RECOMBINANT RESPIRATORY SYNCYTIAL VIRUS FROM WHICH THE ENTIRE SH GENEHAS BEEN DELETED GROWS EFFICIENTLY IN CELL-CULTURE AND EXHIBITS SITE-SPECIFIC ATTENUATION IN THE RESPIRATORY-TRACT OF THE MOUSE

The small hydrophobic protein SH of human respiratory syncytial virus (RSV) is a short transmembrane surface protein of unknown function. A full length cDNA of RSV strain A2 (subgroup A) antigenomic RNA was mod ified such that the entire SH gene, including the transcription signal s and the complete mRNA-encoding sequence, was deleted and replaced by a synthetic intergenic region. This reduced the length of the antigen ome by 398 nucleotides and ablated expression of 1 of the 10 RSV mRNAs . Recombinant virus containing this engineered deletion was recovered, and the absence of the SH gene was confirmed by reverse transcription in conjunction with PCR Northern blot analysis of intracellular RNAs and gel electrophoresis of labeled intracellular proteins confirmed th e lack of expression of the SH mRNA and protein. The absence of the SH gene did not noticeably affect RNA replication, but two effects on tr anscription were noted. First, synthesis of the G, F, and M2 mRNAs was increased, presumably due to their being one position closer to the p romoter in the gene order. Second, transcription of genes downstream o f the engineered site exhibited a steeper gradient of polarity, On mon olayers of HEp-2 cells, the SK-minus virus produced syncytia which wer e at least equivalent in size to those of the wild type and produced p laques which were 70% larger. Furthermore, the SH-minus virus grew som ewhat better (up to 12.6-fold) than wild-type recombinant RSV in certa in cell lines. While the function of the SH protein remains to be dete rmined, it seems to be completely dispensable for growth in tissue cul ture and fusion function, When inoculated intranasally into mice, the SII-minus virus resembled the wild-type recombinant virus in its effic iency of replication in the lungs, whereas it replicated 10-fold less efficiently in the upper respiratory tract. In mice, the SH-minus and wild-type recombinant viruses were similarly immunogenic and effective in inducing resistance to virus challenge.