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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 maintains the levels of unspliced and spliced HIV-1 RNAs.(A) Western blot analysis confirming knockdown in T-Rex-293 cells. (B) Representative Northern blot analysis of HIV-1 RNA classes. T-Rex-293 cells were transfected with pSG3ΔEnv 72 hours after tetracycline induction. At 48 hours post-transfection, total RNA was isolated for electrophoresis and Northern blotting. A DNA probe specific to the HIV-1 LTR detected the ∼9 kb unspliced, ∼4 kb singly spliced, and ∼2 kb fully spliced RNAs. Lane 1 contains RNA from mock-transfected GFP control cells, lane 2 from transfected GFP control cells, and lanes 3 and 4 from transfected Tat-SF1 shRNA cells. The lower panel shows the same membrane, stripped and reprobed for GAPDH. (C) Tat-SF1 depletion alters the levels of HIV-1 RNA classes. Values are reported as the mean proportion of each RNA class, relative to the GFP control cells from three independent Northern blot experiments. Error bars represent standard error. Statistically significant differences between GFP control and Tat-SF1 knockdown conditions are indicated with asterisks. (D) Tat-SF1 depletion does not alter total HIV-1 RNA levels. Levels of the 3 RNA classes quantified from triplicate Northern blots were totaled and normalized to GAPDH levels. Values are reported as the means relative to the GFP control cells from three independent experiments. Error bars represent standard error. (E) Tat-SF1 depletion results in an increase in unspliced HIV-1 transcripts. qRT-PCR was performed on the same RNA samples used for Northern blot experiments. The medians of triplicate amplifications (both unspliced products and all initiated HIV-1 transcripts) were calculated and means of unspliced transcripts/all initiated HIV-1 transcripts ratios from triplicate samples are reported. Error bars represent standard error.
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pone-0005710-g005: Tat-SF1 maintains the levels of unspliced and spliced HIV-1 RNAs.(A) Western blot analysis confirming knockdown in T-Rex-293 cells. (B) Representative Northern blot analysis of HIV-1 RNA classes. T-Rex-293 cells were transfected with pSG3ΔEnv 72 hours after tetracycline induction. At 48 hours post-transfection, total RNA was isolated for electrophoresis and Northern blotting. A DNA probe specific to the HIV-1 LTR detected the ∼9 kb unspliced, ∼4 kb singly spliced, and ∼2 kb fully spliced RNAs. Lane 1 contains RNA from mock-transfected GFP control cells, lane 2 from transfected GFP control cells, and lanes 3 and 4 from transfected Tat-SF1 shRNA cells. The lower panel shows the same membrane, stripped and reprobed for GAPDH. (C) Tat-SF1 depletion alters the levels of HIV-1 RNA classes. Values are reported as the mean proportion of each RNA class, relative to the GFP control cells from three independent Northern blot experiments. Error bars represent standard error. Statistically significant differences between GFP control and Tat-SF1 knockdown conditions are indicated with asterisks. (D) Tat-SF1 depletion does not alter total HIV-1 RNA levels. Levels of the 3 RNA classes quantified from triplicate Northern blots were totaled and normalized to GAPDH levels. Values are reported as the means relative to the GFP control cells from three independent experiments. Error bars represent standard error. (E) Tat-SF1 depletion results in an increase in unspliced HIV-1 transcripts. qRT-PCR was performed on the same RNA samples used for Northern blot experiments. The medians of triplicate amplifications (both unspliced products and all initiated HIV-1 transcripts) were calculated and means of unspliced transcripts/all initiated HIV-1 transcripts ratios from triplicate samples are reported. Error bars represent standard error.

Mentions: Although Tat-SF1 depletion resulted in a decrease in HIV-1 infectivity, it did not affect Tat transactivation. Rather than having a role in transcription elongation of HIV-1 RNA, it seemed possible that Tat-SF1 could post-transcriptionally regulate viral gene expression. To test this hypothesis, we used Northern blots to analyze HIV-1 RNAs in control and knockdown cells. The pSG3ΔEnv plasmid was transfected into GFP control and Tat-SF1 knockdown T-Rex-293 cell lines, and total RNA was harvested 48 hours later. Hybridization was performed using an HIV-1 LTR-specific radiolabeled probe that detects all three classes of viral RNAs: unspliced (∼9 kb), singly spliced (∼4 kb), and fully spliced (∼2 kb). A western blot of T-Rex-293 cell lysates confirms efficient knockdown of Tat-SF1 (Figure 5A). Figure 5B shows the results of a representative Northern blot probing RNA from GFP control and Tat-SF1 knockdown cell lines 48 hours after transfection with the pSG3ΔEnv plasmid. In the mock transfected lane, no viral pre-mRNA signal is detected, indicating that the bands seen in the other lanes are HIV-1-specific. Quantification of each RNA class demonstrates that intron-containing unspliced and singly spliced transcripts were elevated and fully spliced transcripts were reduced when Tat-SF1 was depleted (Figure 5C). This corresponds to an unspliced/fully spliced ratio of ∼0.7 in GFP control cells and ∼1.6 in Tat-SF1 depleted cells. Similar changes in ratios were observed when another viral plasmid, pNL-Luc-HXB, was transfected into Tat-SF1 depleted T-Rex-293 cells (data not shown). These data further suggest a post-transcriptional role for Tat-SF1.


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 maintains the levels of unspliced and spliced HIV-1 RNAs.(A) Western blot analysis confirming knockdown in T-Rex-293 cells. (B) Representative Northern blot analysis of HIV-1 RNA classes. T-Rex-293 cells were transfected with pSG3ΔEnv 72 hours after tetracycline induction. At 48 hours post-transfection, total RNA was isolated for electrophoresis and Northern blotting. A DNA probe specific to the HIV-1 LTR detected the ∼9 kb unspliced, ∼4 kb singly spliced, and ∼2 kb fully spliced RNAs. Lane 1 contains RNA from mock-transfected GFP control cells, lane 2 from transfected GFP control cells, and lanes 3 and 4 from transfected Tat-SF1 shRNA cells. The lower panel shows the same membrane, stripped and reprobed for GAPDH. (C) Tat-SF1 depletion alters the levels of HIV-1 RNA classes. Values are reported as the mean proportion of each RNA class, relative to the GFP control cells from three independent Northern blot experiments. Error bars represent standard error. Statistically significant differences between GFP control and Tat-SF1 knockdown conditions are indicated with asterisks. (D) Tat-SF1 depletion does not alter total HIV-1 RNA levels. Levels of the 3 RNA classes quantified from triplicate Northern blots were totaled and normalized to GAPDH levels. Values are reported as the means relative to the GFP control cells from three independent experiments. Error bars represent standard error. (E) Tat-SF1 depletion results in an increase in unspliced HIV-1 transcripts. qRT-PCR was performed on the same RNA samples used for Northern blot experiments. The medians of triplicate amplifications (both unspliced products and all initiated HIV-1 transcripts) were calculated and means of unspliced transcripts/all initiated HIV-1 transcripts ratios from triplicate samples are reported. Error bars represent standard error.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682658&req=5

pone-0005710-g005: Tat-SF1 maintains the levels of unspliced and spliced HIV-1 RNAs.(A) Western blot analysis confirming knockdown in T-Rex-293 cells. (B) Representative Northern blot analysis of HIV-1 RNA classes. T-Rex-293 cells were transfected with pSG3ΔEnv 72 hours after tetracycline induction. At 48 hours post-transfection, total RNA was isolated for electrophoresis and Northern blotting. A DNA probe specific to the HIV-1 LTR detected the ∼9 kb unspliced, ∼4 kb singly spliced, and ∼2 kb fully spliced RNAs. Lane 1 contains RNA from mock-transfected GFP control cells, lane 2 from transfected GFP control cells, and lanes 3 and 4 from transfected Tat-SF1 shRNA cells. The lower panel shows the same membrane, stripped and reprobed for GAPDH. (C) Tat-SF1 depletion alters the levels of HIV-1 RNA classes. Values are reported as the mean proportion of each RNA class, relative to the GFP control cells from three independent Northern blot experiments. Error bars represent standard error. Statistically significant differences between GFP control and Tat-SF1 knockdown conditions are indicated with asterisks. (D) Tat-SF1 depletion does not alter total HIV-1 RNA levels. Levels of the 3 RNA classes quantified from triplicate Northern blots were totaled and normalized to GAPDH levels. Values are reported as the means relative to the GFP control cells from three independent experiments. Error bars represent standard error. (E) Tat-SF1 depletion results in an increase in unspliced HIV-1 transcripts. qRT-PCR was performed on the same RNA samples used for Northern blot experiments. The medians of triplicate amplifications (both unspliced products and all initiated HIV-1 transcripts) were calculated and means of unspliced transcripts/all initiated HIV-1 transcripts ratios from triplicate samples are reported. Error bars represent standard error.
Mentions: Although Tat-SF1 depletion resulted in a decrease in HIV-1 infectivity, it did not affect Tat transactivation. Rather than having a role in transcription elongation of HIV-1 RNA, it seemed possible that Tat-SF1 could post-transcriptionally regulate viral gene expression. To test this hypothesis, we used Northern blots to analyze HIV-1 RNAs in control and knockdown cells. The pSG3ΔEnv plasmid was transfected into GFP control and Tat-SF1 knockdown T-Rex-293 cell lines, and total RNA was harvested 48 hours later. Hybridization was performed using an HIV-1 LTR-specific radiolabeled probe that detects all three classes of viral RNAs: unspliced (∼9 kb), singly spliced (∼4 kb), and fully spliced (∼2 kb). A western blot of T-Rex-293 cell lysates confirms efficient knockdown of Tat-SF1 (Figure 5A). Figure 5B shows the results of a representative Northern blot probing RNA from GFP control and Tat-SF1 knockdown cell lines 48 hours after transfection with the pSG3ΔEnv plasmid. In the mock transfected lane, no viral pre-mRNA signal is detected, indicating that the bands seen in the other lanes are HIV-1-specific. Quantification of each RNA class demonstrates that intron-containing unspliced and singly spliced transcripts were elevated and fully spliced transcripts were reduced when Tat-SF1 was depleted (Figure 5C). This corresponds to an unspliced/fully spliced ratio of ∼0.7 in GFP control cells and ∼1.6 in Tat-SF1 depleted cells. Similar changes in ratios were observed when another viral plasmid, pNL-Luc-HXB, was transfected into Tat-SF1 depleted T-Rex-293 cells (data not shown). These data further suggest a post-transcriptional role for Tat-SF1.

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