title: The Epstein-Barr virus (EBV) deubiquitinating enzyme BPLF1 reduces EBV ribonucleotide reductase activity.
abstract: A newly discovered virally encoded deubiquitinating enzyme (DUB) is strictly conserved across the Herpesviridae. Epstein-Barr virus (EBV) BPLF1 encodes a tegument protein (3,149 amino acids) that exhibits deubiquitinating (DUB) activity that is lost upon mutation of the active-site cysteine. However, targets for the herpesviral DUBs have remained elusive. To investigate a predicted interaction between EBV BPLF1 and EBV ribonucleotide reductase (RR), a functional clone of the first 246 N-terminal amino acids of BPLF1 (BPLF1 1-246) was constructed. Immunoprecipitation verified an interaction between the small subunit of the viral RR2 and BPLF1 proteins. In addition, the large subunit (RR1) of the RR appeared to be ubiquitinated both in vivo and in vitro; however, ubiquitinated forms of the small subunit, RR2, were not detected. Ubiquitination of RR1 requires the expression of both subunits of the RR complex. Furthermore, coexpression of RR1 and RR2 with BPLF1 1-246 abolishes ubiquitination of RR1. EBV RR1, RR2, and BPLF1 1-246 colocalized to the cytoplasm in HEK 293T cells. Finally, expression of enzymatically active BPLF1 1-246 decreased RR activity, whereas a nonfunctional active-site mutant (BPLF1 C61S) had no effect. These results indicate that the EBV deubiquitinating enzyme interacts with, deubiquitinates, and influences the activity of the EBV RR. This is the first verified protein target of the EBV deubiquitinating enzyme.
authors.affiliation: Departments of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Whitehurst
authors.forename: Shunbin
authors.initials: S
authors.lastname: Ning
authors.forename: Gretchen L
authors.initials: GL
authors.lastname: Bentz
authors.forename: Florent
authors.initials: F
authors.lastname: Dufour
authors.forename: Edward
authors.initials: E
authors.lastname: Gershburg
authors.forename: Julia
authors.initials: J
authors.lastname: Shackelford
authors.forename: Yves
authors.initials: Y
authors.lastname: Langelier
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Pagano
mesh_terms: D002460:Cell Line; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D016679:Genome, Viral / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C495488:BPLF1 protein, Epstein-Barr virus; D021122:Protein Subunits; D012333:RNA, Messenger; D011994:Recombinant Proteins; D054334:Viral Regulatory and Accessory Proteins; D012264:Ribonucleotide Reductases