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Within the sequence motif conserved at the extreme ends of the influenza virus vRNAs, a unique natural variation, U or C, is observed at position 4 of the 3' end. To test the role of this nucleotide, two isogenic A/WSN/33 viruses, carrying either C4 or U4 nucleotide at the 3' end of the neuraminidase (NA) gene, were generated. Compared with the C4 virus, the U4 virus exhibited delayed synthesis of vRNA and stimulation of mRNA synthesis with prolonged accumulation in influenza virus-infected cells. The mRNA/ vRNA ratio was increased up to 20-fold by the C4 --&gt; U4 substitution suggesting that the U4 nucleotide greatly stimulated transcription of the vRNA template. In isolated virion, the U4 virus had higher NA activity than the C4 virus. In MDBK cells, the U4 virus grew to lower haemagglutination (HA) titres but with higher infectivity than the C4 virus, with a corresponding increase in the ratio of p.f.u./HA units of about 10- to 40-fold. Western blot analysis of isolated virion showed that the ratio of two surface proteins, HA/NA, was greatly decreased in the U4 virus. This suggests that the position 4 nucleotide is a genetic determinant for the repertoire of surface antigens and their ratio could be changed without detrimental effects on virus growth. Results could be used to design genetically engineered influenza virus for vaccination. The observed down-regulation of transcription by C4 nucleotide is consistent with its potential role in segment-specific regulation of influenza virus gene expression, especially PB1, PB2 and PA proteins, during virus infection.

journal of general virology

The position 4 nucleotide at the 3' end of the influenza virus neuraminidase vRNA is involved in temporal regulation of transcription and replication of neuraminidase RNAs and affects the repertoire of influenza virus surface antigens.