SEQUENCE-ANALYSIS OF A FUNCTIONAL POLYMERASE (L) GENE OF BOVINE RESPIRATORY SYNCYTIAL VIRUS - DETERMINATION OF MINIMAL TRANS-ACTING REQUIREMENTS FOR RNA REPLICATION

The complete nucleotide sequence of a functional clone of the large po lymerase (L) gene of bovine respiratory syncytial virus (BRSV) strain A51908 was determined by analysis of cloned cDNAs obtained from genomi c and mRNAs, The BRSV L gene is 6573 nt in length and the derived poly peptide has 2162 aa, Alignment of the sequences of the BRSV L gene, an d its encoded protein, with sequences of the L gene and protein of hum an respiratory syncytial virus strain A2 showed 77% identity at the nu cleotide level and 84% identity at the amino acid level, By comparison , the L gene and protein of avian pneumovirus showed only 50% identity at the nucleotide level and 64% identity at the amino acid level, A m inigenome was constructed to encode a BRSV vRNA analogue containing th e gene for chloramphenicol acetyltransferase (CAT) under the control o f putative BRSV transcription motifs and Ranked by the BRSV genomic te rmini, Transfection of plasmids encoding the BRSV minigenome, nucleoca psid protein (N), phosphoprotein (P) and L protein, each under the con trol of T7 promoter, into cells infected with a vaccinia virus recombi nant expressing the T7 RNA polymerase gave rise to CAT activity and pr ogeny with the minigenome, This result indicates that the N, P and L p roteins are necessary and sufficient for transcription and replication of the BRSV minigenome and are functional, Further, inclusion of smal l amounts of the M2 protein along with the N, P and L proteins greatly augmented minigenome transcription.