RESUMEN
HIV-1 is able to infect nondividing cells productively in part because the postentry viral nucleoprotein complexes are actively imported into the nucleus. In this manuscript, we identify a novel nuclear localization signal (NLS) in the viral integrase (IN) protein that is essential for virus replication in both dividing and nondividing cells. The IN NLS stimulates the efficient nuclear accumulation of viral DNA as well as virion-derived IN protein during the initial stages of infection but is dispensable for catalytic function. Because this NLS is required for infection irrespective of target cell proliferation, we suggest that interactions between uncoated viral nucleoprotein complexes and the host cell nuclear import machinery are critical for HIV-1 infection of all cells.
Asunto(s)
Infecciones por VIH/enzimología , Integrasa de VIH/metabolismo , VIH-1 , Señales de Localización Nuclear/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Secuencia de Aminoácidos , Núcleo Celular/virología , Células Cultivadas , ADN Viral/biosíntesis , Integrasa de VIH/química , Integrasa de VIH/genética , Células HeLa , Humanos , Leucocitos Mononucleares/virología , Mutagénesis Sitio-Dirigida , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/fisiología , Estructura Terciaria de Proteína , Alineación de Secuencia , Fracciones Subcelulares/química , Linfocitos T/virologíaRESUMEN
We have developed a rapid-turnover culture system where the life span of a human immunodeficiency virus type 1-infected cell is controlled by periodic addition of a cytotoxic agent, mitomycin C. These mitomycin C-exposed cells are cocultured with a constant number of uninfected cells as new targets for the virus. Passage of the virus-infected cells under these conditions led to the emergence of a viral variant that was able to replicate efficiently in this culture system. After biologic and molecular cloning, we were able to identify a single frameshift mutation in the vpu open reading frame that was sufficient for growth of the mutant virus in the rapid-turnover assay. This virus variant spread more efficiently by cell-to-cell transfer than the parental virus did. Electron micrographs of cells infected with the delta vpu virus revealed a large number of mature viral capsids attached to the plasma membrane. The presence of these mature virus particles on the cell surface led to enhanced fusion and formation of giant syncytia with uninfected cells. Enhanced cell-to-cell transfer of the delta vpu virus provides an explanation for the survival of this mutant virus in the rapid-turnover culture system. The in vitro rapid-turnover culture system is a good representation of the in vivo turnover kinetics of infected cells and their continual replacement by host lymphopoietic mechanisms.
Asunto(s)
VIH-1/fisiología , Replicación Viral , Secuencia de Aminoácidos , Secuencia de Bases , Genes env , Genotipo , Proteínas del Virus de la Inmunodeficiencia Humana , Humanos , Células Jurkat , Datos de Secuencia Molecular , Mutación , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
Plasma HIV RNA is a useful surrogate marker for predicting HIV-1 disease progression in infected individuals but provides no information regarding the infectious viral titer. Traditional assays of infectious HIV-1 are, however, time consuming, insensitive, use non-standardized reagents and are subject to selection bias introduced by prolonged cultivation. In this pilot study infectious HIV-1 was detected directly in patient plasma using the indicator line HeLa-CD4-CCR5-LTR/beta-gal in a centrifugation-culture method. Replication competent HIV-1 was identified within 2 days of tissue culture inoculation in six (26%) of 23 plasma specimens. The capability of a new cell line, MT4-CCR5-tat, to amplify plasma HIV-1 was also tested. HIV was cultivated from ten (71%) of 14 specimens using MT4-CCR5-tat cells before titering the virus with the indicator cell assay. Using these stable cell lines in refined versions of this assay it may be feasible to develop rapid, simple methods for titering infectious plasma HIV-1 and for testing the susceptibility of the virus to antiretroviral drugs.
Asunto(s)
Sangre/virología , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , VIH-1/fisiología , Antígenos CD4/metabolismo , Línea Celular , Centrifugación , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Células HeLa , Humanos , Proyectos Piloto , ARN Viral/sangre , Receptores CCR5/metabolismo , Viremia/virología , Cultivo de Virus , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Some viruses encode proteins that promote cell proliferation while others, such as the human immunodeficiency virus (HIV), encode proteins that prevent cell division. It has been hypothesized that the selective advantage determining which strategy evolves depends on the ability of the virus to induce a cellular environment which maximizes both virus production and cell life span. In HIV, the protein that causes cell cycle arrest is Vpr. In this paper, we develop a mathematical model, based on difference equations, to study the competition between two genotypes of HIV - one that encodes this protein (Vpr+) and one that does not (Vpr-). In particular, we are interested in parameters that could be different between the in vitro condition, where the Vpr- genotype dominates, and the in vivo condition, where the Vpr+ genotype dominates. Our model indicates that the infected cell death-rate, the viral half-life, and the dynamics of the target cell population all effect the competition dynamics between the Vpr+ and Vpr- viral genotypes. Perturbing any of these parameters from the in vitro estimates while holding the others fixed has no affect on the competition outcome, i. e., the Vpr- genotype dominates. Perturbing the infected cell death-rate and the target cell source causes a switch in competitive outcome, although not necessarily at values, which represent the in vivo condition. Adding a perturbation in the viral half-life from in vitro to in vivo condition results in a switch of the competitive advantage from the Vpr- genotype to the Vpr+ genotype with parameters for all three mechanisms set to estimated in vivo values.
Asunto(s)
Simulación por Computador , Productos del Gen vpr/genética , Infecciones por VIH/virología , VIH-1/genética , Modelos Biológicos , División Celular/fisiología , Semivida , Humanos , ARN Viral/química , Factores de Tiempo , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
The advantage that vectors derived from the human immunodeficiency virus offer over other gene-delivery vehicles is their ability to transduce non-proliferative tissues. But a report by Park et al. suggests that HIV-based vectors are not able to infect all non-dividing cells and that host cell activation may influence efficiency of gene delivery.
Asunto(s)
Lentivirus/fisiología , Vectores Genéticos , Lentivirus/genética , Transducción GenéticaRESUMEN
Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) transiently arrests cells in the G2 phase of the cell cycle and is a weak transcriptional transactivator. We found that Vpr increased HIV-1 long terminal repeat (LTR) activity in all cells examined but, when expressed at high levels, decreased HIV-1 LTR expression due to cytotoxic effects. Moreover, Vpr-mediated enhancement of HIV-1 LTR-driven transcription was observed in cycling primary human CD4(+) T cells but not in terminally differentiated, noncycling primary human macrophages. In single-round infection experiments using primary human CD4(+) T cells, proviral clones expressing either wild-type Vpr or Vpr mutants that retained the ability to cause a G2 arrest replicated to higher levels than proviruses lacking Vpr or expressing mutants of Vpr that did not cause an arrest. In support of the hypothesis that enhancement of HIV-1 LTR transcription by Vpr is an indirect effect of the ability of Vpr to delay cells in G2, counterflow centrifugal elutriation of cells into different phases of the cell cycle demonstrated that HIV-1 LTR expression was highest in G2. Finally, the ability of Vpr to upregulate viral transcription was dependent on a minimal promoter containing a functional TATA box and an enhancer.
Asunto(s)
Regulación Viral de la Expresión Génica , Productos del Gen vpr/metabolismo , Duplicado del Terminal Largo de VIH , VIH-1/genética , Linfocitos T/citología , Ciclo Celular , Línea Celular Transformada , Células Cultivadas , Fase G2 , Productos del Gen vpr/genética , VIH-1/fisiología , Células HeLa , Humanos , Células Jurkat , Activación Transcripcional , Células Tumorales Cultivadas , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
It has been hypothesized that protection against human immunodeficiency virus (HIV)-1 infection may result from either acquired host immunity, inheritance of a dysfunctional CCR5 HIV-1 coreceptor, or a low or attenuated virus inoculum. Thirty-seven HIV-1-uninfected persons engaging in repeated high-risk sexual activity with an HIV-1-infected partner were prospectively studied to determine the contribution of these factors in protecting against HIV-1 transmission. More than one-third (13/36) demonstrated HIV-1-specific cytotoxicity, and this activity significantly correlated with the wild type CCR5 genotype (P=.03). Only 1 subject (3%) demonstrated the homozygous CCR5 32-bp deletion (Delta32/Delta32). Median plasma HIV-1 RNA levels from 18 HIV-1-infected sex partners were not statistically different from those of matched infected control patients. These results indicate that inheritance of the Delta32 CCR5 mutation does not account for the majority of persistently HIV-1-resistant cases, and the presence of cellular immunity in these persons suggests either undetected infection or protective immunity.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , Seronegatividad para VIH/genética , Seronegatividad para VIH/inmunología , VIH-1/inmunología , Receptores CCR5/genética , Linfocitos T/inmunología , Adulto , Femenino , Antígenos VIH/inmunología , Heterosexualidad , Homosexualidad Masculina , Humanos , Inmunidad Celular , Inmunidad Innata , Activación de Linfocitos , Masculino , Persona de Mediana Edad , ARN Viral/sangre , Receptores CCR5/fisiología , Asunción de Riesgos , Eliminación de Secuencia , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Apoptosis contributes to the loss of CD4 cells during human immunodeficiency virus type 1 (HIV-1) infection. Although the product of the env gene, gp160/gp120, is known to play a role in cell death mediated by HIV-1, the role of other HIV-1 genes in the process is unclear. We found that HIV-1 lacking the env gene (HIVDeltaenv) still induced apoptosis in T-cell lines and primary CD4 T cells. The ability to induce apoptosis was attributable to Tat, a viral regulatory protein. Tat induction of apoptosis was separate from the transactivation function of Tat, required expression of the second exon of Tat, and was associated with the increased expression and activity of caspase-8 (casp-8), a signaling molecule in apoptotic pathways. Moreover, induction of apoptosis could be prevented by treating cells with an inhibitor of casp-8. In addition, we show that HIV-1Deltaenv infection and Tat expression increased the sensitivity of cells to Fas-mediated apoptosis, an apoptotic pathway that signals via casp-8. The up-regulation of casp-8 by HIV-1 Tat expression may contribute to the increased apoptosis and sensitivity to apoptotic signals observed in the cells of HIV-1-infected persons.
Asunto(s)
Apoptosis , Caspasas/biosíntesis , Productos del Gen tat/fisiología , VIH-1/fisiología , Linfocitos T CD4-Positivos/virología , Caspasa 8 , Caspasa 9 , Proteína Ligando Fas , Humanos , Células Jurkat , Glicoproteínas de Membrana/biosíntesis , Secuencias Repetidas Terminales , Regulación hacia Arriba , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Human immunodeficiency virus type-1 (HIV-1) manipulates fundamental host cell processes in sophisticated ways to achieve optimum replicative efficiency. Recent studies have provided new details on the molecular interactions of HIV-1 with its host cell. For example, HIV-1 encodes a protein that regulates transcriptional elongation by interacting with a cellular cyclin-dependent kinase, another that activates the specific nuclear export of viral RNA, and several others that affect the intracellular trafficking of viral and host cell proteins. Detailed analysis of the interplay between these viral proteins and normal cellular activities has provided new insights into central questions of virology and host cell biology.
Asunto(s)
Genes Virales , Infecciones por VIH/virología , VIH-1/genética , Transactivadores/fisiología , Proteínas Reguladoras y Accesorias Virales/fisiología , Animales , Productos del Gen nef/fisiología , Productos del Gen rev/fisiología , Productos del Gen tat/fisiología , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana , Humanos , Proteínas de la Membrana/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Transactivadores/genética , Transcripción Genética , Proteínas Reguladoras y Accesorias Virales/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaAsunto(s)
Vacunas contra el SIDA , Ensayos Clínicos como Asunto , Vacunas contra el SIDA/inmunología , Síndrome de Inmunodeficiencia Adquirida/prevención & control , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Humanos , National Institutes of Health (U.S.) , Estados UnidosRESUMEN
HIV-1 Vpr promotes nuclear entry of viral nucleic acids in nondividing macrophages and also causes a G2 cell-cycle arrest. Consistent with its role in nuclear transport, we show Vpr localizes to the nuclear envelope in both human and yeast cells. Like the importin-beta subunit of the nuclear import receptor, Vpr also interacts with the yeast importin-alpha subunit and nucleoporins. Moreover, overexpression of either Vpr or importin-beta in yeast blocks nuclear transport of mRNAs. A mutant form of Vpr (Vpr F34I) that does not localize at the nuclear envelope, or bind to importin-alpha and nucleoporins, renders HIV-1 incapable of infecting macrophages efficiently. Vpr F34I, however, still causes a G2 arrest, demonstrating that the dual functions of Vpr are genetically separable. Our data suggest Vpr functionally resembles importin-beta in nuclear import of the HIV-1 pre-integration complex and this function is essential for the role of Vpr in macrophage infection, but not G2 arrest.
Asunto(s)
Núcleo Celular/metabolismo , Productos del Gen vpr/fisiología , Macrófagos/virología , Membrana Nuclear/metabolismo , Núcleo Celular/virología , Células Cultivadas , Técnica del Anticuerpo Fluorescente Indirecta , Fase G2/genética , Productos del Gen vpr/genética , Productos del Gen vpr/metabolismo , VIH-1/genética , VIH-1/patogenicidad , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Mutación Puntual , ARN Mensajero/análisis , Linfocitos T/virología , Levaduras/metabolismo , alfa Carioferinas , beta Carioferinas , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) encodes a protein, called Vpr, that prevents proliferation of infected cells by arresting them in G2 of the cell cycle. This Vpr-mediated cell-cycle arrest is also conserved among highly divergent simian immunodeficiency viruses, suggesting an important role in the virus life cycle. However, it has been unclear how this could be a selective advantage for the virus. Here we provide evidence that expression of the viral genome is optimal in the G2 phase of the cell cycle, and that Vpr increases virus production by delaying cells at the point of the cell cycle where the long terminal repeat (LTR) is most active. Although Vpr is selected against when virus is adapted to tissue culture, we show that selection for Vpr function in vivo occurs in both humans and chimpanzees infected with HIV-1. These results suggest a novel mechanism for maximizing virus production in the face of rapid killing of infected target cells.
Asunto(s)
Ciclo Celular/fisiología , Productos del Gen vpr/biosíntesis , VIH-1/fisiología , Animales , División Celular , Línea Celular , Fase G2 , Productos del Gen vpr/fisiología , Infecciones por VIH/virología , Humanos , Células Jurkat , Cinética , Modelos Biológicos , Pan troglodytes , Reacción en Cadena de la Polimerasa , Provirus/fisiología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T , Transfección , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
Expression of the auxiliary human immunodeficiency virus type 1 (HIV-1) protein Vpr causes arrest of primate host cells in G2. Expression of this protein in budding yeast has been previously reported to cause growth arrest and a large-cell phenotype. Investigation of the effect of Vpr expression in budding yeast, reported here, showed that it causes disruption of the actin cytoskeleton. Expression of HSP42, the gene for a small heat shock protein (sHSP), from a high-copy-number plasmid reversed this effect. The sHSPs are induced by exposure of cells to thermal, osmotic, and oxidative stresses and to mitogens. In animal cells, overexpression of sHSPs causes increased resistance to stress and stabilization of actin stress fibers. Yeast cells subjected to mild stress, such as shifting from 23 to 39 degrees C, arrest growth and then resume cell division. Growth arrest is accompanied by transient disorganization of the cytoskeleton. Yeast in which the HSP42 gene was disrupted and which was subjected to moderate thermal stress reorganized the actin cytoskeleton more slowly than did wild-type control cells. These results demonstrate that in yeast, as in metazoan cells, sHSPs promote maintenance of the actin cytoskeleton.
Asunto(s)
Productos del Gen vpr/fisiología , Proteínas de Choque Térmico/fisiología , Proteínas de Saccharomyces cerevisiae , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Regulación Fúngica de la Expresión Génica , Regulación Viral de la Expresión Génica , Trastornos de Estrés por Calor/patología , Saccharomyces cerevisiaeRESUMEN
The human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents infected cells from passing through mitosis by arresting them in the G2 phase of the cell cycle. Vpr is conserved among all primate lentiviruses, suggesting an important role in the virus life cycle. Moreover, in this study we show that the ability to cause cell cycle arrest is also conserved in Vpr proteins from a wide variety of both tissue culture-passaged and uncultured human (HIV-1 and HIV-2), sooty mangabey (simian immunodeficiency virus SIV(SM)), African green monkey (SIV(AGM)), and Sykes' monkey (SIV(SYK)) isolates. However, this property is cell type specific and appears to depend on the particular primate species from which the cells are derived. SIV(AGM) and SIV(SYK) Vpr proteins are capable of arresting African green monkey cells but are completely inactive in human cells. By contrast, HIV-1, HIV-2, and SIV(SM) Vpr proteins function in both simian and human cell types, although SIV(SM) Vpr functions more efficiently in simian cells than it does in human cells. Neither differential protein stability nor subcellular localization explains the species-specific activities of these proteins. These results thus suggest that Vpr exerts its G2 arrest function by interacting with cellular factors that have evolved differently among the various primate species.
Asunto(s)
Ciclo Celular , Fase G2 , Productos del Gen vpr/fisiología , VIH-1/genética , VIH-2/genética , Virus de la Inmunodeficiencia de los Simios/genética , Animales , Secuencia de Bases , Evolución Biológica , Compartimento Celular , Células Cultivadas , Chlorocebus aethiops , Humanos , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
The Vpr protein encoded by human immunodeficiency virus type 1 (HIV-1) is important for growth of virus in macrophages and prevents infected cells from passing into mitosis (G2 arrest). The cellular target for these functions is not known, but Vpr of HIV-1 and the related Vpr from simian immunodeficiency virus of sooty mangabeys (SIV(SM)) bind the DNA repair enzyme UNG, while the Vpx protein of SIV(SM) does not. Nonetheless, a mutational analysis of Vpr showed that binding to UNG is neither necessary nor sufficient for the effect of Vpr on the cell cycle.
Asunto(s)
Ciclo Celular , ADN Glicosilasas , Productos del Gen vpr/metabolismo , VIH-1/metabolismo , N-Glicosil Hidrolasas/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Secuencia de Aminoácidos , Animales , Cercocebus/microbiología , Fase G2 , Humanos , Datos de Secuencia Molecular , Mutagénesis , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Uracil-ADN Glicosidasa , Proteínas Reguladoras y Accesorias Virales/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
CCR5 and CXCR4 are the two major coreceptors that have been identified for human immunodeficiency virus (HIV) entry. We have modified several beta-galactosidase-based HIV indicator cell lines to express CCR5 and/or CXCR4. Using these new reagents, we have been able to detect all primary isolates tested using one or both of these cell lines. However, there is large variation in the absolute viral infectivity among primary strains. Furthermore, all HIV strains are capable of causing syncytia in the indicator cells when the coreceptor is present regardless of whether they had previously been characterized as "syncytia-inducing" or "non-syncytium-inducing."
Asunto(s)
VIH-1/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Receptores de Citocinas/metabolismo , Receptores del VIH/metabolismo , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Animales , Línea Celular , Células Gigantes , VIH-1/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/genética , Receptores CCR5 , Receptores CXCR4 , Receptores de Citocinas/genética , Receptores del VIH/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismoRESUMEN
The vpr genes of human and simian immunodeficiency viruses (HIV/SIV) encode proteins which are packaged in the virus particle. HIV-1 Vpr has been shown to mediate the nuclear import of viral reverse transcription complexes in non-dividing target cells (e.g. terminally differentiated macrophages), and to alter the cell cycle and proliferation status of the infected host cell. Members of the HIV-2/SIV(SM) group encode, in addition to Vpr, a related protein called Vpx. Because these two proteins share considerable sequence similarity, it has been assumed that they also exhibit similar functions. Here, we report that the functions of Vpr and Vpx are distinct and non-redundant, although both proteins are components of the HIV-2/SIV(SM) virion and reverse transcription complex. Characterizing SIV(SM) proviruses defective in one or both genes, we found that Vpx is both necessary and sufficient for the nuclear import of the viral reverse transcription complex. In contrast, Vpr, but not Vpx, inhibited the progression of infected host cells from the G2 to the M phase of the cell cycle. Thus, two independent functions of the HIV-1 Vpr protein are encoded by separate genes in HIV-2/SIV(SM). This segregation is consistent with the conservation of these genes in HIV-2/SIV(SM) evolution, and underscores the importance of both nuclear transport and cell cycle arrest functions in primate lentivirus biology.