Ectopic USP15 expression inhibits HIV-1 transcription involving changes in YY1 deubiquitination and stability.

Introduction: Protein homeostasis is maintained by the opposing action of ubiquitin ligase and deubiquitinase, two important components of the ubiquitin-proteasome pathway, and contributes to both normal physiological and pathophysiological processes. The current study aims to delineate the roles of ubiquitin-specific protease 15 (USP15), a member of the largest deubiquitinase family, in HIV-1 gene expression and replication. Methods: We took advantage of highly selective and specific ubiquitin variants (UbV), which were recently designed and developed for USP15, and ascertained the inhibitory effects of USP15 on HIV-1 gene expression and production by transfection and Western blotting. We also used real-time RT-PCR, transcription factor profiling, subcellular fractionation, immunoprecipitation followed by Western blotting to determine the transcription factors involved and the underlying molecular mechanisms. Results: We first confirmed the specificity of USP15-mediated HIV-1 gene expression and virus production. We then showed that the inhibition of HIV-1 production by USP15 occurred at the transcription level, associated with an increased protein level of YY1, a known HIV-1 transcription repressor. Moreover, we demonstrated that USP15 regulated YY1 deubiquitination and stability. Lastly, we demonstrated that YY1 siRNA knockdown significantly diminished the inhibition of USP15 on HIV-1 gene expression and virus production. Conclusion: These findings together demonstrate that stabilization of YY1 protein by USP15 deubiquitinating activity contributes to USP15-mediated inhibition of HIV-1 transcription and may help the development of USP15-specific UbV inhibitors as an anti-HIV strategy.