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Tat-SF1 is not required for Tat transactivation but does regulate the relative levels of unspliced and spliced HIV-1 RNAs.

Miller HB, Saunders KO, Tomaras GD, Garcia-Blanco MA - PLoS ONE (2009)

Bottom Line: We achieved efficient depletion of Tat-SF1 and demonstrated that this did not affect cell viability.Neither Tat-dependent nor basal transcription from the HIV-1 LTR was affected by Tat-SF1 depletion, suggesting that the decrease in infectivity was due to a deficiency at a later step in the viral lifecycle.Finally, Tat-SF1 depletion resulted in an increase in the ratio of unspliced to spliced viral transcripts.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT

Background: HIV-1 relies on several host proteins for productive viral transcription. HIV-1 Tat-specific factor 1 (Tat-SF1) is among these cofactors that were identified by in vitro reconstituted transcription reactions with immunodepleted nuclear extracts. At the onset of this work, the prevailing hypothesis was that Tat-SF1 was a required cofactor for the viral regulatory protein, Tat; however, this had not previously been formally tested in vivo.

Methodology/principal findings: To directly address the involvement of Tat-SF1 in HIV-1 gene expression, we depleted Tat-SF1 in HeLa cells by conventional expression of shRNAs and in T- Rex -293 cells containing tetracycline-inducible shRNAs targeting Tat-SF1. We achieved efficient depletion of Tat-SF1 and demonstrated that this did not affect cell viability. HIV-1 infectivity decreased in Tat-SF1-depleted cells, but only when multiple rounds of infection occurred. Neither Tat-dependent nor basal transcription from the HIV-1 LTR was affected by Tat-SF1 depletion, suggesting that the decrease in infectivity was due to a deficiency at a later step in the viral lifecycle. Finally, Tat-SF1 depletion resulted in an increase in the ratio of unspliced to spliced viral transcripts.

Conclusions/significance: Tat-SF1 is not required for regulating HIV-1 transcription, but is required for maintaining the ratios of different classes of HIV-1 transcripts. These new findings highlight a novel, post-transcriptional role for Tat-SF1 in the HIV-1 life cycle.

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Related in: MedlinePlus

Tat-SF1 depletion does not affect basal or Tat-dependent transcription from the HIV-1 LTR in vivo.(A) Tat titration. HeLa cells were cotransfected with the indicated amount of pcTat, an HIV-1 CAT reporter, and an SV40-Luciferase reporter. Lysates were subjected to a CAT assay and normalized with values obtained from a luciferase assay 24 hours after cotransfection. All CAT and Luciferase values were background corrected. Values shown are the means of duplicate transfections. (B) Tat transactivation assay in HeLa cells. HeLa cells were transiently transfected with either an empty vector, a non-silencing shRNA, or two shRNAs targeting Tat-SF1. A western blot confirming knockdown is shown below the chart. Cells were cotransfected with the same reporters as in (A) plus 0.3 ng pcTat or an empty vector. All CAT and Luciferase values were background corrected, and CAT was normalized to Luciferase. Values shown are the means of duplicate transfections. Raw values are shown in Table 1, Exp. 1. (C) Tat transactivation in T-Rex-293 cells. T-Rex-293 cells harboring either an empty vector control, an shRNA targeting GFP, or one of two shRNAs targeting Tat-SF1 were induced with tetracycline for 48 hours. A western blot confirming knockdown is shown below the chart. Cells were cotransfected with the same reporters as in (A) and (B). All CAT and Luciferase values were background corrected, and CAT was normalized to Luciferase. Values shown are the means of triplicate transfections. Raw values are shown in Table 2. Error bars represent standard error.
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pone-0005710-g004: Tat-SF1 depletion does not affect basal or Tat-dependent transcription from the HIV-1 LTR in vivo.(A) Tat titration. HeLa cells were cotransfected with the indicated amount of pcTat, an HIV-1 CAT reporter, and an SV40-Luciferase reporter. Lysates were subjected to a CAT assay and normalized with values obtained from a luciferase assay 24 hours after cotransfection. All CAT and Luciferase values were background corrected. Values shown are the means of duplicate transfections. (B) Tat transactivation assay in HeLa cells. HeLa cells were transiently transfected with either an empty vector, a non-silencing shRNA, or two shRNAs targeting Tat-SF1. A western blot confirming knockdown is shown below the chart. Cells were cotransfected with the same reporters as in (A) plus 0.3 ng pcTat or an empty vector. All CAT and Luciferase values were background corrected, and CAT was normalized to Luciferase. Values shown are the means of duplicate transfections. Raw values are shown in Table 1, Exp. 1. (C) Tat transactivation in T-Rex-293 cells. T-Rex-293 cells harboring either an empty vector control, an shRNA targeting GFP, or one of two shRNAs targeting Tat-SF1 were induced with tetracycline for 48 hours. A western blot confirming knockdown is shown below the chart. Cells were cotransfected with the same reporters as in (A) and (B). All CAT and Luciferase values were background corrected, and CAT was normalized to Luciferase. Values shown are the means of triplicate transfections. Raw values are shown in Table 2. Error bars represent standard error.

Mentions: Although Tat-SF1 has been implicated in vitro as a cofactor for the viral protein Tat, this had yet to be demonstrated in vivo. To examine Tat transactivation when Tat-SF1 was depleted, control and Tat-SF1 knockdown HeLa cell lines were transfected with a chloramphenicol acetyltransferase (CAT) reporter under the control of the HIV-1 LTR. This plasmid, along with an internal luciferase control for transfection efficiency, was cotransfected with either the Tat-expressing plasmid, pcTat [30], or an empty vector. The amount of pcTat transfected was experimentally determined so that Tat transactivation was in the linear range (Figure 4A). As expected, Tat stimulated transcription of the CAT reporter gene by approximately 40-fold in control cells (Figure 4B and Table 1). Surprisingly, similar levels of Tat transactivation were seen when Tat-SF1 was depleted. Furthermore, basal transcription of the LTR (in the absence of Tat) was unaffected by RNAi-mediated depletion of Tat-SF1. In addition, the same results were recapitulated in T-Rex-293 cells (Figure 4C and Table 2). These findings indicate that, contrary to the in vitro results, Tat-SF1 depletion did not affect basal or Tat-mediated transactivation of the HIV-1 LTR in vivo.


Tat-SF1 is not required for Tat transactivation but does regulate the relative levels of unspliced and spliced HIV-1 RNAs.

Miller HB, Saunders KO, Tomaras GD, Garcia-Blanco MA - PLoS ONE (2009)

Tat-SF1 depletion does not affect basal or Tat-dependent transcription from the HIV-1 LTR in vivo.(A) Tat titration. HeLa cells were cotransfected with the indicated amount of pcTat, an HIV-1 CAT reporter, and an SV40-Luciferase reporter. Lysates were subjected to a CAT assay and normalized with values obtained from a luciferase assay 24 hours after cotransfection. All CAT and Luciferase values were background corrected. Values shown are the means of duplicate transfections. (B) Tat transactivation assay in HeLa cells. HeLa cells were transiently transfected with either an empty vector, a non-silencing shRNA, or two shRNAs targeting Tat-SF1. A western blot confirming knockdown is shown below the chart. Cells were cotransfected with the same reporters as in (A) plus 0.3 ng pcTat or an empty vector. All CAT and Luciferase values were background corrected, and CAT was normalized to Luciferase. Values shown are the means of duplicate transfections. Raw values are shown in Table 1, Exp. 1. (C) Tat transactivation in T-Rex-293 cells. T-Rex-293 cells harboring either an empty vector control, an shRNA targeting GFP, or one of two shRNAs targeting Tat-SF1 were induced with tetracycline for 48 hours. A western blot confirming knockdown is shown below the chart. Cells were cotransfected with the same reporters as in (A) and (B). All CAT and Luciferase values were background corrected, and CAT was normalized to Luciferase. Values shown are the means of triplicate transfections. Raw values are shown in Table 2. Error bars represent standard error.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2682658&req=5

pone-0005710-g004: Tat-SF1 depletion does not affect basal or Tat-dependent transcription from the HIV-1 LTR in vivo.(A) Tat titration. HeLa cells were cotransfected with the indicated amount of pcTat, an HIV-1 CAT reporter, and an SV40-Luciferase reporter. Lysates were subjected to a CAT assay and normalized with values obtained from a luciferase assay 24 hours after cotransfection. All CAT and Luciferase values were background corrected. Values shown are the means of duplicate transfections. (B) Tat transactivation assay in HeLa cells. HeLa cells were transiently transfected with either an empty vector, a non-silencing shRNA, or two shRNAs targeting Tat-SF1. A western blot confirming knockdown is shown below the chart. Cells were cotransfected with the same reporters as in (A) plus 0.3 ng pcTat or an empty vector. All CAT and Luciferase values were background corrected, and CAT was normalized to Luciferase. Values shown are the means of duplicate transfections. Raw values are shown in Table 1, Exp. 1. (C) Tat transactivation in T-Rex-293 cells. T-Rex-293 cells harboring either an empty vector control, an shRNA targeting GFP, or one of two shRNAs targeting Tat-SF1 were induced with tetracycline for 48 hours. A western blot confirming knockdown is shown below the chart. Cells were cotransfected with the same reporters as in (A) and (B). All CAT and Luciferase values were background corrected, and CAT was normalized to Luciferase. Values shown are the means of triplicate transfections. Raw values are shown in Table 2. Error bars represent standard error.
Mentions: Although Tat-SF1 has been implicated in vitro as a cofactor for the viral protein Tat, this had yet to be demonstrated in vivo. To examine Tat transactivation when Tat-SF1 was depleted, control and Tat-SF1 knockdown HeLa cell lines were transfected with a chloramphenicol acetyltransferase (CAT) reporter under the control of the HIV-1 LTR. This plasmid, along with an internal luciferase control for transfection efficiency, was cotransfected with either the Tat-expressing plasmid, pcTat [30], or an empty vector. The amount of pcTat transfected was experimentally determined so that Tat transactivation was in the linear range (Figure 4A). As expected, Tat stimulated transcription of the CAT reporter gene by approximately 40-fold in control cells (Figure 4B and Table 1). Surprisingly, similar levels of Tat transactivation were seen when Tat-SF1 was depleted. Furthermore, basal transcription of the LTR (in the absence of Tat) was unaffected by RNAi-mediated depletion of Tat-SF1. In addition, the same results were recapitulated in T-Rex-293 cells (Figure 4C and Table 2). These findings indicate that, contrary to the in vitro results, Tat-SF1 depletion did not affect basal or Tat-mediated transactivation of the HIV-1 LTR in vivo.

Bottom Line: We achieved efficient depletion of Tat-SF1 and demonstrated that this did not affect cell viability.Neither Tat-dependent nor basal transcription from the HIV-1 LTR was affected by Tat-SF1 depletion, suggesting that the decrease in infectivity was due to a deficiency at a later step in the viral lifecycle.Finally, Tat-SF1 depletion resulted in an increase in the ratio of unspliced to spliced viral transcripts.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT

Background: HIV-1 relies on several host proteins for productive viral transcription. HIV-1 Tat-specific factor 1 (Tat-SF1) is among these cofactors that were identified by in vitro reconstituted transcription reactions with immunodepleted nuclear extracts. At the onset of this work, the prevailing hypothesis was that Tat-SF1 was a required cofactor for the viral regulatory protein, Tat; however, this had not previously been formally tested in vivo.

Methodology/principal findings: To directly address the involvement of Tat-SF1 in HIV-1 gene expression, we depleted Tat-SF1 in HeLa cells by conventional expression of shRNAs and in T- Rex -293 cells containing tetracycline-inducible shRNAs targeting Tat-SF1. We achieved efficient depletion of Tat-SF1 and demonstrated that this did not affect cell viability. HIV-1 infectivity decreased in Tat-SF1-depleted cells, but only when multiple rounds of infection occurred. Neither Tat-dependent nor basal transcription from the HIV-1 LTR was affected by Tat-SF1 depletion, suggesting that the decrease in infectivity was due to a deficiency at a later step in the viral lifecycle. Finally, Tat-SF1 depletion resulted in an increase in the ratio of unspliced to spliced viral transcripts.

Conclusions/significance: Tat-SF1 is not required for regulating HIV-1 transcription, but is required for maintaining the ratios of different classes of HIV-1 transcripts. These new findings highlight a novel, post-transcriptional role for Tat-SF1 in the HIV-1 life cycle.

Show MeSH
Related in: MedlinePlus