A functional ubiquitin-specific protease embedded in the large tegument protein (ORF64) of murine gammaherpesvirus 68 is active during the course of infection.
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 amino-terminal 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.
Gredmark
Sara
S
Whitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142, USA.
Schlieker
Christian
C
Quesada
Victor
V
Spooner
Eric
E
Ploegh
Hidde L
HL
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000821:virology; D018142:Gammaherpesvirinae / Q000201:enzymology* / Q000235:genetics; D006566:Herpesviridae Infections / Q000821:virology*; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010802:Phylogeny; D011487:Protein Conformation; D013379:Substrate Specificity; D064570:Ubiquitin-Specific Proteases; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D014764:Viral Proteins; D010450:Endopeptidases; D064570:Ubiquitin-Specific Proteases