Tat-SF1 Is Not Required for Tat Transactivation but Does Regulate the Relative Levels of Unspliced and Spliced HIV-1 RNAs

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
PLoS ONE (Impact Factor: 3.53). 02/2009; 4(5):e5710. DOI: 10.1371/journal.pone.0005710
Source: PubMed

ABSTRACT 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.
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.
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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    • "The association with both elongation and splicing factors has led to the suggestion that factors such as Tat-SF1 can couple these two processes (Chen et al., 2009; Kim et al., 1999; Miller et al., 2009). Tat-SF1 has been shown to be associated with other transcription regulators such as Tat-CT1 and the transcription– splicing coupling factor, CA150 (Zhou et al., 1998). "
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    • "Tat-SF1 depletion results in widespread changes in both transcription and splicing Since Tat-SF1 has been implicated in both transcription and splicing, Affymetrix GeneChip Human Exon 1.0ST Arrays are an attractive technology to use for studying its function because they survey both transcript-level changes (due to altered transcription and/or stability) and exon-level changes (due to alternative transcript initiation, alternative pre-mRNA processing, and/or differential isoform stability) (Gardina et al. 2006). Using these arrays, we explored global changes in the human transcriptome occurring after Tat-SF1 depletion using T-Rex-293 cell lines that stably expressed green fluorescent protein (GFP) and tetracycline-inducible Tat-SF1-specific shRNAs (Miller et al. 2009). To confirm knockdown, cell lysates were collected after 72 h of tetracycline induction and analyzed by Western blotting. "
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