Mapping of the two overlapping genes for polypeptides NS₁ and NS₂ on RNA segment 8 of influenza virus genome

RNA segment 8 of the influenza virus genome is unique in coding for two polypeptides, NS₁ (Mr, ≃25,000) and NS₂ (Mr, ≃11,000). These polypeptides are synthesized from separate mRNA species. By using cloned DNA derived from RNA segment 8 (NS DNA) the two mRNAs have been mapped on segment 8 by hybridization of mRNAs with restriction endonuclease fragments of the DNA and nuclease S1 digestion methods. These data indicate that the body of the NS₁ mRNA (≃850 nucleotides) maps at 0.05-0.95 units of the cloned NS DNA and the body of the NS₂ mRNA (≃340 nucleotides) maps at 0.59-0.95 units, suggesting that the two mRNAs are 3' coterminal and share the same poly(A) addition site. These positions of the mRNAs on the viral genome segment were confirmed in hybrid-arrested translation experiments using fragments of the cloned NS DNA to inhibit the synthesis in vitro of NS₁ or NS₂ polypeptides. In addition, in these translation experiments the use of certain DNA fragments resulted in premature termination of the NS₁ polypeptide. From these data, it could be estimated that the termination of translation of NS₁ is at ≃0.76 map unit. Thus, the coding regions of the two mRNAs overlap by ≃144-159 nucleotides, the equivalent of ≃48-53 amino acids. Peptide mapping experiments indicated that polypeptides NS₁ and NS₂ do not share methionine- or leucine-containing tryptic peptides. The results obtained indicate that translation of the NS₂ mRNA occurs in a reading frame different from that used for NS₁.