ATP is required for correct folding and disulfide bond formation of rotavirus VP7.
Rotavirus is one of very few viruses that utilize the endoplasmic reticulum (ER) for assembly, and therefore it has been used as an attractive model to study ER-associated protein folding. In this study, we have examined the requirements for metabolic energy (ATP) for correct folding of the luminal and ER-associated VP7 of rotavirus. We found that VP7 rapidly misfolds in an energy-depleted milieu and is not degraded within 60 min. We also found that VP7 attained a stable minimum-energy state soon after translation in the ER. Most surprisingly, energy-misfolded VP7 could be recovered and establish correct disulfide bonds and antigenicity following a shift to an ATP-rich milieu. Using a Semliki Forest virus expression system, we observed that VP7 requires ATP and cellular, but not viral, factors for correct disulfide bond formation. Our results show for the first time that the disulfide bond formation of rotavirus VP7 is an ATP-dependent process. It has previously been shown that chaperones hydrolyze ATP during interaction with newly synthesized polypeptides and prevent nonproductive intra- and intermolecular interactions. The most reasonable explanation for the energy requirement of VP7 is thus a close interaction during folding with an ATP-dependent chaperone, such as BiP (Grp78), and possibly with protein disulfide isomerase. Taken together, our observations provide new information about folding of ER-associated proteins in general and rotavirus VP7 in particular.
Department of Virology, Swedish Institute for Infectious Disease Control, Karolinska Institute, 171 82 Solna, Sweden.
A
A
Mirazimi
L
L
Svensson
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000956:Antigens, Viral; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D036022:Capsid Proteins; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008253:Macaca mulatta; D011233:Precipitin Tests; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D012401:Rotavirus / Q000737:chemistry / Q000378:metabolism*
D000956:Antigens, Viral; D036022:Capsid Proteins; D004220:Disulfides; C050779:VP7 protein, Rotavirus; D000255:Adenosine Triphosphate