Abstract
HIV-1 Tat is a key regulator of viral transcription, however little is known about the mechanisms that control its turnover in T cells. Here we use a novel proteomics technique, called DiffPOP, to identify the molecular target of JIB-04, a small molecule compound that potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines. Mass-spectrometry analysis of whole-cell extracts from 2D10 Jurkat T cells revealed that JIB-04 targets Serine Hydroxymethyltransferase 2 (SHMT2), a regulator of glycine biosynthesis and an adaptor for the BRCC36 K63Ub-specific deubiquitinase in the BRISC complex. Importantly, knockdown of SHMT1,2 or BRCC36, or exposure of cells to JIB-04, strongly increased Tat K63Ub-dependent destruction via autophagy. Moreover, point mutation of multiple lysines in Tat, or knockdown of BRCC36 or SHMT1,2, was sufficient to prevent destruction of Tat by JIB-04. We conclude that HIV-1 Tat levels are regulated through K63Ub-selective autophagy mediated through SHMT1,2 and the BRCC36 deubiquitinase.
Highlights
The HIV-1 Tat transactivator stimulates RNAPII transcription elongation at the proviral promoter in response to increased levels of the positive transcription elongation factor-b (P-TEFb/CDK9) complex in activated T cells [1,2,3]
The HIV-1 Tat protein is critical for virus expression and replication, but little is known about the factors that control its expression level in T cells
We identified the Serine Hydroxymethyltransferase 2 (SHMT2) enzyme, a regulator of glycine biosynthesis and an adaptor for the BRCC36 K63-specific deubiquitinase, as a prominent molecular target of JIB-04
Summary
The HIV-1 Tat transactivator stimulates RNAPII transcription elongation at the proviral promoter in response to increased levels of the positive transcription elongation factor-b (P-TEFb/CDK9) complex in activated T cells [1,2,3]. Because Tat protein levels are limiting in latently-infected resting memory CD4+ T cells, events that facilitate the assembly and activation of functional Tat:P-TEFb complexes are generally sufficient to counteract viral latency [5,6,7]. Tat-mediated induction of HIV-1 transcription leads to full-blown infection and virus replication in activated T cells. For these reasons, it is important to fully define the mechanism of Tat transactivation, and identify the factors that control Tat expression levels in infected cells
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