Research

Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription

Tatyana Ammosova¹, Reem Berro⁴, Marina Jerebtsova⁵, Angela Jackson², Sharroya Charles³, Zachary Klase⁴, William Southerland², Victor R Gordeuk¹, Fatah Kashanchi⁴ and Sergei Nekhai^4,1,2*

• * Corresponding author: Sergei Nekhai snekhai@howard.edu

Author Affiliations

¹ Center for Sickle Cell Disease, Howard University College of Medicine, 520 W Street N.W., Washington, DC 20059, USA

² Department of Biochemistry and Molecular Biology, Howard University College of Medicine, 520 W Street N.W., Washington, DC 20059, USA

³ Program in Genetics, Howard University College of Medicine, 520 W Street N.W., Washington, DC 20059, USA

⁴ Department of Biochemistry and Molecular Biology, The George Washington University Medical Center, 2300 I Street N.W., Washington, DC 20037, USA

⁵ Children's National Medical Center, CRI Center III, 111 Michigan Ave., N.W. Washington, D.C. 20010-2970, USA

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Retrovirology 2006, 3:78 doi:10.1186/1742-4690-3-78

Published: 3 November 2006

Abstract

Background

Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription.

Results

We analyzed HIV-1 Tat phosphorylation by CDK2 in vitro and identified Ser¹⁶ and Ser⁴⁶ residues of Tat as potential phosphorylation sites. Tat was phosphorylated in HeLa cells infected with Tat-expressing adenovirus and metabolically labeled with ³²P. CDK2-specific siRNA reduced the amount and the activity of cellular CDK2 and significantly decreased phosphorylation of Tat. Tat co-migrated with CDK2 on glycerol gradient and co-immunoprecipitated with CDK2 from the cellular extracts. Tat was phosphorylated on serine residues in vivo, and mutations of Ser¹⁶ and Ser⁴⁶ residues of Tat reduced Tat phosphorylation in vivo. Mutation of Ser¹⁶ and Ser⁴⁶ residues of Tat reduced HIV-1 transcription in transiently transfected cells. The mutations of Tat also inhibited HIV-1 viral replication and Tat phosphorylation in the context of the integrated HIV-1 provirus. Analysis of physiological importance of the S¹⁶QP(K/R)¹⁹ and S⁴⁶YGR⁴⁹ sequences of Tat showed that Ser¹⁶ and Ser⁴⁶ and R⁴⁹ residues are highly conserved whereas mutation of the (K/R)¹⁹ residue correlated with non-progression of HIV-1 disease.

Conclusion

Our results indicate for the first time that Tat is phosphorylated in vivo; Tat phosphorylation is likely to be mediated by CDK2; and phosphorylation of Tat is important for HIV-1 transcription.