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.

Discovery of Nanomolar DCAF1 Small Molecule Ligands

“…In addition, it could be further developed to Antagonists that disrupt protein−protein interactions between DCAF1 and its interacting partners could be used toward development of therapeutics for cancers when DCAF1 is overexpressed, 26 and HIV when DCAF1 is hijacked by the virus. [15][16][17]23 In these cases, the small molecule prevents the binding of Vpr and Vpx to DCAF1, for example, preventing viral hijacking of the CRL4 DCAF1 or EDVP DCAF1 complexes. Another potential application of antagonizing CRL4 DCAF1 function is in Merlin-deficient tumors.…”

“…Vpr binds to DCAF1 to hijack the CRL4 DCAF1 E3 ligase complex to induce cell cycle arrest . CRL4 DCAF1 -Vpr also targets host proteins such as uracil DNA glycosylase 2 (UNG2), endoribonuclease Dicer, and histone deacetylase SIRT7 for ubiquitination leading to protein degradation. The co-crystal structure of DDB1-DCAF1-Vpr-UNG2 (PDB: 5jk7) reveals the detailed interactions on how Vpr mimics DNA binding to UNG2 for subsequent protein degradation .…”

E3 ligase adaptor FBXO7 contributes to ubiquitination and proteasomal degradation of SIRT7 and promotes cell death in response to hydrogen peroxide

“…Our study presents additional evidence for the canonical role of FBXO7 in the latter case, along with identifying SIRT7 as its novel target. In addition to FBXO7, SIRT7 has also been reported to be polyubiquitinated by HIV-1 Vpr ( 45 ), a multifunctional accessory protein critical for efficient viral infection of target cells. Vpr interacts with the CRL4–DCAF1 ubiquitin E3 ligase complex and induces the proteasome degradation of SIRT7 through CRL4–DCAF1 ( 45 ).…”

“…In addition to FBXO7, SIRT7 has also been reported to be polyubiquitinated by HIV-1 Vpr ( 45 ), a multifunctional accessory protein critical for efficient viral infection of target cells. Vpr interacts with the CRL4–DCAF1 ubiquitin E3 ligase complex and induces the proteasome degradation of SIRT7 through CRL4–DCAF1 ( 45 ). Similar to the FBXO7-ΔU mutant lacking the Ubl domain, and thus disabling the recruitment of substrates for ubiquitination, the PD-linked FBXO7-R498X mutation, which is known to inhibit SCF FBXO7 E3 ligase activity, did not reduce SIRT7 stability.…”

Unlocking DCAFs To Catalyze Degrader Development: An Arena for Innovative Approaches

“…This approach takes advantage of the fact that various types of viruses interact extensively with many CRL components, which allows them to manipulate specific host cell proteins for their own benefit. ,,, For example, CRL4 DCAF1 was initially found to be hijacked by the HIV-1 accessory viral protein R (Vpr), leading to its alternative name as Vpr-binding protein (VprBP). In this context, Vpr targets host cell proteins including uracil DNA glycosylase 2 (UDG2), endoribonuclease Dicer, and deacetylase sirtuin 7 (SIRT7) for proteasomal degradation, thereby prolonging HIV-1 replication. , Additionally, Vpr can also hijack the HECT-type E3 ligase UBR5 (also referred to as EDD)–DYRK2–DDB1 DCAF1 complex, leading to the degradation of host cell proteins CP110 and katanin ((Figure A, B). − Likewise, the accessory viral protein X (Vpx) of HIV-2 and its related simian immunodeficiency virus (SIV) was found to hijack CRL4 DCAF1 for proteasomal degradation of the host cell protein SAMHD1. , In this context, Cullgen, a pure play in TPD, successfully identified ligands that bind to the virally hijacked DDB1 and incorporated them into the design of PROTACs (see A17 and A18 in Figure C). , To date, structural studies have provided valuable insights into the interactions between these viral proteins and the CRL components. − Careful examination of these PPIs may guide the development of small-molecule ligands for CRL components, and we expect to see more of this practice in the future.…”