An Endogenous Inhibitor of Human Immunodeficiency Virus in Human Lymphocytes Is Overcome by the Viral Vif Protein

The vif gene of human immunodeficiency virus type 1 (HIV-1) encodes a basic M_r 23,000 protein that is necessary for production of infectious virions by nonpermissive cells (human lymphocytes and macrophages) but not by permissive cells such as HeLa-CD4. It had been proposed that permissive cells may contain an unidentified factor that functions like the viral Vif protein. To test this hypothesis, we produced pseudotyped wild-type and vif-deleted HIV gpt virions (which contain the HIV-1 genome with the bacterial mycophenolic acid resistance gene gpt in place of the viral env gene) in permissive cells, and we used them to generate nonpermissive H9 leukemic T cells that express these proviruses. We then fused these H9 cells with permissive HeLa cells that express the HIV-1 envelope glycoprotein gp120-gp41, and we asked whether the heterokaryons would release infectious HIV gpt virions. The results clearly showed that the vif-deleted virions released by the heterokaryons were noninfectious whereas the wild-type virions were highly infectious. This strongly suggests that nonpermissive cells, the natural targets of HIV-1, contain a potent endogenous inhibitor of HIV-1 replication that is overcome by Vif.     

A Naturally Occurring rev1-vpu Fusion Gene Does Not Confer a Fitness Advantage to HIV-1

Background

Pandemic strains of HIV-1 (group M) encode a total of nine structural (gag, pol, env), regulatory (rev, tat) and accessory (vif, vpr, vpu, nef) genes. However, some subtype A and C viruses exhibit an unusual gene arrangement in which the first exon of rev (rev1) and the vpu gene are placed in the same open reading frame. Although this rev1-vpu gene fusion is present in a considerable fraction of HIV-1 strains, its functional significance is unknown.

Results

Examining infectious molecular clones (IMCs) of HIV-1 that encode the rev1-vpu polymorphism, we show that a fusion protein is expressed in infected cells. Due to the splicing pattern of viral mRNA, however, these same IMCs also express a regular Vpu protein, which is produced at much higher levels. To investigate the function of the fusion gene, we characterized isogenic IMC pairs differing only in their ability to express a Rev1-Vpu protein. Analysis in transfected HEK293T and infected CD4+ T cells showed that all of these viruses were equally active in known Vpu functions, such as down-modulation of CD4 or counteraction of tetherin. Furthermore, the polymorphism did not affect Vpu-mediated inhibition of NF-кB activation or Rev-dependent nuclear export of incompletely spliced viral mRNAs. There was also no evidence for enhanced replication of Rev1-Vpu expressing viruses in primary PBMCs or ex vivo infected human lymphoid tissues. Finally, the frequency of HIV-1 quasispecies members that encoded a rev1-vpu fusion gene did not change in HIV-1 infected individuals over time.

Conclusions

Expression of a rev1-vpu fusion gene does not affect regular Rev and Vpu functions or alter HIV-1 replication in primary target cells. Since there is no evidence for increased replication fitness of rev1-vpu encoding viruses, this polymorphism likely emerged in the context of other mutations within and/or outside the rev1-vpu intergenic region, and may have a neutral phenotype.     

Conservation of an Intact vif Gene of Human Immunodeficiency Virus Type 1 during Maternal-Fetal Transmission

The human immunodeficiency virus type 1 (HIV-1) vif gene is conserved among most lentiviruses, suggesting that vif is important for natural infection. To determine whether an intact vif gene is positively selected during mother-to-infant transmission, we analyzed vif sequences from five infected mother-infant pairs following perinatal transmission. The coding potential of the vif open reading frame directly derived from uncultured peripheral blood mononuclear cell DNA was maintained in most of the 78,912 bp sequenced. We found that 123 of the 137 clones analyzed showed an 89.8% frequency of intact vif open reading frames. There was a low degree of heterogeneity of vif genes within mothers, within infants, and between epidemiologically linked mother-infant pairs. The distances between vif sequences were greater in epidemiologically unlinked individuals than in epidemiologically linked mother-infant pairs. Furthermore, the epidemiologically linked mother-infant pair vif sequences displayed similar patterns that were not seen in vif sequences from epidemiologically unlinked individuals. The functional domains, including the two cysteines at positions 114 and 133, a serine phosphorylation site at position 144, and the C-terminal basic amino acids essential for vif protein function, were highly conserved in most of the sequences. Phylogenetic analyses of 137 mother-infant pair vif sequences and 187 other available vif sequences from HIV-1 databases revealed distinct clusters for vif sequences from each mother-infant pair and for other vif sequences. Taken together, these findings suggest that vif plays an important role in HIV-1 infection and replication in mothers and their perinatally infected infants.     

Identification of a Human Immunodeficiency Virus Type 2 (HIV-2) Encapsidation Determinant and Transduction of Nondividing Human Cells by HIV-2-Based Lentivirus Vectors

Although previous lentivirus vector systems have used human immunodeficiency virus type 1 (HIV-1), HIV-2 is less pathogenic in humans and is amenable to pathogenicity testing in a primate model. In this study, an HIV-2 molecular clone that is infectious but apathogenic in macaques was used to first define cis-acting regions that can be deleted to prevent HIV-2 genomic encapsidation and replication without inhibiting viral gene expression. Lentivirus encapsidation determinants are complex and incompletely defined; for HIV-2, some deletions between the major 5′ splice donor and the gag open reading frame have been shown to minimally affect encapsidation and replication. We find that a larger deletion (61 to 75 nucleotides) abrogates encapsidation and replication but does not diminish mRNA expression. This deletion was incorporated into a replication-defective, envelope-pseudotyped, three-plasmid HIV-2 lentivirus vector system that supplies HIV-2 Gag/Pol and accessory proteins in trans from an HIV-2 packaging plasmid. The HIV-2 vectors efficiently transduced marker genes into human T and monocytoid cell lines and, in contrast to a murine leukemia virus-based vector, into growth-arrested HeLa cells and terminally differentiated human macrophages and NTN2 neurons. Vector DNA could be detected in HIV-2 vector-transduced nondividing CD34⁺ CD38⁻ human hematopoietic progenitor cells but not in those cells transduced with murine vectors. However, stable integration and expression of the reporter gene could not be detected in these hematopoietic progenitors, leaving open the question of the accessibility of these cells to stable lentivirus transduction.     

APOBEC3G Restricts HIV-1 to a Greater Extent than APOBEC3F and APOBEC3DE in Human Primary CD4+ T Cells and Macrophages

APOBEC3 proteins inhibit HIV-1 replication in experimental systems and induce hypermutation in infected patients; however, the relative contributions of several APOBEC3 proteins to restriction of HIV-1 replication in the absence of the viral Vif protein in human primary CD4⁺ T cells and macrophages are unknown. We observed significant inhibition of HIV-1Δvif produced in 293T cells in the presence of APOBEC3DE (A3DE), APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H haplotype II (A3H HapII) but not APOBEC3B (A3B), APOBEC3C (A3C), or APOBEC3H haplotype I (A3H HapI). Our previous studies showed that Vif amino acids Y⁴⁰RHHY⁴⁴ are important for inducing proteasomal degradation of A3G, whereas amino acids ¹⁴DRMR¹⁷ are important for degradation of A3F and A3DE. Here, we introduced substitution mutations of ⁴⁰YRHHY⁴⁴ and ¹⁴DRMR¹⁷ in replication-competent HIV-1 to generate vif mutants NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 to compare the antiviral activity of A3G to the combined antiviral activity of A3F and A3DE in activated CD4⁺ T cells and macrophages. During the first 15 days (round 1), in which multiple cycles of viral replication occurred, both the NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutants replicated in activated CD4⁺ T cells and macrophages, and only the NL4-3 YRHHY>A5 mutant showed a 2- to 4-day delay in replication compared to the wild type. During the subsequent 27 days (round 2) of cultures initiated with peak virus obtained from round 1, the NL4-3 YRHHY>A5 mutant exhibited a longer, 8- to 10-day delay and the NL4-3 DRMR>A4 mutant exhibited a 2- to 6-day delay in replication compared to the wild type. The NL4-3 YRHHY>A5 and NL4-3 DRMR>A4 mutant proviruses displayed G-to-A hypermutations primarily in GG and GA dinucleotides as expected of A3G- and A3F- or A3DE-mediated deamination, respectively. We conclude that A3G exerts a greater restriction effect on HIV-1 than A3F and A3DE.     

HIV-1 Nef-associated Factor 1 Enhances Viral Production by Interacting with CRM1 to Promote Nuclear Export of Unspliced HIV-1 gag mRNA

HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection.     

A Variant Macaque-Tropic Human Immunodeficiency Virus Type 1 Is Resistant to Alpha Interferon-Induced Restriction in Pig-Tailed Macaque CD4+ T Cells

Human immunodeficiency virus type 1 (HIV-1) antagonizes innate restriction factors in order to infect and persistently replicate in a host. In a previous study, we demonstrated that HIV-1 NL4-3 with a simian immunodeficiency virus mne (SIVmne) vif gene substitution (HSIV-vif-NL4-3) could infect and replicate in pig-tailed macaques (PTM), indicating that APOBEC3 proteins are primary barriers to transmission. Because viral replication was persistent but low, we hypothesized that HSIV-vif-NL4-3 may be suppressed by type I interferons (IFN-I), which are known to upregulate the expression of innate restriction factors. Here, we demonstrate that IFN-α more potently suppresses HSIV-vif-NL4-3 in PTM CD4⁺ T cells than it does pathogenic SIVmne027. Importantly, we identify a variant (HSIV-vif-Yu2) that is resistant to IFN-α, indicating that the IFN-α-induced barrier can be overcome by HSIV-vif chimeras in PTM CD4⁺ T cells. Interestingly, HSIV-vif-Yu2 and HSIV-vif-NL4-3 are similarly restricted by PTM BST2/Tetherin, and neither virus downregulates it from the surface of infected PTM CD4⁺ T cells. Resistance to IFN-α-induced restriction appears to be conferred by a determinant in HSIV-vif-Yu2 that includes env su. Finally, we show that the Yu-2 env su allele may overcome an IFN-α-induced barrier to entry. Together, our data demonstrate that the prototype macaque-tropic HIV-1 clones based on NL4-3 may not sufficiently antagonize innate restriction in PTM cells. However, variants with resistance to IFN-α-induced restriction factors in PTM CD4⁺ T cells may enhance viral replication by overcoming a barrier early in the viral replication cycle.     

Two retroviruses packaged in one cell line can combined inhibit the replication of HIV-1 in TZM-bl cells

The cellular protein tetherin tethers the HIV-1 viral particles on the cellular membrane to inhibit the replication of HIV-1. However, the HIV-1 accessory protein Vpu counteracts the antiviral function of tetherin. In this study, two retroviral vector plasmids were constructed. One inhibited the vpu gene expression; the other one over-expressed the tetherin. Both retroviral vector plasmids could be packaged in the packaging cell line PT67 to obtain the corresponding retroviruses. The retroviral vector plasmids’ functions of tetherin over-expression or vpu-RNAi were detected at the cell level. Retroviral vector plasmids were transfected to PT67 cells at different ratios from 0T3V to 3T0V, and then mixed retroviruses were harvested. The antiviral functions of mixed retroviruses were detected in HIV-1 infected TZM-bl cells. The results showed that packaged mixed retroviruses could repress the replication of HIV-1 in TZM-bl cells.     

Interferon-α and -γ Differentially Reduce Rapid Immature T-Cell Death by Contact with HIV-1 Carrier Cell Clones In Vitro

The non-antigen specific rapid cytotoxic (CT) death of immature TdT⁺CD4⁺CD8⁺ T cells due to contact with HIV-1 carrier T-cell clones we have found recently is a novel phenomenon. The effects of interferons (IFN) on this CT reaction were studied in vitro. Treatment of the HIV-1 carrier clones, referred to as “effectors,” with IFN-α but not IFN-γ, or of the susceptible immature TdT⁺CD4⁺CD8⁺ T cells, referred to as “targets,” with IFN-γ but not IFN-α, for 24 hr prior to CT testing was found to reduce the CT reaction. Simultaneously, a down-regulated CD8 expression and an up-regulated antigen expression of both major histocompatibility antigen complex class I (MHC-I) and HIV-1 gp120/gp160 in the IFN-α treated effector (gp120⁺CD8⁺ HPB-ALL/HIV), and/or simultaneously up-regulated antigen expression of both CD8 and MHC-I in the IFN-γ treated target (CD4⁺CD8⁺ HPB-ALL) were found to be associated with reduced CT reaction. However, altered antigen expression in the IFN-γ treated effectors or IFN-α treated targets did not affect the ultimate degree of CT reaction. This study thus suggests a possible therapeutic efficacy of IFN by reducing the direct elimination of the T-cell precursors in HIV-1 infection.     

Identification of a Cellular Factor That Modulates HIV-1 Programmed Ribosomal Frameshifting

Programmed −1 ribosomal frameshifting (PRF) is a distinctive mode of gene expression utilized by some viruses, including human immunodeficiency virus type 1 (HIV-1), to produce multiple proteins from a single mRNA. −1 PRF induces a subset of elongating ribosomes to shift their translational reading frame by 1 base in the 5′ direction. The appropriate ratio of Gag to Gag-Pol synthesis is tightly regulated by the PRF signal which promotes ribosomes to shift frame, and even small changes in PRF efficiency, either up or down, have significant inhibitory effects upon virus production, making PRF essential for HIV-1 replication. Although little has been reported about the cellular factors that modulate HIV-1 PRF, the cis-acting elements regulating PRF have been extensively investigated, and the PRF signal of HIV-1 was shown to include a slippery site and frameshift stimulatory signal. Recently, a genome-wide screen performed to identify cellular factors that affect HIV-1 replication demonstrated that down-regulation of eukaryotic release factor 1 (eRF1) inhibited HIV-1 replication. Because of the eRF1 role in translation, we hypothesized that eRF1 is important for HIV-1 PRF. Using a dual luciferase reporter system harboring a HIV-1 PRF signal, results showed that depletion or inhibition of eRF1 enhanced PRF in yeast, rabbit reticulocyte lysates, and mammalian cells. Consistent with the eRF1 role in modulating HIV PRF, depleting eRF1 increased the Gag-Pol to Gag ratio in cells infected with replication-competent virus. The increase in PRF was independent of a proximal termination codon and did not result from increased ribosomal pausing at the slippery site. This is the first time that a cellular factor has been identified which can promote HIV-1 PRF and highlights HIV-1 PRF as essential for replication and an important but under exploited antiviral drug target.