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

Abstract

RNA segment 8 of the influenza virus genome is unique in coding for 2 polypeptides, NS1 (MW, .apprxeq. 25,000) and NS2 (MW, .apprxeq. 11,000). These polypeptides are synthesized from separate mRNA species. By using cloned DNA derived from RNA segment 8 (NS DNA) the 2 mRNA were mapped on segment 8 by hybridization of mRNA with restriction endonuclease fragments of the DNA and nuclease S1 digestion methods. Apparently the body of the NS1 mRNA (.apprxeq. 850 nucleotides) maps at 0.05-0.95 units of the cloned NS DNA and the body of the NS2 mRNA (.apprxeq. 340 nucleotides) maps at 0.59-0.95 units, suggesting that the 2 mRNA are 3'' co-terminal and share the same poly(A) addition site. These positions of the mRNA 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 .apprxeq. 0.76 map unit. Thus, the coding regions of the 2 mRNA overlap by .apprxeq. 144-159 nucleotides, the equivalent of .apprxeq. 48-53 amino acids. Peptide mapping experiments indicated that polypeptides NS1 and NS2 do not share methionine- or leucine-containing tryptic peptides. Translation of the NS2 mRNA evidently occurs in a reading frame different from that used for NS1.