Altered growth characteristics of recombinant respiratory syncytial viruses which do not produce NS2 protein

The second gene in the 3'-to-5' gene order in respiratory syncytial virus ( RSV) encodes the nonstructural protein NS2, for which there is no assigned function. To study the function of NS2, we have used a recently developed r everse genetics system to ablate expression of NS2 in recombinant RSV, A fu ll-length cDNA copy of the antigenome of RSV A2 strain under the control of a T7 promoter was modified by introduction of tandem termination codons wi thin the NS2 open reading frame (NS2stop) or by deletion of the entire NS2 gene (Delta NS2), The NS2 knockout antigenomic cDNAs were cotransfected wit h plasmids encoding the N, P, L, and M2-1 proteins of RSV, each controlled by the T7 promoter, into cells infected with a vaccinia virus recombinant e xpressing T7 RNA polymerase. Recombinant NS2stop and Delta NS2 RSVs were re covered and characterized. Both types of NS2 knockout virus displayed pinpo int plaque morphology and grew more slowly than wild-type RSV, The expressi on of monocistronic mRNAs for the five genes examined (NS1, NS2, N, F, and L) was unchanged in cells infected with either type of NS2 knockout virus, except that no NS2 mRNA was detected with the Delta NS2 virus. Synthesis of readthrough mRNAs was affected only for the Delta NS2 virus, where the NS1 -NS2, NS2-N, and NS1-NS2-N mRNAs were replaced with the predicted novel NS1 -N mRNA, Upon passage, the NS2stop virus stock rapidly developed revertants which expressed NS2 protein and grew with similar plaque morphology and ki netics wild-type RSV, Sequence analysis confirmed that the termination codo ns had reverted to sense, albeit not the wild-type assignments, and provide d evidence consistent with biased hypermutation, No revertants were recover ed from recombinant Delta NS2 RSV, These results show that the NS2 protein is not essential for RSV replication, although its presence greatly improve s virus growth in cell culture. The attenuated phenotype of these mutant vi ruses, coupled with the expected genetic stability associated with gene del etions, suggests that the Delta NS2 RSV is a candidate for vaccine developm ent.