A Functional Ubiquitin-Specific Protease Embedded in the Large Tegument Protein (ORF64) of Murine Gammaherpesvirus 68 Is Active during the Course of Infection

Abstract

All herpesviruses contain a ubiquitin (Ub)-specific cysteine protease domain embedded within their large tegument protein, based on homology with the corresponding sequences of UL36 from herpes simplex virus type 1 and M48 from murine cytomegalovirus. This type of activity has yet to be demonstrated for cells infected with a gammaherpesvirus. By activity-based profiling, we show that the large tegument protein of murine gammaherpesvirus (MHV-68) ORF64 (273 kDa) is a functional deubiquitinating protease, as assessed by tandem mass spectrometry of adducts in extracts from MHV-68-infected cells that had been labeled with ubiquitin vinylmethylester, a ubiquitin-based active site-directed probe. The recombinantly expressed aminoterminal segment of ORF64 displays deubiquitinating activity toward Ub C-terminal 7-amido-4-methylcoumarin in vitro. The findings reported here for MHV-68 ORF64 extend those made for the alpha-and betaherpesvirus families and are consistent with an important, conserved enzymatic function of the tegument protein.

A herpesvirus ubiquitin-specific protease is critical for efficient T cell lymphoma formation

“…Nonetheless, sequence comparisons across the Herpesviridae show the presence of a few absolutely conserved residues (Cys, Asp, His, Glu), all of which are consistent with involvement in a potential thiol protease active site. We have, meanwhile, confirmed both the mechanism of action of such ubiquitin-based probes (11)(12)(13)(14)(15) and the identity of the viral cysteine protease domain as an authentic USP by crystallographic analysis of the homologous segment of the murine cytomegalovirus (MCMV) M48 protein (16). Notwithstanding the conservation of the identified USP activity in all herpesviruses, we do not know whether this activity makes a contribution to the replicative success and pathogenicity of herpesviruses in vivo.…”

Histone Deacetylases in Herpesvirus Replication and Virus-Stimulated Host Defense

“…Activity-based protein profiling (ABPP) has been applied to the examination of ubiquitin proteases during herpesvirus infection [136]. Chemoproteomic approaches have also been used to resolve the composition of HDAC complexes and to assess the ability of small molecules to inhibit specific HDACs [26].…”

The Epstein-Barr Virus (EBV) Deubiquitinating Enzyme BPLF1 Reduces EBV Ribonucleotide Reductase Activity

“…The N-terminal fragment of the herpes simplex virus (HSV) homolog (UL36), which encompasses DUB and cleaves polyubiquitin chains, appears to be specific for K48 linkages; cleavage of K63-Ub chains was not detected (25). In addition, DUBs from the alphaherpesviruses and gammaherpesviruses are Ub specific and show little or no preference for other Ub-like molecules, such as Nedd8, SUMO, and ISG15 (21,25).…”