HIV-1 Tat phosphorylation on Ser-16 residue modulates HIV-1 transcription

Andrey Ivanov,Xionghao Lin,Nowah Afangbedji,Andrey V Ilatovskiy,Hatajai Lassiter,Namita Kumari,Sergei Nekhai,Michael G Petukhov,Tatiana Ammosova,Xiaomei Niu

doi:10.1186/s12977-018-0422-5

Abstract

BackgroundHIV-1 transcription activator protein Tat is phosphorylated in vitro by CDK2 and DNA-PK on Ser-16 residue and by PKR on Tat Ser-46 residue. Here we analyzed Tat phosphorylation in cultured cells and its functionality.ResultsMass spectrometry analysis showed primarily Tat Ser-16 phosphorylation in cultured cells. In vitro, CDK2/cyclin E predominantly phosphorylated Tat Ser-16 and PKR—Tat Ser-46. Alanine mutations of either Ser-16 or Ser-46 decreased overall Tat phosphorylation. Phosphorylation of Tat Ser-16 was reduced in cultured cells treated by a small molecule inhibitor of CDK2 and, to a lesser extent, an inhibitor of DNA-PK. Conditional knock-downs of CDK2 and PKR inhibited and induced one round HIV-1 replication respectively. HIV-1 proviral transcription was inhibited by Tat alanine mutants and partially restored by S16E mutation. Pseudotyped HIV-1 with Tat S16E mutation replicated well, and HIV-1 Tat S46E—poorly, but no live viruses were obtained with Tat S16A or Tat S46A mutations. TAR RNA binding was affected by Tat Ser-16 alanine mutation. Binding to cyclin T1 showed decreased binding of all Ser-16 and Ser-46 Tat mutants with S16D and Tat S46D mutationts showing the strongest effect. Molecular modelling and molecular dynamic analysis revealed significant structural changes in Tat/CDK9/cyclin T1 complex with phosphorylated Ser-16 residue, but not with phosphorylated Ser-46 residue.ConclusionPhosphorylation of Tat Ser-16 induces HIV-1 transcription, facilitates binding to TAR RNA and rearranges CDK9/cyclin T1/Tat complex. Thus, phosphorylation of Tat Ser-16 regulates HIV-1 transcription and may serve as target for HIV-1 therapeutics.

Highlights

HIV-1 transcription activator protein Tat is phosphorylated in vitro by CDK2 and DNA-PK on Ser-16 residue and by protein kinase R (PKR) on Tat Ser-46 residue
Our results indicate Tat Ser-16 phosphorylation is an important event in HIV-1 transcription regulation and that it may facilitate binding to TAR RNA and the rearrangement of CDK9/ cyclin T1/Tat complex
PKR that phosphorylates primarily Tat Ser-46 inhibits HIV-1 replication, as shown previously [11] and as our PKR knock down experiment in this study pointed out

Summary

Introduction

HIV-1 transcription activator protein Tat is phosphorylated in vitro by CDK2 and DNA-PK on Ser-16 residue and by PKR on Tat Ser-46 residue. We analyzed Tat phosphorylation in cultured cells and its functionality. Complete eradication of HIV-1 virus in infected individuals is hindered by the presence of latent HIV-1 provirus, which is not affected by the existing anti-HIV-1 drugs [1]. HIV-1 transcription from HIV-1 LTR depends on both host cell factors and HIV-1 transactivation Tat protein [2]. HIV-1 Tat activates viral transcription by recruiting Positive Transcription Elongation Factor b (P-TEFb) that contains CDK9/cyclin T1 to TAR RNA [2]. Inability of Tat to recruit CDK9/cyclin T1 to TAR RNA may contribute to the establishment of latency [1].

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