Abstract
Viral interferon regulatory factor 1 (vIRF1), a Kaposi sarcoma herpesvirus protein, destabilizes p53 by inhibiting p53 acetylation and Hdm2 phosphorylation. This leads to increased ubiquitination and degradation of p53 by Hdm2, which cripples the cellular p53-mediated antiviral response. Ubiquitin-specific protease 7 (USP7) deubiquitinates p53 and Hdm2 and regulates their stability. We identified an EGPS consensus sequence in vIRF1, which is identical to that found in Epstein-Barr virus nuclear antigen 1 (EBNA1) that interacts with the N-terminal domain of USP7 (USP7-NTD). GST pulldown assays demonstrated that vIRF1 interacts with USP7-NTD via its EGPS motif. NMR heteronuclear single quantum correlation (HSQC) analysis revealed chemical perturbations after titration of USP7-NTD with vIRF1 (44)SPGEGPSGTG(53) peptide. In contrast, these perturbations were reduced with a mutant vIRF1 peptide, (44)SPGEGPAGTG(53). Fluorescence polarization analysis indicated that the vIRF1 peptide interacted with USP7-NTD with a Kd of 2.0 μm. The crystal structure of the USP7-NTD·vIRF1 peptide complex revealed an identical mode of binding as that of the EBNA1 peptide to USP7-NTD. We also showed that USP7 interacts with vIRF1 in U2OS cells. Decreased levels of p53, but not Hdm2 or ataxia telangiectasia-mutated (ATM), were seen after expression of vIRF1, but not with a vIRF1 mutant protein. Our results support a new role for vIRF1 through deregulation of the deubiquitinating enzyme USP7 to inhibit p53-mediated antiviral responses.
Highlights
The Viral interferon regulatory factor 1 (vIRF1) DNA binding domain (DBD) has ϳ40% sequence similarity to the DBDs of human IRF3 and IRF7 and contains a helix-turn-helix motif, which is common in interferon regulatory factors (IRFs) and DNA-binding proteins [8]. vIRF1-mediated deregulation of IRF3 and IRF7 leads to disruption of cellular antiviral activity. vIRF1 is a potent inhibitor of the histone acetyltransferase activity of p300
Identification of an EGPS Sequence in Kaposi sarcoma herpesvirus (KSHV) vIRF1—It is well established that many ubiquitin-specific protease 7 (USP7) substrates including Hdm2, p53, and HdmX contain (P/A)XXS motifs for interaction with USP7-NTD through the highly conserved 164DWGF167 motif with dissociation constant (Kd) values ranging from 10 to 45 M (18 –20)
We utilized ScanProsite to search for viral proteins containing the DPGEGPST sequence of Epstein-Barr virus nuclear antigen 1 (EBNA1) and identified 44SPGEGPSG51 from KSHV vIRF1 [37]
Summary
Protein Expression—N-terminal hexahistidine-tagged WT and D164A/W165A mutant USP7-NTD were expressed from the pET15b vector as described previously [18]. FITC-labeled WT (FITC-Acp-44SPGEGPSGTG53-NH2) and mutant (FITC-Acp-44SPGEGPAGTG53-NH2) vIRF1 peptides were synthesized by CanPeptide Inc. Interaction of USP7-NTD with WT (44SPGEGPSGTG53) and mutant (44SPGEGPAGTG53) vIRF1 peptides was monitored by analyzing 1H-15N HSQC spectra. GST Pulldown Assay—Cells expressing GST-tagged fusion proteins were lysed using sonication in 50 mM Tris, pH 7.5, 150 mM NaCl, 1ϫ protease inhibitor cocktail (1 mM benzamidine and 0.5 mM PMSF) and 1ϫ protease inhibitor tablet (Roche Applied Science cOmplete ULTRA Tablets). The beads were washed extensively with 50 mM Tris, pH 7.5, and 150 mM NaCl. GST-tagged WT and deletion mutant vIRF11–90 were kept bound to the glutathione-Sepharose beads. The bound proteins were eluted with 20 mM reduced glutathione and detected by Coomassie Blue staining following SDS-PAGE
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
