Impairment of HIV-1 replication by UBE3A- and HIV-1 Nef-mediated regulation of proteasomal degradation.

To date, regulation of HIV-1 life cycle has been mainly explained by the stage-specific expression of HIV-1 viral genes, even if elimination processes of the synthesized proteins after completion of their duties in the infected cells were quintessential for understanding of the molecular processes of the virus life cycle and thereby HIV-1 pathogenesis. Several lines of our experiments demonstrated that a key pathogenic HIV-1 viral protein, Nef, interacted with ubiquitin (Ub)-protein ligase E3A (UBE3A/E6AP), an important E3 Ub ligase in the proteasomal degradation processes, suggesting that interaction between Nef and UBE3A is integral in regulation of viral and cellular protein decay and thereby competition for survival between the entered HIV-1 and the infected host cells. In fact, Nef and UBE3A degraded reciprocally, and UBE3A-mediated degradation of Nef was significantly more potent than Nef-triggered degradation of UBE3A. Further, UBE3A degraded not only Nef but also HIV-1 structural proteins, Gag, thereby significantly inhibited HIV-1 replication in Jurkat T cells. However, UBE3A failed to induce decay of Gag in ∆nef-HIV-1 replicating cells, indicating that interaction between Nef and UBE3Awas pivotal for UBE3A-mediated degradation of the viral proteins. In contrast, Gag and Env did not degrade UBE3A. Mechanistic study showed that Nef and UBE3A were specific and antagonistic each other in regulating proteasome activity and ubiquitination of cellular proteins in general. Further, excess, not stoichiometric, amount of Nef reduced the amount of intracellular Gag as well as degraded a cardinal transcription regulator, Tat, demonstrating a broad role of Nef in regulation of HIV-1 life cycle. Structure and function analysis of Nef indicated that specific domains of Nef overlapping with LTR were essential for the observed actions. Taken together, these data indicated that the Nef and UBE3A complex play a pivotal role in coordinating viral protein degradation and hence HIV-1 replication, providing insights as to the nature of pathobiologic and defense strategies of HIV-1 and HIV-infected host cells and major clues for physical knockout of key viral pathogenic element, Nef, using UBE3A by the ubiquitin proteasome system.