Construction and characterization of cDNA clones for four respiratory syncytial viral genes.

Abstract

Cytoplasmic poly(A)-containing RNA from respiratory syncytial virus-infected human laryngeal carcinoma HEp-2 cells was used as a template to synthesize oligo(dT)-primed c[complementary]DNA. Discrete size classes of single-stranded cDNA, resolved by alkali agarose gel electrophoresis, were used separately to construct double-stranded cDNA that were subsequently inserted into the plasmid vector pBR322 at the PstI site by means of oligo(dG).cntdot.oligo(dC) tailing. After transfection of Escherichia coli, recombinant plasmids were screened mostly by serial rounds of hybrid selection of mRNA from virus-infected cells and subsequent in vitro translation of the selected mRNA. Comparative peptide mapping of the translation products with those of authentic virion proteins served to establish the viral origin of the cDNA recombinants. In this manner, 4 distinct classes of recombinant plasmids were identified. These encode sequences corresponding to those of respiratory syncytial virus nucleocapsid protein, matrix protein, phosphoprotein and a nonstructural protein.