ZASC1 Stimulates HIV-1 Transcription Elongation by Recruiting P-TEFb and TAT to the LTR Promoter

James W Bruce,Megan Bracken,Elizabeth Mathieu,Paul Ahlquist,John A T Young,Rachel Reddington,Susan R Ross

doi:10.1371/journal.ppat.1003712

Abstract

Transcription from the HIV-1 LTR promoter efficiently initiates but rapidly terminates because of a non-processive form of RNA polymerase II. This premature termination is overcome by assembly of an HIV-1 TAT/P-TEFb complex at the transactivation response region (TAR), a structured RNA element encoded by the first 59 nt of HIV-1 mRNA. Here we have identified a conserved DNA-binding element for the cellular transcription factor, ZASC1, in the HIV-1 core promoter immediately upstream of TAR. We show that ZASC1 interacts with TAT and P-TEFb, co-operating with TAT to regulate HIV-1 gene expression, and promoting HIV-1 transcriptional elongation. The importance of ZASC1 to HIV-1 transcription elongation was confirmed through mutagenesis of the ZASC1 binding sites in the LTR promoter, shRNAs targeting ZASC1 and expression of dominant negative ZASC1. Chromatin immunoprecipitation analysis revealed that ZASC1 recruits Tat and P-TEFb to the HIV-1 core promoter in a TAR-independent manner. Thus, we have identified ZASC1 as novel regulator of HIV-1 gene expression that functions through the DNA-dependent, RNA-independent recruitment of TAT/P-TEFb to the HIV-1 promoter.

Highlights

The Retroviridae family includes human immunodeficiency viruses type-1 and 2 (HIV-1 and HIV-2), the causative agents of acquired immune deficiency syndrome (AIDS)
Consistent with this, a specific high molecular weight mobility shift was observed when ZASC1 was incubated with an human immunodeficiency virus 1 (HIV-1) unique 39 (U3) DNA fragment in an in vitro Electrophoretic mobility assays (EMSA) experiment but not when the same fragment was incubated with a control firefly luciferase protein (Fig. 1B)
To confirm that ZASC1 is bound to the putative ZASC1 binding sites in the HIV-1 promoter in infected cells, we performed chromatin immunoprecipitation (ChIP) experiments from HEK293 and Jurkat cells challenged with the HIV-1 vector, NL43E-R-Luc

Summary

Introduction

The Retroviridae family includes human immunodeficiency viruses type-1 and 2 (HIV-1 and HIV-2), the causative agents of acquired immune deficiency syndrome (AIDS). Retroviruses are unique among RNA viruses in that after virus entry into the cell, the viral RNA is reverse transcribed into double stranded DNA and integrated into the cellular chromosome, generating the provirus. This feature makes retroviruses dependent on the host RNA polymerase II transcription machinery for expressing viral gene products and new genomes. Transcription of integrated proviral DNA is driven from the unique 39 (U3) element in the viral genome This is a strong RNA polymerase II (pol II) promoter that contains many overlapping binding sites for cellular transcription factors that modulate expression in different cell types and response to signaling pathways [1,2]. Subsequent phosphorylation of the negative elongation factor (NELF), the SUPT5 component of DSIF and the C-terminal domain (CTD) of pol II by P-TEFb results in release of stalled polymerase, transfer of TAT/P-TEFb to the extending polymerase and a dramatic increase in transcription elongation [4,5]

Methods

Results

Conclusion