Inhibition of HIV-1 Transcription and Replication by a Newly Identified Cyclin T1 Splice Variant

Guozhen Gao,Xiaoyun Wu,Jieqiong Zhou,Mingfeng He,Johnny J He,Deyin Guo

doi:10.1074/jbc.m112.438465

Abstract

A variety of cellular factors participates in the HIV-1 life cycle. Among them is the well characterized cyclin T1 (CYCT1). CycT1 binds to cyclin-dependent kinase 9 (CDK9) and forms the positive transcription elongation factor-b (P-TEFb). P-TEFb is then recruited by HIV-1 TAT to the HIV-1 long terminal repeat (LTR) promoter and subsequently leads to phosphorylation of the C-terminal domain of RNA polymerase II (pol II), enhanced processivity of pol II, and transcription of a full-length HIV-1 RNA. In this study, we report the identification of a new CYCT1 splice variant, designated as CYCT1b, and accordingly we renamed CYCT1 as CYCT1a. CYCT1b was detected in several cell lines, including primary human CD4 T cells, and its expression was subject to cell cycle regulation. Similar to CYCT1a, CYCT1b was primarily localized in the nucleus. CYCT1b expression was found to be inversely correlated with HIV-1 gene expression and replication. This inverse correlation appeared to involve TAT transactivation, CDK9 binding, and subsequent recruitment of P-TEFb to the HIV-1 LTR promoter and pol II C-terminal domain phosphorylation. In agreement with these findings, CYCT1b expression led to direct inhibition of TAT-transactivated transcription of the HIV-1 LTR promoter. Taken together, these results show that the newly identified CYCT1b splice variant inhibits HIV-1 transcription and may provide new clues for the development of anti-HIV strategies.

Highlights

Production of full-length Human immunodeficiency virus type 1 (HIV-1) mRNA requires interaction of cyclin T1 and cyclin-dependent kinase 9 (CDK9) with HIV-1 TAT protein
Identification of a Novel Cyclin T1 Variant (CYCT1b)— When attempting to amplify the complete coding sequence of cyclin T1 (CYCT1) by reverse transcription-PCR (RT-PCR) with cDNA from 293T cells and the primers targeting the ends of CYCT1 ORF, surprisingly, two DNA bands with slightly different sizes were obtained (Fig. 1A)
The levels of bound hyperphosphorylated polymerase II (pol II) were increased after TAT-mediated transactivation, but significantly decreased when CYCT1b was overexpressed (Fig. 7C). These results suggest that overexpression of CYCT1b inhibits the recruitment of positive transcription elongation factor-b (P-TEFb) to the HIV-1 long terminal repeat (LTR) and inhibits the hyperphosphorylation of pol II on the LTR promoter

Summary

Introduction

Production of full-length HIV-1 mRNA requires interaction of cyclin T1 and cyclin-dependent kinase 9 (CDK9) with HIV-1 TAT protein. CYCT1b expression was found to be inversely correlated with HIV-1 gene expression and replication This inverse correlation appeared to involve TAT transactivation, CDK9 binding, and subsequent recruitment of P-TEFb to the HIV-1 LTR promoter and pol II C-terminal domain phosphorylation. In agreement with these findings, CYCT1b expression led to direct inhibition of TAT-transactivated transcription of the HIV-1 LTR promoter. Taken together, these results show that the newly identified CYCT1b splice variant inhibits HIV-1 transcription and may provide new clues for the development of anti-HIV strategies

Methods

Results

Conclusion