The molecular chaperone calnexin interacts with the NSP4 enterotoxin of rotavirus in vivo and in vitro

Abstract

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.

FIG. 1

Association of calnexin with rotavirus proteins during protein maturation. Cells were mock infected or infected with RRV (MOI, 10). Cells infected with RRV were treated with TM or not (mock) treated. TM (2 μg/ml) was added to the medium of the monolayers at 5 hpi and was maintained throughout the experiment. At 7 hpi, cells were starved for 1 h in methionine- and cysteine-free medium and then metabolically labeled (250 μCi) for 5 min. To examine posttranslational processing, labeled proteins were chased with Eagle’s MEM supplemented with 1 mM cycloheximide and 10 mM methionine for the lengths of time indicated over the lanes. At the end of the chase, the monolayers were incubated with ice-cold PBS containing 40 mM NEM for 2 min. Cells were then harvested in lysis buffer. (A) The cell lysates from the pulse-chase experiments were immunoprecipitated with antibodies to calnexin and NSP4 and analyzed by reducing SDS-PAGE. Numbers at left are kilodaltons. (B) The amount of NSP4 bound to calnexin was measured by densitometry from the fluorograph shown in panel A, and the results are expressed as percentages of coprecipitated NSP4 and calnexin.

FIG. 1

Association of calnexin with rotavirus proteins during protein maturation. Cells were mock infected or infected with RRV (MOI, 10). Cells infected with RRV were treated with TM or not (mock) treated. TM (2 μg/ml) was added to the medium of the monolayers at 5 hpi and was maintained throughout the experiment. At 7 hpi, cells were starved for 1 h in methionine- and cysteine-free medium and then metabolically labeled (250 μCi) for 5 min. To examine posttranslational processing, labeled proteins were chased with Eagle’s MEM supplemented with 1 mM cycloheximide and 10 mM methionine for the lengths of time indicated over the lanes. At the end of the chase, the monolayers were incubated with ice-cold PBS containing 40 mM NEM for 2 min. Cells were then harvested in lysis buffer. (A) The cell lysates from the pulse-chase experiments were immunoprecipitated with antibodies to calnexin and NSP4 and analyzed by reducing SDS-PAGE. Numbers at left are kilodaltons. (B) The amount of NSP4 bound to calnexin was measured by densitometry from the fluorograph shown in panel A, and the results are expressed as percentages of coprecipitated NSP4 and calnexin.

FIG. 2

Calnexin recognizes in vitro-translated NSP4. (A) In vitro translation control with rabbit pancreatic microsomes (lane a) and with gene 10 in the presence or absence of rabbit pancreatic microsomes. (B) Immunoprecipitation of in vitro-translated NSP4 shown in panel A. Numbers at left are kilodaltons.

FIG. 3

Effect of CST on the folding of NSP4. Cells were infected with RRV (MOI, 10), and at 7 hpi, monolayers were either not treated or treated with CST (1 mM) for 1 h before the pulse, during the pulse, and during the chase. Cells were starved for 1 h in methionine- and cysteine-free medium and then metabolically labeled (250 μCi) for 5 min. Labeled proteins were chased in the presence or absence of 1 mM CST in Eagle’s MEM supplemented with 1 mM cycloheximide and 10 mM methionine for the lengths of time indicated over the lanes. At the end of the chase, the monolayers were incubated with ice-cold PBS containing 40 mM NEM for 2 min. Cells were then harvested in lysis buffer. The cell lysates were immunoprecipitated with antibodies to calnexin and NSP4 and analyzed by reducing SDS-PAGE. Numbers at left are kilodaltons.

FIG. 4

Dissociation of calnexin from NSP4. Cells were infected with RRV (MOI, 10). At 7 hpi, cells were starved for 1 h in methionine- and cysteine-free medium and then metabolically labeled (250 μCi) for 5 min. Labeled proteins were chased in the presence or absence of 1 mM CST in Eagle’s MEM supplemented with 1 mM cycloheximide and 10 mM methionine for the lengths of time indicated over the lanes. At the end of the chase, the monolayers were incubated with ice-cold PBS containing 40 mM NEM for 2 min. Cells were then harvested in lysis buffer. The cell lysates were immunoprecipitated with antibodies to calnexin and analyzed by reducing SDS-PAGE. Numbers at left are kilodaltons.