A recombinant influenza A virus expressing an RNA-binding-defective NS1 protein induces high levels of beta interferon and is attenuated in mice
A recombinant influenza A virus expressing an RNA-binding-defective NS1 protein induces high levels of beta interferon and is attenuated in mice
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DOI：
10.1128/jvi.77.24.13257-13266.2003
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发表时间：
2003-12-01
影响因子：
5.4
通讯作者：
García-Sastre, A
中科院分区：
文献类型：
--
作者：
Donelan, NR;Basler, CF;García-Sastre, A
此前，我们发现甲型流感病毒NS 1蛋白的氨基末端区域在阻止病毒感染细胞中β干扰素（IFN-β）的诱导方面起着关键作用。该区域的特征在于其能够结合不同的RNA种类，包括双链RNA（dsRNA），一种已知的IFN的有效诱导剂。为了研究NS 1 RNA结合活性是否是其IFN拮抗剂特性所必需的，我们已经产生了重组甲型流感病毒，其表达dsRNA结合缺陷的突变型NS 1蛋白。为此，我们用丙氨酸取代了NS 1中的两个碱性氨基酸（R38和K41），这两个氨基酸以前被发现是RNA结合所必需的。用所得重组病毒感染的细胞显示IFN-β产生增加，表明这两种氨基酸在病毒感染期间通过NS 1蛋白抑制IFN产生中起关键作用。此外，该病毒在MDCK细胞中生长至比野生型病毒更低的滴度，并且在小鼠中减毒。有趣的是，在MDCK细胞中传代导致选择在NS 1蛋白的氨基酸残基42处含有第三突变（S42 G）的突变病毒。这种突变没有导致NS 1蛋白的dsRNA结合活性增加，如通过体外测定所测量的。然而，NS 1 R38 AK 41 AS 42 G突变体病毒能够在MDCK细胞中复制至接近野生型病毒的滴度。该突变病毒在小鼠中具有介于野生型和亲本NS 1 R38 AK 41 A病毒之间的中等毒力。这些结果表明，NS 1蛋白质的IFN拮抗剂特性不仅取决于其结合dsRNA的能力，而且还可以通过不参与RNA结合的氨基酸残基来调节。
Previously we found that the amino-terminal region of the NS1 protein of influenza A virus plays a key role in preventing the induction of beta interferon (IFN-beta) in virus-infected cells. This region is characterized by its ability to bind to different RNA species, including double-stranded RNA (dsRNA), a known potent inducer of IFNs. In order to investigate whether the NS1 RNA-binding activity is required for its IFN antagonist properties, we have generated a recombinant influenza A virus which expresses a mutant NS1 protein defective in dsRNA binding. For this purpose, we substituted alanines for two basic amino acids within NS1 (R38 and K41) that were previously found to be required for RNA binding. Cells infected with the resulting recombinant virus showed increased IFN-beta production, demonstrating that these two amino acids play a critical role in the inhibition of IFN production by the NS1 protein during viral infection. In addition, this virus grew to lower titers than wild-type virus in MDCK cells, and it was attenuated in mice. Interestingly, passaging in MDCK cells resulted in the selection of a mutant virus containing a third mutation at amino acid residue 42 of the NS1 protein (S42G). This mutation did not result in a gain in dsRNA-binding activity by the NS1 protein, as measured by an in vitro assay. Nevertheless, the NS1 R38AK41AS42G mutant virus was able to replicate in MDCK cells to titers close to those of wild-type virus. This mutant virus had intermediate virulence in mice, between those of the wild-type and parental NS1 R38AK41A viruses. These results suggest not only that the IFN antagonist properties of the NS1 protein depend on its ability to bind dsRNA but also that they can be modulated by amino acid residues not involved in RNA binding.