Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo

The surface glycoproteins of viruses can play important roles in viral atta chment, entry, and morphogenesis. Here, we investigated the role of the att achment G glycoprotein of human respiratory syncytial virus (RSV) in viral infection. RSV G is produced both as a complete, transmembrane form and as an N-terminally truncated form that is secreted. Using reverse genetics, we created mutant recombinant RSVs (rRSV) that do not express G (DeltaG) or e xpress either the secreted or the membrane-bound form of G only (sG and mG, respectively). In Vero cells, the DeltaG virus formed plaques and grew as efficiently as wild-type rRSV and mG. In contrast, DeltaG replicated less e fficiently and did not form distinct plaques in HEp-2 cells. This defect wa s primarily at the level of the initiation of Infection, with only a minor additional effect at the level of packaging. Replication of DeltaG in the r espiratory tract of mice was very highly restricted, indicating that G is i mportant in vivo. Although the G protein expressed by the sG virus was conf irmed to be secreted, this virus grew at least as efficiently as wild-type in HEp-2 cells and was only moderately attenuated in vivo. Thus, the G prot ein was important for efficient replication in HEp-2 cells and in vivo, but this function could be supplied in large part by the secreted form and thu s does not require the cytoplasmic and transmembrane domains. Amino acids 1 84-198 have been identified as the major heparin-binding domain of the G pr otein and were implicated in mediating binding to cells [S. A, Feldman at a l., 1999, J. Virol. 73, 6610-6617]. Heparin-like glycosaminoglycans also ap peared to be important for infection in vitro by direct clinical isolates o f RSV. Deletion of amino acids 187-197 from rRSV did not reduce its sensiti vity to neutralization in vitro by incubation with soluble heparin, did not reduce Its efficiency of growth in vitro, and resulted in only a modest re duction in vivo. Thus, the putative heparin-binding domain is not the sole determinant of heparin sensitivity and Is not a critical functional domain.