Tip110 Protein Binds to Unphosphorylated RNA Polymerase II and Promotes Its Phosphorylation and HIV-1 Long Terminal Repeat Transcription

Weina Zhao,Ying Liu,Khalid Amine Timani,Johnny J He

doi:10.1074/jbc.m113.529784

Abstract

Transcription plays an important role in both HIV-1 gene expression and replication and mandates complicated but coordinated interactions between the host and virus. Our previous studies have shown that an HIV-1 Tat-interacting protein of 110 kDa, Tip110, binds to and enhances Tat function in Tat-mediated HIV-1 gene transcription and replication (Liu, Y., Li, J., Kim, B. O., Pace, B. S., and He, J. J. (2002) HIV-1 Tat protein-mediated transactivation of the HIV-1 long terminal repeat promoter is potentiated by a novel nuclear Tat-interacting protein of 110 kDa, Tip110. J. Biol. Chem. 277, 23854-23863). However, the underlying molecular mechanisms by which this takes place were not understood. In this study, we demonstrated that Tip110 bound to unphosphorylated RNA polymerase II (RNAPII) in a direct and specific manner. In addition, we detected Tip110 at the HIV-1 long terminal repeat (LTR) promoter and found that Tip110 expression was associated with increased phosphorylation of serine 2 of the heptapeptide repeats within the RNAPII C-terminal domain and increased recruitment of positive transcription elongation factor b to the LTR promoter. Consistent with these findings, we showed that Tip110 interaction with Tat directly enhanced transcription elongation of the LTR promoter. Taken together, these findings have provided additional and mechanistic evidence to support Tip110 function in HIV-1 transcription.

Highlights

Tat-interacting protein of 110 kDa (Tip110) synergizes with HIV-1 Tat protein and transactivates Tat-mediated HIV-1 long terminal repeat (LTR) promoter, but the underlying mechanisms have not been understood
We showed that Tip110 bound to unphosphorylated RNA polymerase II (RNAPII) and increased positive transcription factor b (P-TEFb) recruitment to the TAR1⁄7Tat1⁄7P-TEFb transcription complex, which was associated with increased phosphorylation of serine 2 of the heptapeptide repeats within the RNAPII C-terminal domain (CTD) and enhanced Tat-mediated transcription elongation of the LTR promoter
Because Tip110 and P-TEFb co-localize within nuclear speckle structures [35] and P-TEFb is responsible for HIV-1 transcription elongation, we first determined whether Tip110 interacted with CDK9 or cyclin T1 by immunoprecipitation and Western blot analysis

Summary

Introduction

Tip110 synergizes with HIV-1 Tat protein and transactivates Tat-mediated HIV-1 LTR promoter, but the underlying mechanisms have not been understood. We detected Tip110 at the HIV-1 long terminal repeat (LTR) promoter and found that Tip110 expression was associated with increased phosphorylation of serine 2 of the heptapeptide repeats within the RNAPII C-terminal domain and increased recruitment of positive transcription elongation factor b to the LTR promoter. Consistent with these findings, we showed that Tip110 interaction with Tat directly enhanced transcription elongation of the LTR promoter. These findings have provided additional and mechanistic evidence to support Tip110 function in HIV-1 transcription

Methods

Results

Conclusion