Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans

Abstract

Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.

Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

“…However, the G protein is cleaved in only a few cell lines (12). One reason for this difference is that cathepsin L activity is much lower in HeLa cells than in Vero cells (Fig.…”

“…The first indication that Vero cell-derived RSV was different from HEp-2 cell-derived RSV came when we found that HEp-2 cell-derived virus was 20-fold more infectious than Vero cell-derived virus for CHO cells that express HS than for CHO cells that are deficient in HS production (12). This finding prompted us to examine the G protein in virions, and we found that most of it was 55 kDa rather than 90 kDa.…”

“…MRC-5 and WI-38 cells are much less proliferative than Vero cells, and the G protein is also cleaved in MRC-5 cells (12) and WI-38 cells (unpublished data). In addition, Vero cells do not produce interferon (IFN) (24), which is especially appealing if a vaccine candidate is deficient in inhibiting the IFN response.…”

“…HAE cell cultures are an excellent in vitro model for the natural target cells in the human respiratory tract (13,14). Similarly, virions produced by Vero cells, which primarily contain the cleaved G protein, are 5-fold less infectious for HAE cells in culture, suggesting that cleavage severely compromises the attachment function of the G protein (12).…”

“…Although the G protein is not absolutely essential for infection of immortalized cells (10), virus lacking the G protein is 10-fold less infectious for these cells (11). Virus lacking the G protein is a further 10-fold less infectious for primary welldifferentiated human airway epithelial (HAE) cells in culture (12). HAE cell cultures are an excellent in vitro model for the natural target cells in the human respiratory tract (13,14).…”