The molecular chaperone calnexin interacts with the NSP4 enterotoxin of rotavirus in vivo and in vitro.
Calnexin is an endoplasmic reticulum (ER)-associated molecular chaperone proposed to promote folding and assembly of glycoproteins that traverse the secretory pathway in eukaryotic cells. In this study we examined if calnexin interacts with the ER-associated luminal (VP7) and transmembrane (NSP4) proteins of rotavirus. Only glycosylated NSP4 interacted with calnexin and did so in a time-dependent manner (half-life, 20 min). In vitro translation experiments programmed with gene 10 of rhesus rotavirus confirmed that calnexin recognizes only glycosylated NSP4. Castanospermine (a glucosidase I and II inhibitor) experiments established that calnexin associates only with partly deglucosylated (di- or monoglucosylated) NSP4. Furthermore, enzymatic removal of the remaining glucose residues on the N-linked glycan units was essential to disengage the NSP4-calnexin complex. Novel experiments with castanospermine revealed that glucose trimming and the calnexin-NSP4 interaction were not critical for the assembly of infectious virus.
Department of Virology, SMI/Karolinska Institute, 105 21 Stockholm, Sweden.
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Mirazimi
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Nilsson
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Svensson
D000818:Animals; D000956:Antigens, Viral; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D002213:Capsid / Q000378:metabolism; D036022:Capsid Proteins; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000821:virology; D004768:Enterotoxins / Q000737:chemistry / Q000378:metabolism*; D005814:Genes, Viral; D005947:Glucose / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D066298:In Vitro Techniques; D008253:Macaca mulatta; D018832:Molecular Chaperones / Q000378:metabolism*; D014176:Protein Biosynthesis; D012401:Rotavirus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D014774:Virulence; D000520:alpha-Glucosidases / Q000378:metabolism
D000956:Antigens, Viral; D002135:Calcium-Binding Proteins; D036022:Capsid Proteins; D017931:DNA Primers; D004768:Enterotoxins; D006023:Glycoproteins; D018832:Molecular Chaperones; C081369:NS28 protein, rotavirus; D014118:Toxins, Biological; C050779:VP7 protein, Rotavirus; D017361:Viral Nonstructural Proteins; D037281:Calnexin; C034251:4-nitrophenyl-alpha-glucosidase; D000520:alpha-Glucosidases; D005947:Glucose