MESSENGER-RNA SEQUENCE OF 3 RESPIRATORY SYNCYTIAL VIRUS GENES ENCODING 2 NONSTRUCTURAL PROTEINS AND A 22K STRUCTURAL PROTEIN

An mRNA sequence of 2 human respiratory syncytial viral nonstructural protein genes and of a gene for a 22,000 MW (22K) protein was obtained by cDNA cloning and DNA sequencing. Sequences corresponding to the 5'' ends of the respective transcripts were deduced directly by primer extension and dideoxy nucleotide sequencing of the mRNA. The availability of a bicistronic cloned (pRSC6) confirmed the gene order for this portion of the genome. Contrary to other unsegmented negative-stranded RNA viruses, a 19-nucleotide intercistronic sequence was present between the NS1 and NS2 genes. The translation of cloned viral sequences in the bicistronic and monocistronic clones (pRSNS1 and pRSNS2) revealed 2 moderately hydrophobic proteins of 15,568 and 14,703 daltons. Their similarity in molecular size explained the earlier inability to resolve these proteins. A DNA sequence of an additional recombinant plasmid (pRSA2) revealed a long open reading frame encoding a 22,156-dalton protein containing 194 amino acids. It was relatively basic and moderately hydrophobic. A protein of this size was readily translated in vitro from a viral mRNA hybrid selected by this plasmid and corresponded to an unglycosylated 22K protein seen in purified extracellular virus but not associated with detergent- and salt-resistant cores. A 2nd open reading frame of 90 amino acids partially overlapping with the C terminus of the 22K protein was also present within this sequence. This was reminiscent of the viral matrix gene which was previously shown to contain 2 overlapping reading frames. The finding of 3 additional viral transcripts encoding at least 3 identifiable proteins in human respiratory syncytial virus was a novel departure from the usual genetic organization of paramyxoviruses. The 5'' ends of all 3 transcripts had a 5''NGGGCAAAU sequence that is common to all viral transcripts analyzed so far. Although there was no obvious homology immediately upstream of the poly(A) tail, an AGUUA (AGUAA in the case of NS2) was present 1-4 nucleotides upstream of the poly(A) end of NS1 and 22K protein mRNA species.