HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7

Abstract

Background: Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1. Methods: HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein-protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results: We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, Conclusions: Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether , our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

HIV-1 Vpr Induces Degradation of Nucleolar Protein CCDC137 as a Consequence of Cell Cycle Arrest

“…The use of cell-cycle arrest null mutants has been used extensively to decouple Vpr's protein degradation and cell cycle arrest functions. For example, MUS81 (DePaula-Silva et al, 2015), helicase-like transcription factor (HLTF) (Lahouassa et al, 2016), tet methylcytosine dioxygenase 2 (TET2) (Lv et al, 2018), and SIRT7 (Zhou et al, 2021) are targeted for depletion by WT Vpr and by cell-cycle arrest null mutants, such as R80A, indicating that degradation of these cellular targets are not associated with cell-cycle arrest. However, steps leading the degradation of CCDC137 by cell cycle null R80A Vpr mutant are unclear.…”

“…At the G2/M transition, CDK1 is activated, rRNA transcription resumes, and the nucleolus reassembles (Hayashi et al, 2018). Given the finding that Vpr expression is associated with degradation of nucleolar proteins CCDC137 (Zhang and Bieniasz, 2020) and SIRT7 (Zhou et al, 2021), it is plausible that Vpr cell cycle arrest may favor viral replication and transactivation through delayed assembly of the nucleolus as a consequence of CDK1 inhibitory phosphorylation. By analogy to adenovirus E1A protein antagonism of CCDC137 to enhance RAR transactivation, it is also possible that Vpr binding and degradation of CCDC137 displaces a protein, such as a transcription factor, that is necessary for enhanced viral transcription and replication.…”

“…CCDC137 is antagonized by another viral protein, adenovirus E1A protein, that was shown to disrupt CCDC137 association with RAR, which results in release of RAR into the nucleoplasm and increased transcription of RAR target genes (Um et al, 2014). Recently, Vpr has been shown to target sirtuin 7 (SIRT7), a protein that resides predominantly in the nucleolus (Zhou et al, 2021).…”