RESUMO
The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F.
Assuntos
Regiões 5' não Traduzidas/metabolismo , Produtos do Gene vif/metabolismo , HIV-1/genética , HIV-1/metabolismo , RNA Viral/metabolismo , Regiões 5' não Traduzidas/química , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Repetição Terminal Longa de HIV , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA Viral/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência HumanaRESUMO
The main function of Vif is to limit the antiviral activity of APOBEC3G by counteracting its packaging into HIV-1 virions. In this work, we examine the possible functional interactions between Vif, APOBEC3G, and two Src family tyrosine kinases, Fyn and Hck, present in T lymphocytes and in monocyte-macrophages, respectively. By GST pull-down, we show that the SH3 domains of Fyn and Hck, and the corresponding full-length proteins bind Vif of HIV-1. One consequence of this interaction is a reduction in their catalytic activity. Interestingly, we also observed that APOBEC3G can be phosphorylated on tyrosine in the presence of Fyn or Hck, suggesting that both kinases may regulate APOBEC3G function. Accordingly, we demonstrate that in the presence of Fyn or Hck and in the absence of Vif, the overall level of APOBEC3G incorporated into HIV-1 particles is decreased, whereas the level of encapsidation of its phosphorylated form is significantly enhanced.
Assuntos
Produtos do Gene vif/metabolismo , HIV-1/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Tirosina/metabolismo , Quinases da Família src/metabolismo , Desaminase APOBEC-3G , Sítios de Ligação , Citidina Desaminase , Células HeLa , Humanos , Rim/metabolismo , Rim/virologia , Monócitos/metabolismo , Monócitos/virologia , Nucleosídeo Desaminases , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-fyn , Proteínas Proto-Oncogênicas c-hck , Proteínas Repressoras , Linfócitos T/metabolismo , Linfócitos T/virologia , Vírion/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência HumanaRESUMO
APOBEC3G is a cytidine deaminase that limits the replication of many retroviruses. This antiviral host factor is packaged into retrovirus particles, where it targets single-stranded DNA generated during reverse transcription and induces up to 2% of G-to-A mutations, which are lethal for the HIV-1 provirus. Vif protein counteracts this antiviral factor by decreasing its packaging into lentivirus particles. Here, we demonstrate that Nedd4-1, an HECT E3 ubiquitin ligase, interacts with APOBEC3G, through its WW2 and WW3 domains. As a result of this interaction, APOBEC3G undergoes post-translational modification by addition of ubiquitin moieties. Accordingly, we demonstrate that the dominant negative Nedd4-1 C/S form prevents APOBEC3G ubiquitination. Moreover, the packaging of APOBEC3G into Pr55 Gag virus-like particles and into HIV-1 virions is reduced when Nedd4-1 C/S is expressed. During HIV-1 viral production in the presence of APOBEC3G, Nedd4-1 C/S restores partially the infectivity of Deltavif HIV-1. We conclude that the ubiquitination of APOBEC3G by Nedd4-1 favors its targeting to the virus assembly site where APOBEC3G interacts with Gag and is packaged into HIV-1 particles in the absence of Vif.
Assuntos
Proteínas de Transporte/metabolismo , HIV-1/metabolismo , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Ubiquitina/metabolismo , Montagem de Vírus , Desaminase APOBEC-3G , Citidina Desaminase , HIV-1/fisiologia , Células HeLa , Humanos , Imunoprecipitação , Nucleosídeo Desaminases , Proteínas RepressorasRESUMO
To design strategies to purge latent reservoirs of human immunodeficiency virus type 1 (HIV-1), we investigated mechanisms by which a non-tumor-promoting phorbol ester, prostratin, inhibits infection of CD4(+) T lymphocytes and at the same time reactivates virus from latency. CD4(+) T lymphocytes from primary blood mononuclear cells (PBMC) and in blocks of human lymphoid tissue were stimulated with prostratin and infected with HIV-1 to investigate the effects of prostratin on cellular susceptibility to the virus. The capacity of prostratin to reactivate HIV from latency was tested in CD4(+) T cells harboring preintegrated and integrated latent provirus. Prostratin stimulated CD4(+) T cells in an aberrant way. It induced expression of the activation markers CD25 and CD69 but inhibited cell cycling. HIV-1 uptake was reduced in prostratin-stimulated CD4(+) T PBMC and tissues in a manner consistent with a downregulation of CD4 and CXCR4 receptors in these systems. At the postentry level, prostratin inhibited completion of reverse transcription of the viral genome in lymphoid tissue. However, prostratin facilitated integration of the reverse-transcribed HIV-1 genome in nondividing CD4(+) T cells and facilitated expression of already integrated HIV-1, including latent forms. Thus, while stimulation with prostratin restricts susceptibility of primary resting CD4(+) T cells to HIV infection at the virus cell-entry level and at the reverse transcription level, it efficiently reactivates HIV-1 from pre- and postintegration latency in resting CD4(+) T cells.
Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/efeitos dos fármacos , Tecido Linfoide/virologia , Ésteres de Forbol/farmacologia , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos CD4/biossíntese , Divisão Celular/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Lectinas Tipo C , Provírus/efeitos dos fármacos , Infecções por Vírus de RNA , Receptores CXCR4/biossíntese , Receptores de Interleucina-2/biossíntese , Transcrição Gênica/efeitos dos fármacos , Ativação Viral , Integração Viral/efeitos dos fármacosRESUMO
The cytidine deaminase hAPOBEC3G is an antiviral human factor that counteracts the replication of HIV-1 in absence of the Vif protein. hAPOBEC3G is packaged into virus particles and lethally hypermutates HIV-1. In this work, we examine the mechanisms governing hAPOBEC3G packaging. By GST pull-down and co-immunoprecipitation assays, we show that hAPOBEC3G binds to HIV-1 Pr55 Gag and its NC domain and to the RT and IN domains contained in Pr160 Gag-Pol. We demonstrate that the expression of HIV-1 Gag is sufficient to induce the packaging of hAPOBEC3G into Gag particles. Gag-Pol polypeptides containing RT and IN domains, as well as HIV-1 genomic RNA, seem not to be necessary for hAPOBEC3G packaging. Lastly, we show that hAPOBEC3G and its murine ortholog are packaged into HIV-1 and MLV Gag particles. We conclude that the Gag polypeptides from distant retroviruses have conserved domains allowing the packaging of the host antiviral factor APOBEC3G.
Assuntos
Produtos do Gene gag/metabolismo , Vírus da Leucemia Murina/metabolismo , Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Vírion/metabolismo , Desaminase APOBEC-3G , Animais , Citidina Desaminase , Produtos do Gene gag/genética , HIV-1/genética , HIV-1/fisiologia , Células HeLa , Humanos , Vírus da Leucemia Murina/genética , Camundongos , Nucleosídeo Desaminases , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/genética , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras , Vírion/química , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Produtos do Gene pol do Vírus da Imunodeficiência HumanaRESUMO
The viral infectivity factor (Vif), one of the six HIV-1 auxiliary genes, is absolutely necessary for productive infection in primary CD4-positive T lymphocytes and macrophages. Vif overcomes the antiviral function of the host factor APOBEC3G. To better understand this mechanism, it is of interest to characterize cellular proteins that interact with Vif and may regulate its function. Here, we show that Vif binds to hNedd4 and AIP4, two HECT E3 ubiquitin ligases. WW domains present in those HECT enzymes contribute to the binding of Vif. Moreover, the region of Vif, which includes amino acids 20-128 and interacts with the hNedd4 WW domains, does not contain proline-rich stretches. Lastly, we show that Vif undergoes post-translational modifications by addition of ubiquitin both in cells overexpressing Vif and in cells expressing HIV-1 provirus. Vif is mainly mono-ubiquitinated, a modification known to address the Gag precursor to the virus budding site.
Assuntos
Produtos do Gene vif/metabolismo , HIV-1/química , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Western Blotting , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Expressão Gênica , Produtos do Gene vif/genética , Glutationa Transferase/metabolismo , Infecções por HIV/virologia , Células HeLa , Humanos , Ubiquitina-Proteína Ligases Nedd4 , Testes de Precipitina , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Transfecção , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência HumanaRESUMO
Truncated proviruses of variable sizes are present in peripheral blood mononuclear cells (PBMC) of human immunodeficiency virus type 1 (HIV-1)-infected persons and simian immunodeficiency virus (SIV)-infected rhesus macaques. Here, we investigated whether the highly deleted HIV and SIV proviruses are present in infected organisms as multiple copies or whether each truncated provirus is unique. Using end-point dilution, multiple long-distance (LD) DNA PCR assays were run in parallel using DNA extracted from PBMC of seropositive, treatment-naive persons and from lymph nodes of a rhesus monkey inoculated with cloned, full-length SIVmac239 DNA. The PCR products were titrated and mapped. Most truncated proviruses were present in the DNA samples tested as single, nonintegrated molecules that differed from one another in size and/or nucleotide sequence. These results indicate that truncated primate lentiviral sequences found in infected tissues are unique or rare quasispecies that do not replicate significantly.
Assuntos
Soropositividade para HIV/virologia , HIV-1/genética , Provírus/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Animais , Sequência de Bases , DNA Viral/genética , DNA Viral/isolamento & purificação , Deleção de Genes , Variação Genética , Soropositividade para HIV/sangue , Humanos , Leucócitos Mononucleares/virologia , Linfonodos/virologia , Macaca mulatta , Dados de Sequência Molecular , Provírus/isolamento & purificação , Síndrome de Imunodeficiência Adquirida dos Símios/sangue , Integração ViralRESUMO
Persistence of human immunodeficiency virus type 1 (HIV-1) constitutes a major obstacle in the control of HIV-1 infection. Here we investigated whether the CpG methylation of the HIV-1 promoter can directly influence the expression of the HIV-1 genome and thereby contribute to the persistence and latency of HIV-1. The levels of CpG methylation in the promoter of HIV-1 were studied after bisulfite-induced modification of DNA in five Jurkat clonal cell lines transduced by an HIV-1 long terminal repeat (LTR)-driven retroviral vector and expressing enhanced green fluorescent protein (GFP) and in primary resting memory T cells challenged with HIV-1 or with an HIV-1-derived retroviral vector. Basal HIV-1 promoter activities were low or undetectable in three tested HIV-1 LTR-GFP clones, one of which encoded the Tat protein, and they reached medium or high levels in two other clones. The CpG dinucleotide that occurred in a latently infected clonal cell line 240 nucleotides upstream of the transcription start remained methylated after reactivation of HIV-1 transcription with 10 nM phorbol-12-myristate-13-acetate. In two clones showing a medium promoter activity and in resting memory T cells, the HIV-1 LTR was generally not methylated. Our results show that the methylation profiles of the HIV-1 LTR, including those present in latently infected cells, are low and do not correlate with the transcriptional activity. We suggest that, in a noncloned cellular population in which the HIV-1 proviruses are randomly integrated in the human genome, HIV-1 latency is imperfectly controlled by CpG methylation and is inherently accompanied by residual replication.
Assuntos
DNA Viral/genética , DNA Viral/metabolismo , HIV-1/genética , HIV-1/metabolismo , Sequência de Bases , Ilhas de CpG , Metilação de DNA , Vetores Genéticos , Proteínas de Fluorescência Verde , Repetição Terminal Longa de HIV , Humanos , Memória Imunológica , Técnicas In Vitro , Células Jurkat , Proteínas Luminescentes/genética , Regiões Promotoras Genéticas , Provírus/genética , Provírus/metabolismo , Supressão Genética , Linfócitos T/imunologia , Transdução Genética , Ativação Viral/genética , Integração Viral/genéticaRESUMO
Infection of epithelial colon carcinoma cell line HT29 with human immunodeficiency virus type 1 (HIV-1) NDK, a subtype D virus highly cytopathic for CD4-positive lymphocytes, results in the selection of HIV-1 variants, 1000 times more infectious for CD4(-) intestinal cells than the parental virus. Here, we demonstrate that the envelope gene of intestinal cell-adapted virus conferred to recombinant clone HIV-1 iNDK the ability to utilize CXCR4 without CD4 while retaining its tropism for CD4 lymphocytes. Among the major genetic changes required for infection of intestinal cells and CD4 independence, two potential N-glycosylation sites appeared as a result of the extension of five amino acids in the V1/V2 region and three amino acid changes ((296)KYT --> (296)NNI) were identified in the V3 loop of HIV-1 iNDK gp120. Our studies suggest that CD4-independent use of CXCR4 can be mediated by different adaptive changes related to the microenvironment of CD4(-) cell.
Assuntos
Antígenos CD4/fisiologia , HIV-1/fisiologia , Intestinos/virologia , Receptores CXCR4/fisiologia , Replicação Viral , Adaptação Fisiológica , Sequência de Aminoácidos , Linhagem Celular , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Humanos , Dados de Sequência Molecular , Fenótipo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/fisiologiaRESUMO
OBJECTIVE: The mechanisms of HIV-triggered immunodeficiency were examined by determining the segregation of R5 and X4 HIV-1 variants into memory T cell subsets expressing differentially a homing receptor, CD62L-selectin, in human lymphoid tissue. METHODS: Subpopulations of CD3 and intracellular p24 gag-positive cells in human lymphoid tissue infected ex vivo with X4 HIV-1 variant NL4-3 and R5 HIV-1 variant AD8 were analysed for expression of the T cell memory markers CD45RO and CD45RA, the T cell homing receptor for lymphoid tissue CD62L, and the HIV-1 coreceptors CCR5 and CXCR4. RESULTS: Memory CD4 T cells were the predominant targets for productive infection of lymphoid tissue ex vivo with both R5 and X4 HIV-1. R5 HIV-1 predominantly infected CD62L-negative memory T cells, which selectively express CCR5. In contrast, X4 HIV-1 variants predominantly infected CD62L+ memory T cells, although CXCR4 coreceptor was equally expressed by memory T cells of both CD62L-positive and CD62L-negative subsets. A high proportion of X4 HIV-1, but not of R5 HIV-1, productively infected T cells, displayed a CD45RA+CD45RO+ phenotype. CONCLUSION: The selective expression of the CCR5 coreceptor by CD62L-negative terminally differentiated memory T cells correlates with the preferential productive infection of these cells with the R5 HIV-1 variant. The predominance of X4 HIV-1 variants in less-differentiated memory T cells may be related to their recent activation state, as suggested by the coexpression of both CD45RA and CD45RO molecules on their surface. Differential homing of CD62L-positive and CD62L-negative cells suggests different routes of dissemination of X4 and R5 viruses.
Assuntos
HIV-1/genética , HIV-1/imunologia , Memória Imunológica , Selectina L/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/virologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Citometria de Fluxo , HIV-1/classificação , HIV-1/isolamento & purificação , Humanos , Selectina L/análise , Antígenos Comuns de Leucócito/análise , Receptores CCR5/análise , Subpopulações de Linfócitos T/metabolismoRESUMO
Interactions of human immunodeficiency virus type 1 (HIV-1) Vif protein with various forms of Gag and Gag-Pol precursors expressed in insect cells were investigated in vivo and in vitro by co-encapsidation, co-precipitation and viral protease (PR)-mediated Gag processing assays. Addressing of Gag to the plasma membrane, its budding as extracellular virus-like particles (VLP) and the presence of the p6 domain were apparently not required for Vif encapsidation, as non-N-myristoylated Deltap6-Gag and Vif proteins were co-encapsidated into intracellular VLP. Encapsidation of Vif occurred at significantly higher copy numbers in extracellular VLP formed from N-myristoylated, budding-competent Gag-Pol precursors harbouring an inactive PR domain or in chimaeric VLP composed of Gag and Gag-Pol precursors compared with the Vif content of Pr55Gag VLP. Vif encapsidation efficiency did not seem to correlate directly with VLP morphology, since these chimaeric VLP were comparable in size and shape to Pr55Gag VLP. Vif apparently inhibited PR-mediated Pr55Gag processing in vitro, with preferential protection of cleavage sites at the MA-CA and CA-NC junctions. Vif was resistant to PR action in vitro under conditions that allowed full Gag processing, and no direct interaction between Vif and PR was detected in vivo or in vitro. This suggested that inhibition by Vif of PR-mediated Gag processing resulted from interaction of Vif with the Gag substrate and not with the enzyme. Likewise, the higher efficiency of Vif encapsidation by Gag-Pol precursor compared with Pr55Gag was probably not mediated by direct binding of Vif to the Gag-Pol-embedded PR domain, but more likely resulted from a particular conformation of the Gag structural domains of the Gag-Pol precursor.