Respiratory syncytial virus that lacks open reading frame 2 of the M2 gene (M2-2) has altered growth characteristics and is attenuated in rodents

The M2 gene of respiratory syncytial virus (RSV) encodes two putative proteins: M2-1 and M2-2; both are believed to be involved in the RNA transcription or replication process. To understand the function of the M2-2 protein in virus replication, we deleted the majority of the M2-2 open reading frame from an infectious cDNA clone derived from the human RSV A2 strain. Transfection of HEp-2 cells with the cDNA clone containing the M2-2 deletion, together with plasmids that encoded the RSV N, P, and L proteins, produced a recombinant RSV that lacked the M2-2 protein (rA2 Delta M2-2). Recombinant virus rA2 Delta M2-2 was recovered and characterized. The levels of viral mRNA expression for 10 RSV genes examined were unchanged in cells infected with rA2 Delta M2-2, except that a shorter M2 mRNA was detected. However, the ratio of viral genomic or antigenomic RNA to mRNA was reduced in rA2 Delta M2-2-infected cells, By use of an antibody directed against the bacterially expressed M2-2 protein, the putative M2-2 protein was detected in cells infected with wild-type RSV but not in cells infected with rA2 Delta M2-2. rA2 Delta M2-2 displayed a small-plaque morphology and grew much more slowly than wild-type RSV in HEp-2 cells. In infected Vero cells, rA2 Delta M2-2 exhibited very large syncytium formation compared to that of wild-type recombinant RSV, rA2 Delta M2-2 appeared to be a host range mutant, since it replicated poorly in HEp-2, HeLa, and MRCS cells but replicated efficiently in Vero and LLC-MK2 cells, Replication of rA2 Delta M2-2 in the upper and lo,ver respiratory tracts of mice and cotton rats was highly restricted. Despite its attenuated replication in rodents, rA2 Delta M2-2 was able to provide protection against challenge with wild-type RSV A2, The genotype and phenotype of the M2-2 deletion mutant were stably maintained after extensive in vitro passages. The attenuated phenotype of rA2 Delta M2-2 suggested that rA2 Delta M2-2 may be! a potential candidate for use as a live attenuated vaccine.