In early biochemical studies, the large RSV glycoprotein, eventually named the G protein, was found in virions produced from infected HeLa cells (34
) but not in virions from BSC-1 cells (52
). BSC-1 cells were derived from African green monkey kidneys, as are Vero cells. A study to resolve this dramatic difference between American- and Scottish-grown RSVs found that both cell lines produced the 85-kDa form, though the BSC-1 cells produced less (42
). In the present report, we have identified an associated phenotype: RSV produced in HEp-2 cells (or HeLa cells [data not shown]) are highly dependent on GAGs for virus entry, while RSV grown in Vero, PMK, MRC-5, A549, or BSC-1 cells is much less dependent on GAGs for infection.
We have found that while virions from Vero cells have low levels of the 90-kDa G protein, they contain much more of a smaller, 55-kDa version of the G protein. Using our GAG dependency assay, we have determined that this Vero cell-grown RSV infects GAG-expressing CHO cells fourfold less efficiently than virus produced in HEp-2 cells and containing primarily the 90-kDa full-length G protein. It appears that the 90-kDa form of the G protein uses GAGs on the surface of immortalized cells to initiate infection much more efficiently than the 55-kDa form.
Estimating the cleavage position in the 90-kDa G protein that would generate a 55-kDa G protein is challenging. Identifying the site is helped by knowing that the cleavage site is conserved between the A2 and Long strains of RSV because virions from Long also contain primarily the 55-kDa G protein when produced in Vero cells (data not shown). The real difficulty in estimating the position is due to the large amount of carbohydrates attached to this protein. The G protein contains seven potential N-linked glycosylation sites, but the difference of 9 kDa between the 45-kDa intermediate and its size following Endo H treatment, 36 kDa, suggests that only three or four of these sites are used. Approximately half of the final 90-kDa molecular mass is due to O-linked glycosylation. We used the program NetNGlyc 1.0 to predict the three or four N-linked sites most likely to be used and the NetOGlyc 3.1 (25
) program to predict the most likely O-glycosylation sites (ExPASy Proteomics Server, http://ca.expasy.org/tools/#ptm
). Using these programs to predict molecular weight, we estimate that the cleavage site would be C terminal to the central disulfide-bonded region (13
), within or just C terminal to the HBD (Fig. ). HBDs are defined by a high concentration of basic amino acids. Cleavage in or near the HBD might well destroy the ability of the G protein to bind GAGs. One or more of the many lysines in this area could be a target for a plasmin-like enzyme, or the lone arginine could be the substrate for a trypsin-like enzyme.
The original, formalin-inactivated RSV vaccine that led to increased pathology rather than protection upon challenge was produced in cells derived from the same source as Vero cells, African green monkey kidneys (17
). Vero cells have been used to produce similar vaccines for many of the animal studies that have focused on the cause of the increased pathology. Along with the increased pathology, the vaccine induced nonfunctional antibodies. Deletions from the G protein C terminus result in a loss of recognition by human, rabbit, and murine antibodies (32
), suggesting that the C terminus of the G protein contains its major antigenic determinants. The reduced amount of the C-terminal antigenic determinants and the presence of a possibly misfolded, truncated G protein may have also contributed to the decrease in neutralizing antibodies and the increase in nonfunctional antibodies in children who received the formalin-inactivated RSV vaccine (37
While GAG interactions are important for infection of immortalized cells in culture, it is unlikely that they are critical for infection of airway cells in vivo. The well-differentiated HAE cell cultures that appear and function like normal airway epithelium do not express HS on their apical surface (53
). We anticipate, therefore, that the ability of the RSV G protein to bind to GAGs will not be important for the initiation of RSV infection of HAE cell cultures or of airway cells in vivo. However, the loss of the C-terminal region of the G protein might well lead to a loss of attachment function. Indeed, we found that the infectivity of Vero cell-grown virus for HAE cell cultures is much lower (600- to 1,800-fold) than that of HEp-2 cell-grown virus (Fig. , day p.i.).
Since HAE cell cultures closely model the human airway epithelium, attenuated vaccines grown in Vero cells would also be expected to infect humans inefficiently. In trials of attenuated virus vaccines, escalating doses are used to identify the lowest dose that induces an adequate antibody response. Vero cell-grown attenuated vaccine candidates do induce neutralizing antibodies in infants. However, these vaccine candidates have caused respiratory symptoms in a small number of infants and these symptoms have been deemed unacceptable (28
We predict that if such vaccines were produced in a different cell line, less inoculum would be needed to induce the same antibody response. In addition to increased efficiency of vaccine production, a lower inoculum would also reduce exposure to nonreplicating virus antigens, cell culture-derived cytokines, chemokines, and other possible contaminants in the inoculum. Exposure to local high levels of these proteins could contribute to inflammation and symptoms in the minority of infants who have been vaccinated. After the initial infection of HAE cell cultures, the source of the virus would not play a role, as progeny virus from the infected HAE cells would contain primarily intact G protein (Fig. ). This virus spreads at a rate similar to infection initiated by HEp-2 cell-grown RSV (Fig. ), after a 3-day lag. The same pattern would likely be true in vaccinees.
The G protein is important for infection in vivo, since RSV lacking its G
gene replicates poorly in rodents and nonhuman primates (8
). In model animal experiments, as in vaccine trials, Vero cell-grown virus could reduce the efficiency of the initial infection. In fact, Vero cell-grown virus has been shown to infect mice poorly compared to HeLa cell-grown virus (44
), consistent with our data and predictions.
In summary, efficient RSV infection requires the intact G protein, particularly infection of primary respiratory epithelial target cells. Most of the G protein in virions produced from Vero cells is truncated by cleavage. This truncated form of the G protein does not appear to be functional in either GAG attachment to immortalized cells or attachment to primary HAE cell cultures, since the initiation of infection is reduced in both cases. This loss of attachment function would most likely also result in poor infection initiation in vivo, negatively impacting on both animal experiments and attenuated-vaccine studies with volunteers.