No abstract

We established a quantitative hybridization system by which three types of influenza virus RNAs (vRNA, mRNA, and cRNA) for the 8 genome segments were measured individually. As the hybridization probes, 32P-labeled RNAs of both plus and minus polarity were produced employing an SP-6 transcription system and used in a large molar excess, sufficient to overcome complementary RNAs present in the viral RNA samples. Employing the system, we studied the control of the synthesis of each viral RNA species in MDCK cells infected with A/Udorn/72 (H3N2). Our new observations were as follows. 1) Segment-specific transcription was observed at the primary transcription. 2) Replication of the virus genome began simultaneously for all segments. No delay was observed in the replication of the segments carrying late genes. 3) In addition to control at the transcriptional levels, the expression of viral late genes was regulated at some post-transcriptional step(s). These results are not compatible with the concepts reported previously, and lead us to propose unique regulations operating on the expression of the viral late genes.

No abstract

The non-structural protein NS1, encoded by genome segment 8 of influenza A virus, was expressed in Escherichia coli from cloned cDNA and purified. The NS1 protein had a specific RNA-binding activity, binding to influenza A virus minus-sense but not plussense RNA synthesized in vitro from cloned DNA using phage RNA polymerase. NS1 bound preferentially to the regions of RNA containing either 5'-or 3'-terminal common sequences of the genomic RNA. Binding was inhibited by virion RNA, but not by singlestranded minus-sense cDNA and oligo DNAs having the common sequences. In addition, binding was also inhibited by 28S rRNA but not 18S rRNA prepared from MDCK cells.

Temperature-sensitive (ts) mutants of influenza virus strain A/Udorn/72 (H3N2 subtype) with lesions in RNA segment 8 exhibited intrasegmental complementation, and were divided in two complementation groups (HI and H2) on MDCK cells. The nucleotide sequence of segment 8 was determined for three of these mutants. The H1 strains, ICR1629 and SPC45, have a single amino acid substitution in the coding region of the non-structural protein NS 1, whereas the H2 strain, ICR516, has a substitution in the NS2-coding region. With both NS1 ts mutants, the synthesis of two late proteins, the matrix protein (M1) and haemagglutinin (HA), was greatly reduced and NS1 synthesis also decreased at 40 °C (non-permissive temperature) compared to that at 34 °C (permissive temperature). The synthesis of each virus-specific RNA was analysed using a quantitative hybridization method. However, at 40 °C, the levels of individual mRNAs including those for the late proteins, were almost the same as those at 34 °C, and attained the wild-type levels later in the infection (5 h postinfection) when the synthesis of the late proteins and the NS1 protein was severely reduced. The observations suggest that the NS 1 protein, which is a nuclear protein, is involved in some post-transcriptional processes in the synthesis of the late proteins and the NS 1 protein.