Vaccinia virus G4L glutaredoxin is an essential intermediate of a cytoplasmic disulfide bond pathway required for virion assembly.
Our previous studies provided evidence that E10R, a vaccinia virus protein belonging to the ERV1/ALR family, has a redox function and is required for virion assembly. Repression of E10R prevented the formation of intramolecular disulfide bonds of the G4L glutaredoxin, the L1R membrane protein, and the structurally related F9L protein. Here, we demonstrate an oxidation pathway (E10R(SS) --> G4L(SS) --> L1R(SS), F9L(SS)) in which G4L occupies an intermediate position. By reacting free thiols with 4-acetamido-4'-malemideylstilbene-2,2'-disulfonic acid, alkylated and nonalkylated disulfide-bonded forms of G4L could be resolved from each other by polyacrylamide gel electrophoresis. The cysteines of intracellular G4L were in both disulfide and reduced forms, whereas those of E10R, L1R, and F9L and virion-associated G4L were mostly disulfide bonded. Repression of G4L expression prevented the formation of disulfide bonds in both L1R and F9L but not E10R. Both cysteines of G4L were required for L1R and F9L disulfide bond formation or for trans-complementation of virus infectivity when G4L expression was repressed. No role in the E10R-G4L redox pathway was found for O2L, a nonessential glutaredoxin encoded by vaccinia virus. We suggest that cytoplasmic G4L is a redox shuttle between membrane-associated E10R and L1R or F9L.
White
Christine L
CL
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0445, USA.
Senkevich
Tatiana G
TG
Moss
Bernard
B
D000478:Alkylation; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000821:virology*; D004220:Disulfides / Q000378:metabolism*; D005816:Genetic Complementation Test; D054477:Glutaredoxins; D016366:Open Reading Frames / Q000235:genetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D011506:Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly
D004220:Disulfides; D054477:Glutaredoxins; D011506:Proteins; D014764:Viral Proteins; D012694:Serine; D010088:Oxidoreductases; D003545:Cysteine