Research

Modulating HIV-1 replication by RNA interference directed against human transcription elongation factor SPT5

Yueh-Hsin Ping^1,3 , Chia-ying Chu¹ , Hong Cao¹ , Jean-Marc Jacque² , Mario Stevenson² and Tariq M Rana¹

¹  Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA

²  Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA

³  Department and Institute of Pharmacology National Yang-Ming University Shih-Pai, Taipei 11221 Taiwan

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Retrovirology 2004, 1:46doi:10.1186/1742-4690-1-46

Published:	27 December 2004

Abstract

Background

Several cellular positive and negative elongation factors are involved in regulating RNA polymerase II processivity during transcription elongation in human cells. In recruiting several of these regulatory factors to the 5' long terminal repeat (LTR) promoter during transcription elongation, HIV-1 modulates replication of its genome in a process mediated by the virus-encoded transactivator Tat. One particular cellular regulatory factor, DSIF subunit human SPT5 (hSpt5), has been implicated in both positively and negatively regulating transcriptional elongation but its role in Tat transactivation in vivo and in HIV-1 replication has not been completely elucidated.

Results

To understand the in vivo function of hSpt5 and define its role in Tat transactivation and HIV-1 replication, we used RNA interference (RNAi) to specifically knockdown hSpt5 expression by degrading hSpt5 mRNA. Short-interfering RNA (siRNA) designed to target hSpt5 for RNAi successfully resulted in knockdown of both hSpt5 mRNA and protein levels, and did not significantly affect cell viability. In contrast to hSpt5 knockdown, siRNA-mediated silencing of human mRNA capping enzyme, a functionally important hSpt5-interacting cellular protein, was lethal and showed a significant increase in cell death over the course of the knockdown experiment. In addition, hSpt5 knockdown led to significant decreases in Tat transactivation and inhibited HIV-1 replication, indicating that hSpt5 was required for mediating Tat transactivation and HIV-1 replication.

Conclusions

The findings presented here showed that hSpt5 is a bona fide positive regulator of Tat transactivation and HIV-1 replication in vivo. These results also suggest that hSpt5 function in transcription regulation and mRNA capping is essential for a subset of cellular and viral genes and may not be required for global gene expression.