| Respiratory syncytial virus (RSV) infection of HeLa cells induces fusion, but transient expression of the three viral glycoproteins induces fusion poorly if at all. We have found that neuraminidase treatment of RSV-infected cells or cells transfected to express the fusion (F) protein of RSV alone or with the other glycoproteins, increased dramatically. Neuraminidase treatment of the effector but not the target cells enhanced fusion. Likewise, neuraminidase treatment of virions but not target cells increased infectivity. We compared the effects of neuraminidase treatment on a mutant virus expressing F as the only glycoprotein, rgRSV-F with that on a mutant virus expressing both the G and F proteins, rgRSV-GF. Neuraminidase more dramatically affected infectivity of rgRSV-F, suggesting that tight packing of the G protein in rgRSV-GF hinders the access of the neuraminidase to the critical SA residues. Neuraminidase treatment increased virus binding slightly, but not enough to account for the observed increase in infectivity. Binding remained heparan sulfate-dependent, indicating that a novel attachment mechanism is not revealed by SA removal.; The RSV F protein is cleaved at two sites by furin. The intervening 27-amino acid peptide (pep27) is not associated with virions and, therefore, is not involved in mediating fusion. The role of pep27 is unknown. We mutated the human RSV F gene to remove pep27, along either one of the cleavage sites. We were able to rescue virus, but in both cases the deletion severely affected growth. While one of the mutants (RARR as the sole cleavage site) was poorly cleaved and reached the cell surface inefficiently, the other (KKRKRR as the sole cleavage site) was cleaved and reached the cell surface at rates comparable to the wild type (wt) F. Therefore, without pep27, the F protein was impaired in supporting virus growth, even in the mutant that reached the cell surface. To test the importance of both cleavage sites in the F protein, each site was separately replaced with a single basic residue. We could not recover virus when either cleavage site was knocked out, indicating that cleavage at both sites is required for infectivity. |
