Mutations at Alternative 5′ Splice Sites of M1 mRNA Negatively Affect Influenza A Virus Viability and Growth Rate
Mutations at Alternative 5′ Splice Sites of M1 mRNA Negatively Affect Influenza A Virus Viability and Growth Rate
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DOI：
10.1128/jvi.00506-08
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发表时间：
2008-11-01
影响因子：
5.4
通讯作者：
Shih, Shin-Ru
中科院分区：
文献类型：
--
作者：
Chiang, Chiayn;Chen, Guang-Wu;Shih, Shin-Ru
流感病毒蛋白的不同氨基酸序列导致不同的病毒表型。然而，这些序列的多样性及其对非编码区或剪接位点的影响尚未得到深入研究。本研究的重点是M1 mRNA上其他5'剪接位点的序列。在剪接位点引入6个不同的突变，并检测了12个质粒反向遗传产生的突变体的病毒生长特性，其中G12C （mRNA3 5’剪接位点内含子第一个核苷酸的G-to-C突变）、C51G （M2 mRNA 5’剪接位点外显子3’末端）和G146C （mRNA4内含子第一个核苷酸）是致死性突变。另一方面，具有突变G11C（位于mRNA3 5‘剪接位点外显子3’端），G52C （M2 mRNA内含子的第一个核苷酸）或G145A（位于mRNA4外显子3'端）的突变体被挽救，尽管它们的生长速率显着减弱。值得注意的是，这些突变没有改变M1或M2蛋白中的任何氨基酸。进一步分析重组突变病毒感染细胞的前体（M1 mRNA）和剪接产物（M2 mRNA、mRNA3和mRNA4）的水平。与野生型重组病毒感染的细胞相比，G11C、G52C和G145A突变病毒感染的细胞中mRNA3的产生水平降低。G11C突变型病毒感染细胞产生的M2 mRNA多于野生型病毒感染细胞，而G145A和G52C突变型病毒感染细胞产生的M2 mRNA很少，完全没有。本研究的结果表明，将这些突变引入备选的5'剪接位点会干扰M1 mRNA剪接，从而可能降低病毒的生长速度。
Different amino acid sequences of influenza virus proteins contribute to different viral phenotypes. However, the diversity of the sequences and its impact on noncoding regions or splice sites have not been intensively studied. This study focuses on the sequences at alternative 5' splice sites on M1 mRNA. Six different mutations at the splice sites were introduced, and viral growth characteristics for those mutants generated by reverse genetics with 12 plasmids were examined, for which G12C (the G-to-C mutation at the first nucleotide of the intron for the mRNA3 5' splice site), C51G (at the 3' end of the exon of the M2 mRNA 5' splice site), and G146C (for the first nucleotide of the intron for mRNA4) are lethal mutations. On the other hand, mutants with the mutation G11C (at the 3' end of exon of the mRNA3 5' splice site), G52C (for the first nucleotide of the intron for M2 mRNA), or G145A (at the 3' end of the exon of mRNA4) were rescued, although they had significantly attenuated growth rates. Notably, these mutations did not change any amino acids in M1 or M2 proteins. The levels of precursor (M1 mRNA) and spliced products (M2 mRNA, mRNA3, and mRNA4) from the recombinant mutant virus-infected cells were further analyzed. The production levels of mRNA3 in cells infected with G11C, G52C, and G145A mutant viruses were reduced in comparison with that in wild-type recombinant virus-infected ones. More M2 mRNA was produced in G11C mutant virus-infected cells than in wild-type-virus-infected cells, and there was little M2 mRNA and none at all in G145A and G52C mutant virus-infected ones, respectively. Results obtained here suggest that introducing these mutations into the alternative 5' splice sites disturbed M1 mRNA splicing, which may attenuate viral growth rates.