RESUMO
HIV-1 Vpr is a viral accessory protein that activates ATR through the induction of DNA replication stress. ATR activation results in cell cycle arrest in G2 and induction of apoptosis. In the present study, we investigate the role of the ubiquitin/proteasome system (UPS) in the above activity of Vpr. We report that the general function of the UPS is required for Vpr to induce G2 checkpoint activation, as incubation of Vpr-expressing cells with proteasome inhibitors abolishes this effect. We further investigated in detail the specific E3 ubiquitin ligase subunits that Vpr manipulates. We found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase. The carboxy-terminal domain Vpr(R80A) mutant, which is able to bind DCAF1, is inactive in checkpoint activation and has dominant-negative character. In contrast, the mutation Q65R, in the leucine-rich domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative behavior. Thus, the interaction of Vpr with DCAF1 is required, but not sufficient, for Vpr to cause G2 arrest. We propose that Vpr recruits, through its carboxy terminal domain, an unknown cellular factor that is required for G2-to-M transition. Recruitment of this factor leads to its ubiquitination and degradation, resulting in failure to enter mitosis.
Assuntos
Produtos do Gene vpr/fisiologia , HIV-1/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Linhagem Celular , Fase G2 , Produtos do Gene vpr/isolamento & purificação , HIV-1/efeitos dos fármacos , Células HeLa/citologia , Células HeLa/fisiologia , Células HeLa/virologia , Humanos , Rim , Oligopeptídeos/farmacologia , RNA Interferente Pequeno/genética , RNA Viral/genética , Transfecção , Replicação Viral , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Human immunodeficiency virus (HIV) Vpr contributes to nuclear import of the viral pre-integration complex and induces G(2) cell cycle arrest. We describe the production of synthetic Vpr that permitted the first studies on the structure and folding of the full-length protein. Vpr is unstructured at neutral pH, whereas under acidic conditions or upon addition of trifluorethanol it adopts alpha-helical structures. Vpr forms dimers in aqueous trifluorethanol, whereas oligomers exist in pure water. (1)H NMR spectroscopy allows the signal assignment of N- and C-terminal amino acid residues; however, the central section of the molecule is obscured by self-association. These findings suggest that the in vivo folding of Vpr may require structure-stabilizing interacting factors such as previously described interacting cellular and viral proteins or nucleic acids. In biological studies we found that Vpr is efficiently taken up from the extracellular medium by cells in a process that occurs independent of other HIV-1 proteins and appears to be independent of cellular receptors. Following cellular uptake, Vpr is efficiently imported into the nucleus of transduced cells. Extracellular addition of Vpr induces G(2) cell cycle arrest in dividing cells. Together, these findings raise the possibility that circulating forms of Vpr observed in HIV-infected patients may exert biological effects on a broad range of host target cells.
Assuntos
Núcleo Celular/metabolismo , Fase G2 , Produtos do Gene vpr/química , Produtos do Gene vpr/metabolismo , HIV-1/química , Sequência de Aminoácidos , Western Blotting , Núcleo Celular/virologia , Dicroísmo Circular , Dimerização , Produtos do Gene vpr/síntese química , Produtos do Gene vpr/isolamento & purificação , HIV-1/metabolismo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/citologia , Macrófagos/metabolismo , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Dobramento de Proteína , Estrutura Quaternária de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Transporte Proteico , Espalhamento de Radiação , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trifluoretanol/farmacologia , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
The Vpr protein, encoded by the human immunodeficiency virus type 1 (HIV-1) genome, is one of the nonstructural proteins packaged in large amounts into viral particles. We have previously reported that Vpr associates with the DNA repair enzyme uracil DNA glycosylase (UDG). In this study, we extended these observations by investigating whether UDG is incorporated into virions and whether this incorporation requires the presence of Vpr. Our results, with highly purified viruses, show that UDG is efficiently incorporated either into wild-type virions or into Vpr-deficient HIV-1 virions, indicating that Vpr is not involved in UDG packaging. Using an in vitro protein-protein binding assay, we reveal a direct interaction between the precursor form of UDG and the viral integrase (IN). Finally, we demonstrate that IN-defective viruses fail to incorporate UDG, indicating that IN is required for packaging of UDG into virions.
Assuntos
DNA Glicosilases , Reparo do DNA , Produtos do Gene vpr/metabolismo , HIV-1/metabolismo , N-Glicosil Hidrolases/metabolismo , Linhagem Celular , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Produtos do Gene vpr/isolamento & purificação , Integrase de HIV/metabolismo , Humanos , N-Glicosil Hidrolases/isolamento & purificação , Ligação Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Uracila-DNA Glicosidase , Vírion , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
The Vpr protein of human immunodeficiency virus type 1 (HIV-1) is incorporated into the virion by the Gag polyprotein precursor Pr55gag. The importance of the p6gag sequence at the C-terminal end of Pr55gag has a crucial role in Vpr incorporation. To identify the Gag sequences directly involved in Vpr binding, we compared the Vpr binding affinities of the 71 amino acid nucleocapsid protein p7, the C-terminal peptide (35-71) p7C and p6gag by affinity chromatography. p7 and p7C have the strongest Vpr binding activities compared to p6gag. These results suggest that the nucleocapsid protein and its C-terminal domain may be important for the incorporation of Vpr into the mature HIV-1 virion and the subsequent localisation of viral nucleic acid to the cell nucleus by Vpr.
Assuntos
Capsídeo/metabolismo , Produtos do Gene vpr/metabolismo , HIV-1/metabolismo , Proteínas do Core Viral/metabolismo , Western Blotting , Capsídeo/química , Capsídeo/isolamento & purificação , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Produtos do Gene gag/isolamento & purificação , Produtos do Gene gag/metabolismo , Produtos do Gene vpr/química , Produtos do Gene vpr/isolamento & purificação , Humanos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas do Core Viral/química , Proteínas do Core Viral/isolamento & purificação , Vírion/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Mutations of the cK-ras gene which confer oncogenic properties to the corresponding encoded small G protein, occur in 30 to 50% of the human colonic adenocarcinomas. Overexpression of Rap 1A, a member of the Ras family, in K-ras transformed fibroblasts, reverts the transforming properties of the oncogene. This indicates that Rap 1A may exert antagonistic properties towards the K-Ras protein. In this respect, we have been interested in comparing Rap 1 expression in the human adenocarcinoma cell line HT29 and in safe or pathological tissues deriving from the human colonic epithelium. In the human adenocarcinoma cells HT29, several immunoreactive forms of Rap 1 of 71 kDa, 47 kDa, 40 kDa and 24 kDa are detected. Extraction in Triton X-114 allows separation of HT29 cell proteins on the basis of their differential hydrophobic properties. Parallelly, proteins were separated in crude cytosolic or membrane fractions. Most of the immunoreactive material corresponding to the 24 kDa band may contain hydrophobic and membrane associated components. The molecular nature of the higher size components is also discussed here. In the tissues, the 47 kDa form which is common in all of the safe and pathological samples considered, appears to be specifically expressed in the colon. Besides, the 71, 68 and 24 kDa were found in pathological tissues. High expression of Rap 1 was demonstrated to be correlated with cell differentiation in the safe colonic epithelium and in the adenomas. Cloning of the Rap 1A cDNA is now in progress in the laboratory, using PCR detection in an HT29 expression library.
Assuntos
Colo/citologia , Produtos do Gene vpr/isolamento & purificação , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , DNA Complementar/metabolismo , Células Epiteliais , Epitélio/metabolismo , Epitélio/patologia , Humanos , Reação em Cadeia da PolimeraseAssuntos
Produtos do Gene vpr/isolamento & purificação , Proteínas ras/isolamento & purificação , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Cromatografia de Afinidade , Epitopos/genética , Produtos do Gene vpr/genética , Produtos do Gene vpr/imunologia , Guanosina Trifosfato/metabolismo , Humanos , Dados de Sequência Molecular , Octoxinol , Polietilenoglicóis , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Spodoptera , Proteínas ras/genética , Proteínas ras/imunologiaRESUMO
The human immunodeficiency virus, type 1 (HIV-1) genome encodes a 15-kDa accessory gene product, Vpr, that is essential for virus replication in primary monocytes/macrophages. Being present in the virion, Vpr is believed to function in the early phases of HIV-1 replication, including nuclear migration of the pre-integration complex and/or transcription of the provirus genome. By gel filtration analysis of highly purified Vpr protein and its mutants, we demonstrate that HIV-1 Vpr exists as an oligomer. The N-terminal domain of Vpr (amino acids (aa) 1-42) is sufficient for oligomerization; however, deletion of aa 36-76 from Vpr disrupts oligomerization, suggesting that aa 36-42 are critical for Vpr oligomerization. As a result of Vpr oligomerization, basic aa residues within Vpr aa 1-73 are highly resistant to trypsin digestion, while those within Vpr aa 74-96 are easily accessible. Mutations within the leucine-/isoleucine-rich domain (aa 60-81), which was previously identified to be involved in Vpr interaction with a host cellular protein, rendered Arg62 more susceptible to trypsin digestion. Thus, the Vpr oligomeric structure must be extended into this domain. These results suggest a novel feature of HIV-1 Vpr that may be important for its functions.
Assuntos
Produtos do Gene vpr/metabolismo , HIV-1/metabolismo , Sequência de Aminoácidos , Biopolímeros , Western Blotting , Cromatografia em Gel , Produtos do Gene vpr/química , Produtos do Gene vpr/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Tripsina/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
vpr is an accessory gene of human immunodeficiency virus I (HIV-I). Although unnecessary for viral replication in T cell lines, growing evidence suggests that it is essential for virus replication in monocytes/macrophages and for replication in vivo. We expressed HIV-I vpr in Escherichia coli and purified Vpr by affinity chromatography. In a coprecipitation assay, the purified Vpr interacted specifically with a cellular protein designated as Vpr-interacting protein, or RIP. Mutational analysis suggested that this interaction required a domain rich in leucine/isoleucine residues and highly conserved between HIV-I and SIVmac Vprs. During transient expression in mammalian cells, HIV-I Vpr was localized in the nucleus. However, mutational analysis failed to identify in Vpr a typical nuclear localization signal rich in basic amino acid residues. Instead, Vpr nuclear localization seemed to correlate with Vpr interaction with RIP. Mutations in the C-terminal 20-amino acid region containing a cryptic nuclear localization signal did not abolish Vpr nuclear localization or interaction with RIP, whereas point mutations in the leucine/isoleucine-rich domain abolished Vpr interaction with RIP and rendered Vpr unstable during transient expression. These results suggest that RIP may be involved in Vpr function.