Abstract
HIV viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/replication stress signalling pathway through engagement of the DDB1-CUL4A-DCAF1 E3 ubiquitin ligase via a direct binding to the substrate specificity receptor DCAF1. Since no high resolution structures of the DDB1-DCAF1-Vpr substrate recognition module currently exist, we used a mutagenesis approach to better define motifs in DCAF1 that are crucial for Vpr and DDB1 binding. Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD). Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1. While we could not identify elements specifically involved in Vpr binding, overall, the mutagenesis data suggest that the predicted β-propeller conformation of DCAF1 is likely to be critical for Vpr association. Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex. Lastly, we show that expression of DCAF1 WD in the absence of endogenous DCAF1 was not sufficient to enable Vpr-mediated G2 arrest activity. Overall, our results reveal that Vpr and DDB1 binding on DCAF1 can be genetically separated and further suggest that DCAF1 contains determinants in addition to the Vpr and DDB1 minimal binding domain, which are required for Vpr to enable the induction of a G2 arrest.
Highlights
Human immunodeficiency virus type 1 (HIV-1) encodes several accessory proteins – Vif, viral protein R (Vpr), Vpu, and Nef – that have been implicated in the modulation of the host environment to promote viral replication and evasion of innate and acquired immunity
To confirm these results and delineate the minimal domain of DCAF1 required to recruit both Vpr and endogenous damaged DNA binding protein 1 (DDB1), we tested the ability of three different Myc-tagged DCAF1 variants, including full-length DCAF1 (1-1507), DCAF1 WD (1041-1393) and DCAF1 1377 (1041-1377) (Fig. 1A), to form a ternary complex when coexpressed with HA-Vpr in HEK293T cells
The minimal domain of DCAF1 capable of recruiting both Vpr and endogenous DDB1 is predicted to fold as a b-propeller, a structure which is similar to that already described for DDB1 and thought to be involved in protein-protein interactions [28]
Summary
Human immunodeficiency virus type 1 (HIV-1) encodes several accessory proteins – Vif, Vpr, Vpu, and Nef – that have been implicated in the modulation of the host environment to promote viral replication and evasion of innate and acquired immunity. Viral protein R (Vpr) was shown to engage a cullin 4A-ring E3 ubiquitin ligase (CRL4A) composed of cullin4A (CUL4A), the E3 ligase Roc, damaged DNA binding protein 1 (DDB1) and a member of the DCAF (DDB1–cullin4-associated-factor) family called DCAF1 or VprBP (viral protein R binding protein) [3,4,5,6,7,8,9]. By analogy with how other lentiviral accessory proteins usurp host E3 ubiquitin ligases, it was proposed that Vpr would recruit the CRL4A (DCAF1) E3 ligase to trigger poly-ubiquitination and subsequent degradation of a putative cellular protein(s) resulting in the activation of a G2 checkpoint. Whether Vpr acts as an adaptor to recruit a new substrate(s) to the CRL4A (DCAF1) E3 ligase for ubiquitination and proteasomal degradation or whether Vpr enhances the ubiquitination of a natural substrate(s) targeted by DCAF1 remains unresolved
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