Activation of HIV Transcription by the Viral Tat Protein Requires a Demethylation Step Mediated by Lysine-specific Demethylase 1 (LSD1/KDM1)

Naoki Sakane,Kara G Lassen,Katrin Kaehlcke,Sara Pagans,Martina Schnoelzer,Yasuhiro Mizusawa,Hye-Sook Kwon,Masafumi Kamada,Melanie Ott,Jonathan Chan,Warner C Greene

doi:10.1371/journal.ppat.1002184

Abstract

The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear.We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells.Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation of K51 in Tat. Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells.

Highlights

Epigenetic processes are critical in the regulation of gene expression from the integrated HIV provirus and have become a focal point of research in therapeutics for HIV latency
Viral transcription can be reactivated in latently infected cells, a process that rekindles HIV infection after antiretroviral therapy is discontinued
In HIV infection, it acts as a transcriptional activator because downregulation of LSD1 expression or inhibition of its enzymatic activity suppresses reactivation of HIV from latency

Summary

Introduction

Epigenetic processes are critical in the regulation of gene expression from the integrated HIV provirus and have become a focal point of research in therapeutics for HIV latency. Two Tat species naturally exist in HIV-infected cells: a full-length Tat protein of ,101 aa length encoded by both tat exons and a shorter splice variant of 72 aa length encoded by the first tat exon. Both Tat forms are transcriptionally active and form a trimolecular complex with the cyclin T1 subunit of P-TEFb and TAR RNA to recruit the kinase activity of CDK9 to elongating HIV transcripts. The bulk of Tat is produced after successful integration of the provirus into the host genome where it activates its own production via a feed-forward mechanism [19]

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