HIV-2 strains.     

Genetic and biological characterization of recombinant HIV type 1 with Env derived from long-term nonprogressor (LTNP) viruses

Multiple factors are known to contribute to nonprogressive disease in long-term nonprogressors (LTNP). We previously selected LTNPs, in which broadly neutralizing antibodies against HIV-1 very likely contribute to disease prevention. Here, we characterize those LTNPs further. We analyzed sequences of the viral genes env, nef, vpr, tat, and rev as well as the cellular ccr5, HLA-B*5701, and HLA-B*27 genes derived from eight LTNPs, as mutations in these genes have been associated with the LTNP status in some studies. Furthermore, we compared the replication rates of recombinant reporter viruses carrying envelope proteins from LTNPs to control viruses from patients with similar CD4 count and viral load. Concerning the cellular factors, none of the eight LTNPs showed the 32-base pair deletion in the ccr5 gene, and HLA-B*5701 and HLA-B*27 alleles were detected in only one LTNP, respectively. The reading frames for the regulatory genes nef, vpr, tat, and rev were all open. Although Env sequences from LTNPs differed from those of control patients with respect to the length of variable domains and the number of N-glycosylation sites, these differences were not statistically significant and did not lead to differences in infectivity of recombinant reporter viruses.     

Recombinant simian immunodeficiency virus expressing green fluorescent protein identifies infected cells in rhesus monkeys

We engineered recombinant derivatives of simian immunodeficiency virus (SIV) to express enhanced green fluorescent protein (EGFP). Replacement of vpr sequences with EGFP resulted in a genome that did not produce detectable levels of replication-competent virus. Replication-competent virus and bright fluorescence of infected cells were obtained with two other constructs, one in which SIV nef sequences were replaced by EGFP and another in which EGFP was inserted into the SIV nef locus and HIV-1 nef sequences were expressed by downstream placement of an internal ribosomal entry site. These strains were infectious in rhesus monkeys and green fluorescing cells were detected in the tissues of infected monkeys by FACS analysis and by direct microscopic visualization. EGFP sequences were absent from recovered virus by 8 weeks following infection. We conclude that recombinant SIV that is engineered to express EGFP can be used to directly detect productively infected cells and aid in the immunophenotypic characterization of these cells within the first 2 weeks of infection of rhesus monkeys.     

Construction of chimeric simian and human immunodeficiency viruses that produce interleukin 12

Chimeric simian and human immunodeficiency viruses (SHIVs) are useful for evaluating vaccine candidates against HIV-1 and for investigating the pathogenesis of HIV-1 in vivo. In addition, SHIVs are candidates for a vaccine against HIV-1 because attenuated SHIVs can induce long-lasting anti-HIV-1 Env humoral and cell-mediated immunity in monkeys without AIDS-like diseases. In this study, we inserted IL-12 genes in a nef-deleted SHIV to increase the ability of the SHIV to induce cell-mediated immunity against HIV-1. The SHIV vector was constructed by deleting the nef gene and replacing it with restriction enzyme sites. Since IL-12 consists of two subunit genes, p35 and p40, SHIVs with one or both of these genes were constructed. SHIVs with either one of the subunit genes could replicate without a deletion of the inserted gene, but SHIVs with two subunit genes replicated poorly and the inserted genes were rapidly deleted. Production of IL-12 was detected when both of the single-subunit SHIVs were coinfected. The production of IL-12 by the coinfection reached 800 pg/ml, and IL-12 was detected after serial passage in cell cultures, although this amount of IL-12 heterodimer was 150-1500 times less than that of the p40 subunits. These IL-12-producing SHIVs are candidates for a live-attenuated vaccine to induce effective cellular immunity against HIV-1.     

The glucocorticoid receptor type II complex is a target of the HIV-1 vpr gene product.

The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 15-kDa virion-associated protein that functions as a regulator of cellular processes linked to the HIV life cycle. We report the interaction of a 41-kDa cytosolic viral protein R interacting protein 1 (Rip-1) with Vpr in vitro. Rip-1 displays a wide tissue distribution, including relevant targets of HIV infection. Vpr protein induced nuclear translocation of Rip-1, as did glucocorticoid receptor (GR)-II-stimulating steroids. Importantly, Vpr and Rip-1 coimmunoprecipitated with the human GR as part of an activated receptor complex. Vpr complementation of a vpr mutant virus was also mimicked by GR-II-stimulating steroids. Vpr and GR-II actions were inhibited by mifepristone, a GR-II pathway inhibitor. Together these data directly link the activity of the vpr gene product to the glucocorticoid steroid pathway and provide a biochemical mechanism for the cellular and viral activity of Vpr, as well as suggest that a unique class of antivirals, which includes mifepristone (RU486), may influence HIV-1 replication.     

Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIV-specific cytotoxic T-lymphocyte response in vitro

Dendritic cells (DCs) genetically modified to continually express and present antigens may be potent physiologic adjuvants for induction of prophylactic or therapeutic immunity. We have previously shown that an env and nef deleted HIV-1 vector (HIV-1 delta EN) pseudotyped with VSV-G transduced monocyte-derived macrophages as well as CD34(+) precursors of DCs. Here we extended these findings with HIV-1 delta EN to highly differentiated human DCs derived in culture from circulating monocytes (DCs). In addition, a new vector derived from HIV-1 delta EN but further deleted in its remaining accessory genes vif, vpr, and vpu (HIV-1 delta EN V(3)) was also tested. Both vectors efficiently transduced DCs. Transduction of DCs did not significantly alter their viability or their immunophenotype when compared with untransduced DCs. Furthermore, the phagocytic potential of immature DCs, as well as their ability to differentiate into mature DCs capable of stimulating T-cell proliferation, was not affected. Finally, DCs transduced by the HIV-1 delta EN vector were able to elicit a primary antiviral cytotoxic T-cell response in autologous CD8 T cells. These results suggest that HIV-1-based vectors expressing viral antigens may be useful for in vivo active immunization as well as ex vivo priming of cytotoxic T cells for adoptive T-cell therapy. (Blood. 2000;96:1327-1333)     

Vaccine effect using a live attenuated nef-deficient simian immunodeficiency virus of African green monkeys in the absence of detectable vaccine virus replication in vivo

Immunization of adult macaques with live attenuated simian immunodeficiency viruses (SIVs) lacking the nef genes has been shown to protect against challenge with full-length pathogenic SIV. To test live attenuated virus vaccines for the first time in a natural host we have constructed a mutant SIV from African green monkeys (SIVagm) with a deletion of 125 bp in the nef gene (SIVagm3Δnef). This mutant showed moderately delayed in vitro replication in the T cell line MOLT-4/8 and in primary peripheral blood mononuclear cells from African green monkeys (Cercopithecus aetiops) and pig-tailed macaques (Macaca nemestrina) compared with cloned wild-type SIVagm3. In contrast, in vivo replication of SIVagm3Δnef in African green monkeys was severely impaired or undetectable and did not induce seroconversion. After challenge with wild-type SIVagm3 the SIVagm3Δnef preinoculated African green monkeys showed a memory antibody response that declined after week 2. In three of four African green monkeys the cell-associated virus load and in two of four African green monkeys the plasma virus load was dramatically decreased after the challenge compared with naive control animals. The remaining animal showed no evidence of productive challenge virus replication. This study demonstrates that a strong vaccine effect or protection in the SIVagm/African green monkey system is possible using a live attenuated vaccine in the absence of a productive infection and corresponding humoral immune response.     

Human immunodeficiency virus 1 reservoir in CD4+ T cells is restricted to certain V beta subsets.

The human immunodeficiency virus 1 (HIV-1) replicates more efficiently in T-cell lines expressing T-cell receptors derived from certain V beta genes, V beta 12 in particular, suggesting the effects of a superantigen. The targeted V beta 12 subset was not deleted in HIV-1-infected patients. It was therefore possible that it might represent an in vivo viral reservoir. Viral load was assessed by quantitative PCR with gag primers and with an infectivity assay to measure competent virus. It was shown that the tiny V beta 12 subset (1-2% of T cells) often has a higher viral load than other V beta subsets in infected patients. Selective HIV-1 replication in V beta 12 cells was also observed 6-8 days after in vitro infection of peripheral blood lymphocytes from normal, HIV-1 negative donors. Viral replication in targeted V beta subsets may serve to promote a biologically relevant viral reservoir.     

Genetic and biological comparisons of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus (SIVmac).

Simian immunodeficiency virus (SIV) is a designation for a group of related but unique lentiviruses identified in several primate species. A viral isolate from a rhesus macaque (i.e., SIVmac) causes a fatal AIDS-like disease in experimentally infected macaques, and several infectious molecular clones of this virus have been characterized. This report presents the complete nucleotide sequence of molecularly cloned SIVmac1A11, and comparisons are made with the sequence of molecularly cloned SIVmac239. SIVmac1A11 has delayed replication kinetics in lymphoid cells but replicates as well as uncloned SIVmac in macrophage cultures. Macaques infected with virus from the SIVmac1A11 clone develop antiviral antibodies, but virus does not persist in peripheral blood mononuclear cells and no disease signs are observed. SIVmac239 infects lymphoid cells, shows restricted replication in cultured macrophages, and establishes a persistent infection in animals that leads to a fatal AIDS-like disease. Both viruses are about 98% homologous at the nucleotide sequence level. In SIVmac1A11, the vpr gene as well as the transmembrane domain of env are prematurely truncated, whereas the nef gene of SIVmac239 is prematurely truncated. Sequence differences are also noted in variable region 1 (V1) in the surface domain of the env gene. The potential implications of these and other sequence differences are discussed with respect to the phenotypes of both viruses. This animal model is critically important for investigating the roles of specific viral genes in viral/host interactions that cannot be studied in individuals infected with the human immunodeficiency virus (HIV).