Carbohydrates facilitate correct disulfide bond formation and folding of rotavirus VP7

Abstract

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.

FIG. 1

Role of glycosylation and oligosaccharide processing in folding of NSP4 and VP7. Cells infected with RRV (MOI of 10) were mock or BFA (2 μg/ml) treated at 1 hpi. At 5 hpi, TM (2 μg/ml) was added to the medium of the same monolayers (as indicated) and was maintained through the experiment. At 7 hpi, cells were starved for 1 h in methionine-cysteine-free media and then subsequently metabolically labeled (50 μCi) for 30 min. To examine posttranslational processing, labeled proteins were chased 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 pulse or chase, monolayers were incubated with ice-cold PBS with 40 mM NEM for 2 min, and cells were then harvested in SDS lysis buffer and mixed with nonreducing sample buffer (A) or reducing sample buffer (B), boiled, and analyzed by SDS-PAGE.

FIG. 2

To identify the component of the disulfide aggregate presented in Fig. 1A, the 78-kDa and NSP4 bands from the gel presented in Fig. 1A (TM+BFA, 60-min chase) were excised and incubated with elution buffer (10 mM Tris-HCl [pH 7], 0.1% SDS) for 2 h at 37°C. The eluted 78-kDa protein was analyzed by nonreducing (lane b) and reducing (lane c) SDS-PAGE. The eluted NSP4 protein was analyzed by nonreducing SDS-PAGE (lane a). The lysate from the pulse-chase experiment presented in Fig. 1A (TM+BFA, 60-min chase) was immunoprecipitated with a VP7 MAb (M60) (lane d). M, molecular mass markers. The molecular masses (kilodaltons) of the markers are shown in the middle of the panel.

FIG. 3

(A) Effect of a reducing milieu on folding of TM- and/or BFA-treated VP7. Infected cells were TM and/or BFA treated as described in the legend to Fig. 1. At 7 hpi, cells were starved for 1 h in methionine-cysteine-free media. Twenty minutes before a 30-min metabolical ³⁵S pulse, 2 mM DTT was added to the cells for the duration of the experiment. After a 30-min pulse with 50 μCi of ³⁵S Trans-label, cells were chased for 60 min in Eagle’s MEM supplemented with 1 mM cycloheximide and 10 mM methionine. At the end of the pulse or chase, cells were washed twice with MEM and incubated with ice-cold PBS containing 40 mM NEM for 2 min to alkylate free thiol groups. Cells were subsequently harvested in SDS lysis buffer and mixed with nonreducing sample buffer, boiled, and analyzed by SDS-PAGE. The arrowheads indicate disulfide aggregates. The molecular masses (kilodaltons) of the markers are shown on the right side of the panel. (B) Effect of intracellular reoxidation on folding of non-N-linked glycosylated VP7. Infected cells were BFA and TM treated as described in the legend to Fig. 1. At 7 hpi, cells were starved for 1 h in methionine-cysteine-free media. Twenty minutes before a 30-min pulse, 4 mM DTT was added, and it remained in the media through the pulse and chase. After a 30-min pulse with 50 μCi of ³⁵S Trans-label, cells were chased for 60 min in Eagle’s MEM supplemented with 1 mM cycloheximide, 10 mM methionine, and 4 mM DTT. The monolayers were then washed with MEM and incubated with chase medium without DTT for different periods of time as indicated. At the end of the pulse or chase, cells were washed twice with MEM and incubated with ice-cold PBS containing 40 mM NEM for 2 min to alkylate free thiol groups. Cells were subsequently harvested in SDS lysis buffer and mixed with nonreducing sample buffer, boiled, and analyzed by SDS-PAGE. a, TM- and BFA-treated infected cells chased for 120 min in the presence of 4 mM DTT. The molecular masses (kilodaltons) of the markers are shown on the left side of the panel.

FIG. 4

Cell lysates from the pulse-chase experiment presented in Fig. 1 immunoprecipitated with a VP7 MAb (M60) and analyzed by nonreducing SDS-PAGE.

FIG. 5

Cells infected with RRV (MOI of 10) were mock (A) or BFA (2 μg/ml) treated (B) at 1 hpi. At 5 hpi, TM (2 μg/ml) was added to the medium of the monolayers (as indicated) and was maintained through the experiment. At 7 hpi, cells were starved for 1 h in methionine-cysteine-free media and then metabolically labeled (200 μCi) for 15 min. 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 pulse or chase, monolayers were incubated with ice-cold PBS with 40 mM NEM for 2 min, and cells were harvested in gentle lysis buffer. The cell lysates were immunoprecipitated with MAbs to VP7 and NSP4 and rabbit polyclonal antibodies to PDI.