INFLUENZA VIRUS A/PR/8/34 GENES: SEQUENCING BY A SHOTGUN APPROACH

ABSTRACT We are using a shotgun approach to sequence the genome of influenza virus strain A/PR/8/34, and have now completed the sequences of the genes encoding the haemagglutinin, neuraminidase, matrix protein and non-structural proteins. We prepare double-stranded cDNA corresponding to all the virion RNA segments using synthetic primers for both first and second strand synthesis. The cDNA is then digested with restriction enzymes and the fragments cloned into derivatives of the bacteriophage M13 for sequence analysis by the dideoxynucleotide chain terminator technique. Highlights of our results so far include: 1)identifying a close sequence relationship between the haemagglutinins of the antigenically unrelated HI and H2 subtypes; 2)the discovery of a long hydrophobic sequence in the neuraminidase which may orient the molecule with its N-terminus buried in the viral membrane; and 3)finding a second long reading frame in segment 7 which we suggest encodes a novel influenza virus polypeptide.