Carbohydrates facilitate correct disulfide bond formation and folding of rotavirus VP7.
It is well established that glycosylation is essential for assembly of enveloped viruses, but no information is yet available as to the function of carbohydrates on the nonenveloped but glycosylated rotavirus. We show that tunicamycin and, more pronouncedly, a combination of tunicamycin and brefeldin A treatment caused misfolding of the luminal VP7 protein, leading to interdisulfide bond aggregation. While formation of VP7 aggregates could be prevented under reducing conditions, they reoccurred in less than 30 min after a shift to an oxidizing milieu. Furthermore, while glycosylated VP7 interacted during maturation with protein disulfide isomerase, nonglycosylated VP7 did not, suggesting that glycosylation is a prerequisite for protein disulfide isomerase interaction. While native NSP4, which does not possess S-S bonds, was not dependent on N-linked glycosylation or on protein disulfide isomerase assistance for maturation, nonglycosylated NSP4 was surprisingly found to interact with protein disulfide isomerase, further suggesting that protein disulfide isomerase can act both as an enzyme and as a chaperone. In conclusion, our data suggest that the major function of carbohydrates on VP7 is to facilitate correct disulfide bond formation and protein folding.
Mirazimi
A
A
Department of Virology, SMI/Karolinska Institute, Stockholm, Sweden.
Svensson
L
L
D000818:Animals; D000956:Antigens, Viral / Q000276:immunology / Q000378:metabolism*; D002213:Capsid / Q000276:immunology / Q000378:metabolism*; D036022:Capsid Proteins; D050260:Carbohydrate Metabolism; D002460:Cell Line; D004220:Disulfides; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D008253:Macaca mulatta; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D017510:Protein Folding; D012401:Rotavirus / Q000276:immunology / Q000378:metabolism*; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
D000956:Antigens, Viral; D036022:Capsid Proteins; D004220:Disulfides; D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D014118:Toxins, Biological; C050779:VP7 protein, Rotavirus; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D019704:Protein Disulfide-Isomerases