RESUMEN
Tat expression is required for efficient human immunodeficiency virus type 1 (HIV-1) reverse transcription. In the present study, we generated a series of 293 cell lines that contained a provirus with a tat gene deletion (Deltatat). Cell lines that contained Deltatat and stably transfected vectors containing either wild-type tat or a number of tat mutants were obtained so that the abilities of these tat genes to stimulate HIV-1 gene expression and reverse transcription could be compared. tat genes with mutations in the amino terminus did not stimulate either viral gene expression or HIV-1 reverse transcription. In contrast, tat mutants in the activation, core, and basic domains of Tat did not stimulate HIV-1 gene expression but markedly stimulated HIV-1 reverse transcription. No differences in the levels of virion genomic RNA or tRNA3Lys were seen in the HIV-1 Deltatat viruses complemented with either mutant or wild-type tat. Finally, overexpression of the Tat-associated kinases CDK7 and CDK9, which are involved in Tat activation of HIV-1 transcription, was not able to complement the reverse transcription defects associated with the lack of a functional tat gene. These results indicate that the mechanism by which tat modulates HIV-1 reverse transcription is distinct from its ability to activate HIV-1 gene expression.
Asunto(s)
Quinasas Ciclina-Dependientes , Productos del Gen tat/fisiología , VIH-1/genética , Transcripción Genética , Secuencia de Aminoácidos , Productos del Gen tat/química , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas Serina-Treonina Quinasas/fisiología , ARN Viral/análisis , Relación Estructura-Actividad , Transfección , Virión/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
The ability of human immunodeficiency virus-1 (HIV-1) to undergo efficient reverse transcription is dependent on a number of parameters. These include the binding of the tRNA(3)(Lys) to the HIV-1 primer binding site and the subsequent interaction with the heterodimeric reverse transcriptase. Recently, we demonstrated that TAR RNA was also necessary for efficient HIV-1 reverse transcription. Given the fact that the Tat protein is involved in the activation of HIV-1 gene expression in conjunction with TAR, we wished to determine whether Tat might also be involved in the control of HIV-1 reverse transcription. HIV-1 virions deleted in the tat gene were unable to initiate reverse transcription efficiently upon infection of peripheral blood mononuclear cells (PBMCs). This defect was not due to decreased amounts of genomic RNA, reverse transcriptase or other HIV-1 proteins which were incorporated into the virion. Following transfection of wild-type but not mutant tat genes into cell lines producing HIV-1 lacking tat, the virions produced could be complemented for defects in reverse transcription upon subsequent infection of PBMCs. In contrast, the defect in reverse transcription seen with HIV-1 lacking the tat gene could not be complemented when the target cells rather than the producer cells contained tat. Viruses lacking tat were also defective in endogenous assays of reverse transcription, although these viruses contained similar levels of reverse transcriptase. These results indicate that the Tat protein, in addition to regulating the level of gene expression, is also important for efficient HIV-1 reverse transcription.
Asunto(s)
Productos del Gen tat/metabolismo , VIH-1/genética , VIH-1/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , ADN Viral/genética , Virus Defectuosos/genética , Eliminación de Gen , Regulación Viral de la Expresión Génica , Productos del Gen tat/genética , Genes tat , Prueba de Complementación Genética , Humanos , Datos de Secuencia Molecular , Transcripción Genética , Transfección , Replicación Viral/genética , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Mutation of either of two critical human immunodeficiency virus type 1 (HIV-1) regulatory proteins, Tat and Rev, results in marked defects in viral replication. Thus, inhibition of the function of one or both of these proteins can significantly inhibit viral growth. In the present study, we constructed a novel transdominant Tat mutant protein and compared its efficiency in inhibiting HIV-1 replication with that of transdominant mutant Rev M10 when these proteins were stably expressed either alone or in combination in T-lymphocyte cell lines. The transdominant Tat mutant protein alone resulted in a modest inhibition of HIV replication, but it was able to enhance the ability of the M10 Rev mutant protein to inhibit HIV-1 replication. These results suggest a possible synergistic effect of these transdominant mutant proteins in inhibiting HIV-1 replication.
Asunto(s)
Productos del Gen rev/fisiología , Productos del Gen tat/fisiología , VIH-1/fisiología , Replicación Viral/fisiología , Secuencia de Aminoácidos , Sitios de Unión , Antígenos CD4/metabolismo , Productos del Gen rev/genética , Productos del Gen tat/genética , Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Linfocitos T/citología , Células Tumorales Cultivadas , Replicación Viral/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression is dependent on the transactivator protein Tat and an RNA element extending from the transcription initiation site to +57 known as TAR. TAR forms a stable RNA secondary structure which is critical for high levels of HIV-1 gene expression and efficient viral replication. Using a genetic approach, we isolated HIV-1 mutants in TAR that were competent for high levels of gene expression but yet were markedly defective for viral replication. Single-cycle infections with these viruses demonstrated that they were defective in the initiation of reverse transcription. Additional mutational analysis revealed a variety of other HIV-1 TAR mutants with the same defective phenotype. Thus, in addition to the well-characterized role of the primer binding site, other RNA elements within the HIV-1 genome are also critical in the regulation of reverse transcription. These studies demonstrate that HIV-1 TAR RNA is a key regulator of the reverse transcription and illustrate how a unique RNA structure can modulate diverse regulatory processes in the HIV-1 life cycle crucial for efficient viral replication.
Asunto(s)
Duplicado del Terminal Largo de VIH , VIH-1/genética , ARN Viral/fisiología , Transcripción Genética , Secuencia de Bases , Línea Celular , Humanos , Datos de Secuencia Molecular , ARN Viral/análisis , ARN Viral/química , Proteínas Virales/análisis , Replicación ViralRESUMEN
The U1 and ACH-2 cell lines are subclones of human monocytic and T-lymphoid cells, respectively, persistently infected with human immunodeficiency virus type 1. These cell lines harbor the viral genome but produce only very low levels of viral progeny, which can be increased by stimulation with agents such as phorbol ester and cytokines. As such, they provide an in vitro model for human immunodeficiency virus type 1 latency. In order to examine the basis for their latent state, we have analyzed the activity of endogenous Tat protein in these cells and investigated the effect on viral replication of the addition of exogenous Tat protein. We find that U1 cells seem to have levels of Tat protein that are suboptimal for long terminal repeat (LTR) transcription, because transcription from a transfected LTR-chloramphenicol acetyltransferase plasmid can be enhanced by cotransfection of a Tat expression plasmid. Furthermore, viral replication can be stimulated in this cell line by incubation with purified Tat protein. In contrast, ACH-2 cells are not limited for LTR-chloramphenicol acetyltransferase transcription by endogenous levels of Tat, and virus production is not increased by the addition of exogenous Tat protein. By semiquantitative PCR analysis of viral RNA, we have demonstrated that Tat protein caused an increase in human immunodeficiency virus RNA expression in U1 cells but had no effect in ACH-2 cells. This suggests that a different mechanism underlies the latent state in U1 and ACH-2 cells.