cDNA cloning and transcriptional mapping of nine polyadenylylated RNAs encoded by the genome of human respiratory syncytial virus

Abstract

We have isolated cDNA clones representing nine unique poly(A)+ RNAs transcribed from the genome of human respiratory syncytial virus, a paramyxovirus. A cDNA library was constructed by using poly(A)+ RNA from virus-infected cells as template and the Escherichia coli plasmid pBR322 as vector. Viral cDNA clones were identified by hybridization with cDNA probes prepared from viral genomic RNA. The viral clones were grouped into nine different families by hybridization with individual size-selected reverse transcripts representing the major classes of poly(A)+ RNA from virus-infected cells. The largest clone from each family was selected for analysis. These nine clones, molecular sizes ranging from 520 to 2,600 base pairs, were shown to be unrelated on the basis of reciprocal hybridization using dot-blots. These cDNA clones were then used as hybridization probes to analyze intracellular viral RNAs that had been separated by gel electrophoresis and transferred to diazobenzyloxymethyl-paper. All nine clones hybridized with intracellular viral genomic RNA, confirmation of virus specificity. Nine unique intracellular viral poly(A)+ RNAs were identified [molecular sizes ranging from 720 to 7,500 nucleotides, including poly(A)]. Comparison of the sizes of these major RNAs and the cDNA clones indicated that a number of the clones represented nearly complete copies of the corresponding RNAs. Several other intracellular viral poly(A)+ RNAs appeared to be polycistronic by the criteria of molecular weights and homologies to various combinations of cDNA clones. The sizes and sequence contents of these polycistronic RNAs were used to prepare a transcriptional map whose significance is discussed.