The effect of the regulatory mechanism of splicing on influenza C virus replication

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We recently established reverse genetics of influenza C virus with seven Pol I plasmids expressing PB2, PB 1, P3, HE, NP, M and NS viral RNA and nine plasmids expressing PB2, PB1, P3, HE, NP, M1, CM2, NS1 and NS2 proteins and succeeded in generation of a recombinant influenza C virus. To determine whether NS gene products of influenza C virus play a role in pre-mRNA splicing, we attempted to obtain the virus, in which Leu 20 was substituted into termination codon to rescue the virus with little expressing of NS1 and NS2. However, the virus was not rescued, which suggested that NS gene products, NS1 and/or NS2, are required for virus replication. In the transient expression system, coexpression of M gene cDNA with NS1 protein showed that splicing rate of M pre-mRNA increased by 30%, suggesting that NS1 may have the activity to enhance splicing. We then investigated whether C-terminus region (63-246 residues) specific for NS1 possesses the functional domain to enhance splicing. First, we obtained the recombinant virus which lost almost 70% of C-terminus by substituting Phe at residue 91 into termination codon (F91 stop). HMV-II cells infected with the recombinant virus containing F91 stop mutation showed that the ratio of M1 derived from spliced M mRNA / CM2 from unspliced M mRNA was reduced by 20%, a finding which suggests that the functional domain may be present in C-terminal region. The functional domain for enhancing splicing remains to be elucidated by generation NS1 mutants containing the various deletions in C-terminus.