Abstract
Transcription complexes that assemble at the HIV-1 promoter efficiently initiate transcription but generate paused RNA polymerase II downstream from the start site. The virally encoded Tat protein hijacks positive transcription elongation factor b (P-TEFb) to phosphorylate and activate this paused polymerase. In addition, Tat undergoes a series of reversible post-translational modifications that regulate distinct steps of the transcription cycle. To identify additional functionally important Tat cofactors, we performed RNAi knockdowns of sixteen previously identified Tat interactors and found that a novel E3 ligase, PJA2, ubiquitinates Tat in a non-degradative manner and specifically regulates the step of HIV transcription elongation. Interestingly, several different lysine residues in Tat can function as ubiquitin acceptor sites, and variable combinations of these lysines support both full transcriptional activity and viral replication. Further, the polyubiquitin chain conjugated to Tat by PJA2 can itself be assembled through variable ubiquitin lysine linkages. Importantly, proper ubiquitin chain assembly by PJA2 requires that Tat first binds its P-TEFb cofactor. These results highlight that both the Tat substrate and ubiquitin modification have plastic site usage, and this plasticity is likely another way in which the virus exploits the host molecular machinery to expand its limited genetic repertoire.
Highlights
Tat is regulated by several post-translational modifications that greatly expand the activity of this small viral protein
A very small quantity of Tat plasmid that was in the linear range of activation for the HIV promoter was used in the screen, ensuring physiological Tat protein levels
Examining the functions of these nine positives revealed an unexpected enrichment for ubiquitin signaling proteins, with three E3 ligases (ZFP91, PJA2, and UBE2O)[37,38,39,40,41], two substrate receptors for multi-subunit E3 ligases (DCAF16, FBX3)[42,43], and one de-ubiquitinating enzyme (USP11)[44] (Fig. 1c)
Summary
Tat is regulated by several post-translational modifications that greatly expand the activity of this small viral protein. The tumor suppressor, p53, and proto-oncogene, Myc, are both tightly regulated by numerous ubiquitin ligases that modify multiple lysine residues in each protein Ubiquitin modification of these transcription factors can lead to proteasomal degradation[27,28,29,30], protein stabilization[31,32], or protein re-localization[33,34]. PJA2, is a RING finger E3 ligase that polyubiquitinates Tat. One of these, PJA2, is a RING finger E3 ligase that polyubiquitinates Tat These modifications are non-degradative and rather serve to regulate the transcriptional activity of Tat. Tat ubiquitination by PJA2 is an incredibly diverse signal, both at the substrate level, where multiple Tat lysines can function as ubiquitin acceptor sites, and at the level of the modification itself, where polyubiquitin chains can be generated through multiple linkages. This immense signal plasticity likely allows the virus to potently activate transcription in the face of a high mutation rate
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