We have shown here that the binding of SARS-CoV S protein to the cellular molecular chaperone calnexin plays a critical role in SARS-CoV infection. Calnexin strictly managed the folding of glycosylated S protein in viral production, with the result that daughter viruses acquired the infectious ability.
The interaction between S protein and calnexin was analyzed here, because calnexin provided the strongest signal in our pulldown assays using S2-N. These pulldown assays, however, showed that S2-N also bound to other cellular proteins containing actin. Recently, palmitoylated calnexin, which forms a supercomplex with the ribosome-translocon complex, was reported to interact with actin (25
), suggesting that S protein may indirectly interact with actin through calnexin. Calnexin bound to all truncated S proteins except for S2-C, consistent with a previous report that 23 putative N-linked glycosylation sites are scattered throughout S protein (34
). Calnexin associates with monoglycosylated N-glycan chains on polypeptides/proteins. The presence of many glycosylation sites in S protein is also consistent with our Western blot results with cell lysates, showing that S protein is present as two major bands with smearing. This result is consistent with a previous report using a full glycan analysis (39
We found in the present study that S-pseudovirus produced by calnexin knockdown cells was significantly less infectious than S-pseudoviruses produced by untreated and control siRNA-treated cells. The S protein in S-pseudovirus produced by calnexin knockdown cells differed in quality and quantity from the S proteins in the other two S-pseudoviruses produced by untreated and control siRNA-treated cells. In the absence of both PNGase F and endo H, the S protein from calnexin knockdown cells consisted of multiple bands with smearing, whereas the S proteins from untreated and control siRNA-treated cells were each single bands. However, with PNGase F treatment, the band mobilities of those three S proteins were identical, indicating that the S protein from calnexin knockdown cells differed in posttranslational modification rather than truncation. In addition, following PNGase F treatment, the band signal of S protein from calnexin knockdown cells, relative to p24 protein, was stronger than those from untreated and control siRNA-treated cells, suggesting that excess amounts of S proteins were incorporated into the former virus particles. Furthermore, when the S-pseudoviruses produced by untreated and control siRNA-treated cells were digested by PNGase F or endo H, the band mobilities of these S proteins differed. This result suggested that the majority of sugar chains with which the S proteins are modified are complex N-glycan, which is consistent with a previous report (39
). Sucrose density gradient analysis showed that S-pseudovirus produced by calnexin knockdown cells consisted of highly dense particles with abundant S protein and normally dense particles with little S protein. These results indicated that in the absence of calnexin, excessive amounts of improperly glycosylated S protein are incorporated into virus particles, resulting in the latter being more dense and noninfectious. Based on these results, it is speculated that a decrease of calnexin led to an impairment of surveillance capability in the ER quality control, with the result that the S protein with improper glycosylation is not displaced from the ER. This is consistent with the observation that the secretion of immature glycoprotein is enhanced in calnexin-deficient Saccharomyces cerevisiae
). In the process of maturation of newly synthesized proteins, posttranslational modification with N-glycan is closely involved in protein folding, which is intrinsically difficult and error prone (18
). Therefore, S protein from calnexin knockdown cells would be misfolded because calnexin is one of the central players in the ER quality control.
Removal of N-glycan chains from correctly matured S protein by PNGase F treatment diminished the infectivity of S-pseudovirus, indicating that the glycosylation of S protein is critical for the infectivity of S-pseudovirus not only during but also after the process of viral production. Since the RBD in S protein is required for binding to ACE2 in SARS-CoV infection (45
), the PNGase F-induced decrease in S-pseudovirus infectivity may be due to conformational changes of the RBD itself or to masking of the RBD by conformational changes of the entire S protein resulting from the removal of N-glycan chains.
α-Glucosidase inhibitors disrupt the interactions between calnexin and its substrates in the ER by interrupting α-glucosidase I and II (12
). However, we found here that calnexin immunoprecipitated with S protein in the presence of α-glucosidase inhibitors. As mentioned above, there are several N-glycosylation sites in S protein (40
). Therefore, calnexin can bind to S protein that possesses at least one monoglucosylated N-glycan despite of the presence of α-glucosidase inhibitors. α-Glucosidase inhibitors affected the amount of S protein in S-pseudovirus, in the ER, and on the cell surface but not in cell lysates. S protein, which shifted upward on electrophoresis, was precipitated with calnexin, suggesting that calnexin binds to S protein possessing monoglycosylated N-glycans and monitors S protein folding. This monitoring would remove improperly glycosylated and incorrectly folded S proteins, as reported previously (6
). Therefore, the amounts of S protein in the ER and at the cell surface would be reduced in the presence of α-glucosidase inhibitors. SARS-CoV was significantly less infectious when produced in Vero E6 cells in the presence than in the absence of NN-DNJ, due primarily to a decrease in viral production. The upward shift of S protein in these cell lysates was similar to that of S-pseudovirus in α-glucosidase inhibitor-treated HEK293 cells, suggesting that the decrease in SARS-CoV infectivity may be due to the virus containing less S protein in addition to decreased viral production.
Although S-pseudoviruses produced by both calnexin siRNA- and α-glucosidase inhibitor-treated cells had lower infectivity, the mechanisms underlying these decreases differed. In the absence of calnexin, incorrectly glycosylated S protein, which is likely misfolded, remained in the ER, resulting in the incorporation of nonfunctional S protein into daughter virions. However, in the presence of α-glucosidase inhibitors, incorrectly glycosylated S protein is sorted by calnexin and sent for ER-associated degradation (ERAD), resulting in the production of daughter virions without S protein.
Protein glycosylation is performed in a step-by-step manner (20
). Nascent polypeptide is cotranslationally transferred to the ER, along with the large oligosaccharide Glc3
, by oligosaccharyltransferase. ER α-glucosidases I and II, the targets of the α-glucosidase inhibitors, remove terminal glucose residues stepwise from N-glycan chains attached to nascent glycoproteins. Disappearance of the upper band in the S protein doublet and upward shifting of the lower band were dependent on the concentration of α-glucosidase inhibitors. The decreased incorporation of S protein into virus particles was also dependent on the concentration of α-glucosidase inhibitors. These findings suggested that the upper band in the S protein doublet may be mature S protein with infectious ability, whereas the lower band may be an intermediate form of S protein. Moreover, in the presence of α-glucosidase inhibitors, the amounts of bi- and triglycosylated S protein should increase, reducing its mobility and showing an upward shift, similar to the mobility shifts of hepatitis C virus gp E1 and E2 (7
). α-Glucosidase inhibitors have been found to disrupt infection by several enveloped viruses, including dengue virus, human hepatitis C virus, and mouse hepatitis virus (12
). The present study showed that α-glucosidase inhibitors also decreased infection by SARS-CoV. Viral entry into target cells is one of the most important steps in virus propagation. Therefore, the association between S protein and calnexin may be a new target in the treatment of SARS. Future studies of α-glucosidase inhibitors or their derivatives may lead to the development of anti-SARS-CoV drugs.