Subtle mutations in the cysteine region of HIV-1 Vif drastically alter the viral replication phenotype

Mutations were introduced into the region encoding the two cysteine and nearby amino acid residues of human immunodeficiency virus type 1 (HIV-1) Vif protein and, 12 single-amino-acid viral mutants were constructed. Determination of their growth characteristics in two lymphocytic cell lines revealed that only a single amino acid change in the cysteine region greatly altered the replication phenotype. In particular, the four mutants of amino acid 132 of Vif were grouped into three categories on the basis of their growth potentials. These results indicate that the cysteine region of Vif is critical for the cell-dependent replication efficiency of HIV-1.     

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.     

Apparent lack of trans-dominant negative effects of various vif mutants on the replication of HIV-1

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for virus growth in non-permissive cells such as H9. To elucidate the mechanism of action of the Vif protein, vif mutants, which show trans-dominant negative effects on the replication of HIV-1, would be useful tools. In this study, a new assay system to identify the mutants of this category was established. For this new system, various reporter clones carrying both mutant and authentic vif sequences were generated. By determining the growth ability of the viruses derived from the reporter constructs, the potential negative effect of the mutant vif sequence was readily and sensitively monitored. Ten vif mutant sequences tested were found not to exert the trans-dominant negative effect on the replication of HIV-1.     

Cell-dependent requirement of human immunodeficiency virus type 1 Vif protein for maturation of virus particles.

A highly sensitive single-round infection assay using a bacterial chloramphenicol acetyltransferase was developed to analyze an early stage of human immunodeficiency virus type 1 replication. By a combination of transfection and single-round infection assay, a virus with a vif mutation, depending on host cells from which the virus was derived, was demonstrated to be defective at the early phase of infection cycle. Analysis of viral proteins synthesized in cells indicated that incorporation of the Env surface protein into virions of the vif mutant, again in a cell-dependent way, was greatly restricted. Taken together, it is concluded that the Vif protein acts through modulation of the Env protein in the virions, directly or indirectly, to enhance viral infectivity in a certain cell type.     

Cell-dependent replication potentials of HIV-1 gag mutants

An infectious molecular clone of human immunodeficiency virus type 1 (HIV-1), designated pNLaiKH, which is tropic for both lymphocytic and monocytic cells, was constructed. To study the early function of HIV-1 Gag proteins in two types of cells, the mutations known to give host cell-dependent early defects were introduced into pNLaiKH, and the replication potentials and defective replication sites in the cells of the resultant mutants were monitored. All mutants grew in some lymphocytic cells, but not at all in monocytic cells. A nucleocapsid mutant was found to be defective at an early replication phase in all the cell lines to various extent, as expected. In contrast, a matrix mutant and a capsid mutant displayed a replication defect in a producer-cell-dependent manner. These results demonstrated that complex interactions of cell factors and Gag proteins are involved in an early process of HIV-1 replication.     

Identification of amino acid residues in HIV-1 Vif critical for binding and exclusion of APOBEC3G/F

To define a region(s) in human immunodeficiency virus type 1 (HIV-1) Vif that involves binding to its target APOBEC3G (A3G), we have generated a series of site-specific proviral vif mutants. Of 30 mutants examined, 15 did not grow at all or grew more poorly than wild-type virus in non-permissive cells. Eight clones with N-terminal mutations located outside of the HCCH motif and BC-box, which are known to be directly crucial for the degradation of A3G, were chosen from these growth-defective mutants and mainly analyzed in detail for functional activity of their mutant Vif proteins. By single-cycle replication and immunoprecipitation/immunoblotting analyses, mutants designated W21A, S32A, W38A, Y40A, and H43A were demonstrated to hardly or poorly bind to and neutralize A3G. Upon transfection, these mutants produced progeny virions containing much more A3G than wild-type clone. Interestingly, while mutants designated E76A and W79A acted normally to inactivate A3G, they were found to exhibit a Vif-defective phenotype against A3F. Another unique mutant designated Y69A incompetent against both of A3G/F was also identified. Our results here have indicated that at least two distinct regions in the N-terminal half of HIV-1 Vif are critical for binding and exclusion of A3G/F.     

Expression of HIV-1 accessory protein Vif is controlled uniquely to be low and optimal by proteasome degradation

While the Vif protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in non-permissive cells, it is rapidly degraded intracellularly. We have previously suggested that the rapid turn-over of Vif is biologically meaningful to prevent detrimental effects of this protein at high expression levels. We now studied the mechanism of Vif degradation by examining the blocking effect of protease inhibitors in pulse/chase experiments and by monitoring the extent of Vif ubiquitination. The rapid turn-over of Vif could be blocked by proteasome inhibitors, and Vif was highly ubiquitinated. Cytoskeletal Vif was found to be more stable than soluble cytosolic Vif. These degradation characteristics of Vif were cell type-independent and observed in both non-permissive and permissive cells. Characterization of a series of vif deletion mutants showed that amino acids predicted to be important for formation of beta-strand structures (amino acid nos. 63-70 and 86-89) were critical for maintaining a normal expression level of Vif and for viral infectivity. Finally, we performed comparative stability analysis of the four HIV-1 accessory proteins. Vif was unique in its short half-life and in the magnitude of the degradation. Taken together, we conclude that the proteasome degradation of HIV-1 Vif is a virologically important process and crucial for the function of Vif.     

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.     

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.     

Amino acid residues 88 and 89 in the central hydrophilic region of human immunodeficiency virus type 1 Vif are critical for viral infectivity by enhancing the steady-state expression of Vif

A hydrophilic region consisting of strikingly clustered charged amino acids is present at the center of human immunodeficiency virus type 1 (HIV-1) Vif. In this study, the role for this central hydrophilic region (E(88)WRKKR(93)) in the virus replication in nonpermissive H9 cells was investigated by extensive deletion and substitution analysis. A total of 31 mutants were constructed. Deletion of the E(88) or W(89) residue alone abolished viral infectivity in H9 cells and impaired virus replication in primary macrophage cultures. Substitution analysis indicated that the hydrophilicity and charge of the central region are insignificant for the function of Vif. Of the 16 substitution mutants, 3 mutants with substitution of E(88) and W(89) with an A residue did not grow in H9 cells. Upon transfection, four mutants (i.e., two mutants with deletion of E(88) or W(89); a mutant with substitution of E(88) and W(89) with A; and a mutant with substitution of E(88), W(89), and R(90) with A) were found to express Vif at a very reduced level relative to that by the wild-type clone. These results have thus demonstrated that amino acid residues 88 and 89 of Vif are critical for the replication of HIV-1 in target cells by enhancing the steady-state expression of Vif. In addition, E(88) and W(89) residues were found to be extremely conserved among the Vif proteins of naturally occurring HIV-1 field isolates as well as those of laboratory HIV-1 strains.     

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.     

Functional analysis of Vif protein shows less restriction of human immunodeficiency virus type 2 by APOBEC3G

Viral infectivity factor (Vif) is one of the human immunodeficiency virus (HIV) accessory proteins and is conserved in the primate lentivirus group. This protein is essential for viral replication in vivo and for productive infection of nonpermissive cells, such as peripheral blood mononuclear cells (PBMC). Vif counteracts an antiretroviral cellular factor in nonpermissive cells named CEM15/APOBEC3G. Although HIV type 1 (HIV-1) Vif protein (Vif1) can be functionally replaced by HIV-2 Vif protein (Vif2), its identity is very small. Most of the functional studies have been carried out with Vif1. Characterization of functional domains of Vif2 may elucidate its function, as well as differences between HIV-1 and HIV-2 infectivity. Our aim was to identify the permissivity of different cell lines for HIV-2 vif-minus viruses. By mutagenesis specific conserved motifs of HIV-2 Vif protein were analyzed, as well as in conserved motifs between Vif1 and Vif2 proteins. Vif2 mutants were examined for their stability, expression, and cellular localization in order to characterize essential domains of Vif2 proteins. Viral replication in various target cells (PBMC and H9, A3.01, U38, and Jurkat cells) and infectivity in single cycle assays in the presence of APOBEC3G were also analyzed. Our results of viral replication show that only PBMC have a nonpermissive phenotype in the absence of Vif2. Moreover, the HIV-1 vif-minus nonpermissive cell line H9 does not show a similar phenotype for vif-negative HIV-2. We also report a limited effect of APOBEC3G in a single-cycle infectivity assay, where only conserved domains between HIV-1 and HIV-2 Vif proteins influence viral infectivity. Taken together, these results allow us to speculate that viral inhibition by APOBEC3G is not the sole and most important determinant of antiviral activity against HIV-2.     

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.     

Mutational analysis of the human immunodeficiency virus vpr open reading frame.

Mutations were introduced by recombinant DNA techniques into the vpr open reading frame of an infectious molecular clone of human immunodeficiency virus type 1. The effect of these changes on the replicative and cytopathologic properties of the virus recovered from transfected cells was studied in several human CD4+ lymphocyte cell lines. In all cases, mutant viruses were infectious and cytopathic. However, when a low-input dose was used, mutants grew significantly more slowly than the wild-type virus. The growth kinetics of vpr mutants were distinct from those of vif and vpu mutants.     

Complementation of vif-defective human immunodeficiency virus type 1 by primate, but not nonprimate, lentivirus vif genes.

The productive infection of many susceptible human cells, including lymphocytes and macrophages derived from peripheral blood, by the pathogenic lentivirus human immunodeficiency virus type 1 requires expression of the virally encoded vif (for virion infectivity factor) gene. Interestingly, this gene appears to have been conserved among all of the lentiviruses of primates and almost all of the lentiviruses of nonprimates. Using T cells constitutively expressing vif genes derived from diverse sources and virus replication assays, we show that the vif gene of a second primate lentivirus, simian immunodeficiency virus from macaques, complements vif-defective human immunodeficiency virus type 1 but that those of three distinct nonprimate lentiviruses do not. Although the molecular basis for Vif function has yet to be defined, the potential implications of this noted restriction of vif complementarity are discussed.