Cysteine residues in the Vif protein of human immunodeficiency virus type 1 are essential for viral infectivity.

The infectivity factor of human immunodeficiency virus type 1 (HIV-1), Vif, contains two cysteine residues which are highly conserved among animal lentiviruses. We introduced substitutions of leucine for cysteine residues in the vif gene of a full-length HIV-1 clone to analyze their roles in viral infection. Mutant viruses containing substitutions in either Cys-114, Cys-133, or both displayed a vif-negative infection phenotype similar to that of an isogeneic vif deletion mutant, namely, a cell-dependent complete to partial loss of infectivity. The vif defect could be complemented by cotransfection of mutant viral DNA with a Vif expression vector, and there was no evidence that recombination contributed to the repair of the vif deficiency. The viral protein profile, as determined by immunoblotting, in cells infected with cysteine substitution mutants and that in wild-type virus were similar, including the presence of the 23-kDa Vif polypeptide. In addition, immunoblotting with an antiserum directed against the carboxyl terminus of gp41 revealed that gp41 was intact in cells infected with either wild-type or vif mutant HIV-1, excluding that Vif cleaves the C terminus of gp41. Our results indicate that the cysteines in HIV-1 Vif are critical for Vif function in viral infectivity.     

Aberrant Gag protein composition of a human immunodeficiency virus type 1 vif mutant produced in primary lymphocytes.

Productive, spreading infection of peripheral blood lymphocytes (PBL) with human immunodeficiency virus type 1 (HIV-1) requires the viral protein Vif. To study the requirement for vif in this system, we infected PBL with a phenotypically complemented HIV-1 clone mutated in vif. Progeny virus was produced which was noninfectious in PBL but replicated in SupT1 cells. Analysis of metabolically labeled proteins of sedimentable extracellular particles made in PBL by radioimmunoprecipitation with either serum from a patient with AIDS or a monoclonal antibody reactive with HIV-1 Gag proteins revealed that vif-negative but not wild-type particles carry higher levels of p55, p41, and p38 Gag-specific proteins compared with those of p24. Similar results were obtained with sucrose-purified virions. Our data indicate that vif plays a role in Gag protein processing or in incorporation of processed Gag products into mature virions. The presence of unprocessed precursor Gag polyprotein (Pr55gag) and other Gag processing intermediates in PBL-derived vif-negative extracellular particles may contribute to the reduced infectivity of this virus.     

Phenotypically Vif- human immunodeficiency virus type 1 is produced by chronically infected restrictive cells.

The permissivity of CD4+ transformed T cells for the replication of human immunodeficiency virus type 1 (HIV-1) vif mutants varies widely between different cell lines. Mutant vif-negative viruses propagate normally in permissive CD4+ cell lines but are unable to establish a productive infection in restrictive cell lines such as H9. As a consequence, elucidation of the function of Vif has been considerably hampered by the inherent difficulty in obtaining a stable source of authentically replication-defective vif-negative viral particles produced by restrictive cells. vif-negative, vpr-negative HIV-1 strain NDK stock, produced by the permissive SupT1 cell line, was used to infect restrictive H9 cells. By using a high multiplicity, infection of H9 cells was achieved, leading to persistent production of viral particles displaying a dramatically reduced infectious virus titer when measured in a single-cycle infectivity assay. Although these viral particles were unable to further propagate in H9 cells, they could replicate normally in CEM and SupT1 cells. Comparison of unprocessed and processed Gag proteins in the persistently produced vif-negative viral particles revealed no defect in the processing of polypeptide precursors, with no inversion of the Pr55gag/p24 ratio. In addition, there was no defect in Env incorporation for the vif-negative viral particles. Despite their apparently normal protein content, these particles were morphologically abnormal when examined by transmission electron microscopy, displaying a previously described abnormally condensed nucleoid. Chronically infected restrictive cell lines producing stable levels of phenotypically vif-negative HIV-1 particles could prove particularly useful in further studies on the function of Vif in the virus life cycle.     

Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.     

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.     

Differential glycosylation, virion incorporation, and sensitivity to neutralizing antibodies of human immunodeficiency virus type 1 envelope produced from infected primary T-lymphocyte and macrophage cultures.

Two primary cell targets for human immunodeficiency virus type 1 (HIV-1) infection in vivo are CD4+ T lymphocytes and monocyte-derived macrophages (MDM). HIV-1 encodes envelope glycoproteins which mediate virus entry into these cells. We have utilized infected and radiolabelled primary peripheral blood mononuclear cell (PBMC) and MDM cultures to examine the biochemical and antigenic properties of the HIV-1 envelope produced in these two cell types. The gp120 produced in MDM migrates as a broad, diffuse band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels compared with that of the more homogeneous gp120 released from PBMCs. Glycosidase analyses indicated that the diffuse appearance of the MDM gp120 is due to the presence of asparagine-linked carbohydrates containing lactosaminoglycans, a modification not observed with the gp120 produced in PBMCs. Neutralization experiments, using isogeneic PBMC and MDM-derived macrophage-tropic HIV-1 isolates, indicate that 8- to 10-fold more neutralizing antibody, directed against the viral envelope, is required to block virus produced from MDM. These results demonstrate that HIV-1 released from infected PBMC and MDM cultures differs in its biochemical and antigenic properties.     

Conservation of an intact human immunodeficiency virus type 1 vif gene in vitro and in vivo.

Replication of vif-negative human immunodeficiency virus type 1 (HIV-1) is attenuated in certain cell lines and highly impaired in peripheral blood lymphocytes in vitro. To determine whether intact vif is positively selected during natural HIV-1 infection and to determine vif sequence variability, we employed PCR amplification, cloning, and sequencing to investigate the vif region of replicating virus in short-term-passage HIV-1 primary isolates from five asymptomatic individuals and from five persons with AIDS. A total of 46 vif clones were obtained and analyzed. Recombinant proviruses were constructed from selected vif clones from one patient and found to be fully infectious. We found that 38 of the 46 clones sequenced carried open vif reading frames and that there was a low degree of heterogeneity of vif genes within isolates from the same individual and among isolates from different donors. The cysteines previously found to be essential for vif protein function were conserved in all clones. A phylogenetic tree constructed from all available vif nucleotide sequences resulted in a virus grouping similar to those of gag and env. Direct sequencing of vif amplified by PCR from uncultured lymphocytes of 15 individuals at various stages of progression toward AIDS demonstrated vif open reading frames in 13 of 15 samples tested. There was no obvious correlation between disease status and the presence of an intact vif within this sample group at the time of sample procurement. The conservation of the vif open reading frame in vitro and in vivo and its limited variability following virus transmission in vitro are consistent with a role for vif in natural HIV-1 infection.     

Construction and characterization of an infectious DNA clone and of mutants of simian immunodeficiency virus isolated from the African green monkey.

We constructed a full-length molecular clone of simian immunodeficiency virus from an African green monkey. Upon transfection, this clone directed the production of virus particles cytopathic and infectious to human CD4+ leukemia cell lines. Mutations were introduced by recombinant DNA techniques into eight open reading frames of simian immunodeficiency virus from the African green monkey thus far identified. The phenotypes of mutant viruses, i.e., infectivity, cytopathogenicity, transactivation of gene expression controlled by a long terminal repeat, and viral RNA and protein syntheses, were examined by transfection and infection experiments. Three structural (gag, pol, and env) and two regulatory (tat and rev) gene mutants were not infectious, whereas vif, vpx, and nef were dispensable for infectivity and mutant viruses were highly cytopathic. In transient transfection assays, a rev mutant produced mainly small mRNA species and no detectable virus protein and particles. The transactivation potential of a tat mutant was about 10-fold less than that of wild-type DNA, generating small amounts of virus.     

Roles of the auxiliary genes and AP-1 binding site in the long terminal repeat of feline immunodeficiency virus in the early stage of infection in cats.

To examine the roles of auxiliary genes and the AP-1 binding site in the long terminal repeat of feline immunodeficiency virus (FIV) in vivo, three mutant viruses, which are defective in the vif gene ([delta]vif), ORF-A gene (deltaORF-A), and AP-1 binding site (deltaAP-1), and wild-type virus as a positive control were separately inoculated into three specific-pathogen-free cats. These cats were assessed by measuring the number of proviral DNA copies in peripheral blood mononuclear cells (PBMCs), the CD4/CD8 ratio and antibody responses to FIV for 16 weeks and then examining histological changes at necropsy. Although viral DNAs were detected in PBMCs from all 12 cats to various degrees until 16 weeks postinoculation, no virus was recovered from PBMCs of cats infected with (delta)vif virus during the observation period. However, a very weak antibody response was induced in one cat infected with the (delta)vif virus. In contrast, despite the successful recovery of virus from both groups of cats infected with deltaORF-A and deltaAP-1 virus, antibody responses and decrease in the CD4/CD8 ratio in the groups were milder than those in cats infected with wild-type virus. Furthermore, the numbers of proviral DNA copies in PBMCs from the two groups were not able to reach the level in cats infected with wild-type virus during the observation period. From these results, we conclude that these mutant viruses are still infectious for cats but failed in efficient viral replication and suggest that these auxiliary genes and enhancer element are important or essential to full viral replication kinetics and presumably to full pathogenicity during the early stage of infection in vivo.     

Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells.

We constructed five chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVMAC) and four SIVMAC mutants by recombinant DNA techniques. Three chimeric clones and all mutants with an alteration in either the vif, vpx, vpr, or nef gene were infectious to human CD4-positive cell lines. The susceptibility of macaque monkey peripheral blood mononuclear cells (PBMC) to infection by these mutants and chimeras was examined in vitro. Macaque PBMC supported the replication of wild-type and vpx, vpr, and nef mutant SIVMAC strains. A chimera carrying the long terminal repeats (LTRs), gag, pol, vif, and vpx of SIVMAC and tat, rev, vpu, and env of HIV-1 was also replication competent in PBMC. In contrast, HIV-1, the vif mutant of SIVMAC, a chimera containing rev and env of SIVMAC, and a chimera containing vpx, vpr, tat, rev, and env of SIVMAC did not grow in PBMC. Western immunoblotting analysis of the replicating chimera in PBMC confirmed the hybrid nature of the virus. These data strongly suggested that the sequence important for macaque cell tropism lies within the LTR, gag, pol, and/or vif sequences of the SIVMAC genome.     

Human immunodeficiency virus type 1 replication in the absence of integrase-mediated dna recombination: definition of permissive and nonpermissive T-cell lines

Functional retroviral integrase protein is thought to be essential for productive viral replication. Yet, previous studies differed on the extent to which integrase mutant viruses expressed human immunodeficiency virus type 1 (HIV-1) genes from unintegrated DNA. Although one reason for this difference was that class II integrase mutations pleiotropically affected the viral life cycle, another reason apparently depended on the identity of the infected cell. Here, we analyzed integrase mutant viral infectivities in a variety of cell types. Single-round infectivity of class I integration-specific mutant HIV-1 ranged from <0.03 to 0.3% of that of the wild type (WT) across four different T-cell lines. Based on this approximately 10-fold influence of cell type on mutant gene expression, we examined class I and class II mutant replication kinetics in seven different cell lines and two primary cell types. Unexpectedly, some cell lines supported productive class I mutant viral replication under conditions that restricted class II mutant growth. Cells were defined as permissive, semipermissive, or nonpermissive based on their ability to support the continual passage of class I integration-defective HIV-1. Mutant infectivity in semipermissive and permissive cells as quantified by 50% tissue culture infectious doses, however, was only 0.0006 to 0.005% of that of WT. Since the frequencies of mutant DNA recombination in these lines ranged from 0.023 to <0.093% of the WT, we conclude that productive replication in the absence of integrase function most likely required the illegitimate integration of HIV-1 into host chromosomes by cellular DNA recombination enzymes.     

Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and simian immunodeficiency virus SIV (AGM).

We constructed an infectious molecular clone of the human immunodeficiency virus type 2 (HIV-2) and generated nine frameshift mutants corresponding to nine open reading frames identified so far. Three structural (gag, pol, env) and two regulative (tat, rev) gene mutants were not infectious, whereas vif, vpx, vpr, and nef genes were dispensable for infectivity. All of the mutants except env and rev were cytopathic in CD4+ human leukemia cells. In transfection assays, the expression of HIV-2 long terminal repeat was activated by infectious clones of HIV-1, HIV-2, and simian immunodeficiency virus from African green monkey but not by the tat mutants. However, an HIV-2 tat mutant could produce small amounts of virus proteins and particles in contrast to a rev mutant, which directed no detectable synthesis of virus proteins and virions.     

Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes.

The viral infectivity factor gene vif of human immunodeficiency virus type 1 has been shown to affect the infectivity but not the production of virus particles. In this study, the effect of vif in the context of the HXB2 virus on virus replication in several CD4+ T-cell lines was investigated. vif was found to be required for replication in the CD4+ T-cell lines CEM and H9 as well as in peripheral blood T lymphocytes. vif was not required for replication in the SupT1, C8166, and Jurkat T-cell lines. The infectivity of vif-defective viruses depended on the cell type in which the virus was produced. In CEM cells, vif was required for production of virus capable of initiating infection in all cell lines studied. vif-defective virus produced by SupT1, C8166, and Jurkat cells and the monkey cell line COS-1 could initiate infection in multiple cell lines, including CEM and H9. These results suggest that vif can compensate for cellular factors required for production of infectious virus particles that are present in some cell lines such as SupT1, C8166, and Jurkat but are absent in others such as CEM and H9 as well as peripheral blood T lymphocytes. The effect of vif was not altered by deletion of the carboxyl terminus of gp41, a proposed target for vif (B. Guy, M. Geist, K. Dott, D. Spehner, M.-P. Kieny, and J.-P. Lecocq, J. Virol. 65:1325-1331, 1991). These studies demonstrate that vif enhances viral infectivity during virus production and also suggest that vif is likely to be important for natural infections.