RECOVERY OF INFECTIOUS RESPIRATORY SYNCYTIAL VIRUS EXPRESSING AN ADDITIONAL, FOREIGN GENE

A previous report described the recovery from cDNA of infectious recom binant respiratory syncytial virus (RSV) strain A2 (P. L. Collins, M. G. Hill, E. Camargo, H. Grosfeld, R. M. Chanock, and B. R. Murphy, Pro c. Natl. Acad. Sci, USA, 92:11563-11567, 1995). Here, the system was u sed to construct recombinant RSV containing an additional gene encodin g chloramphenicol acetyltransferase (CAT). The CAT coding sequence was flanked by RSV-specific gene-start and gene-end motifs; the transcrip tion signals for the viral RNA-dependent RNA polymerase. The RSV-CAT c himeric transcription cassette,vas inserted into the region between th e G and F genes of the complete cDNA-encoded positive sense RSV antige nome, and infectious CAT-expressing recombinant RSV was recovered. Tra nscription of the inserted gene into the predicted subgenomic polyaden ylated mRNA was demonstrated by Northern (RNA) blot hybridization anal ysis, and the encoded protein was detected by enzyme assay and by radi oimmunoprecipitation. Quantitation of intracellular CAT, SH, G, and F mRNAs showed that the CAT mRNA was efficiently expressed and that the levels of the G and F mRNAs (which represent the genes on either side of the inserted CAT gene) were comparable to those expressed by a wild -type recombinant RSV. Consistent with this finding, the CAT-containin g acid mild-type viruses were very similar with regard to the levels o f synthesis of the major viral proteins, Each of 25 RSV isolates obtai ned by plaque purification following eight serial passages expressed C AT, shoeing that the foreign gene was faithfully maintained in functio nal form. Analysis by reverse transcription and PCR did not reveal evi dence of deletion of the foreign sequence. This finding demonstrated t hat the RSV genome can accept and maintain an increase in length of 76 2 nucleotides of foreign sequence and can be engineered to encode an a dditional, 11th mRNA. The presence of the additional gene resulted in a 10% decrease in plaque diameter and was associated with delay in vir us growth and 20-fold decrease in virus yield in vitro. Thus, introduc tion of an additional gene into the RSV genome might represent a metho d of attenuation. The ability to express foreign genes by recombinant RSV has implications for basic studies as well as for the development of live recombinant vaccines.