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

最近，我们用表达PB2、PB1、P3、HE、NP、M和NS病毒RNA的7个Pol I质粒和表达PB2、PB1、P3、HE、NP、M1、CM2、NS1和NS2蛋白的9个质粒，建立了丙型流感病毒的反向遗传学，并成功地构建了重组丙型流感病毒。为了确定C亚型流感病毒NS基因产物是否在前-mRNA剪接中起作用，我们试图获得将Leu 20替换为终止密码子的病毒，以挽救NS1和NS2几乎不表达的病毒。然而，病毒没有被拯救，这表明病毒复制需要NS基因产物NS1和/或NS2。在瞬时表达系统中，M基因与NS1蛋白的共表达表明，M-Pre-mRNA的剪接率提高了30%，提示NS1可能具有促进剪接的活性。然后，我们研究了NS1特异的C-末端区域(63-246个残基)是否具有增强剪接的功能域。首先，我们通过将第91位的Phe替换到终止密码子(F91Stop)中，获得了失去近70%C末端的重组病毒。含有F91终止突变的重组病毒感染HMV-II细胞后，剪接的M-mRNA1/未剪接的M-mR-2的比例降低了20%，提示该功能结构域可能存在于C-末端。增强剪接的功能域还有待于含有C末端各种缺失的NS1代突变体来阐明。

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.