Human Staufen1 Protein Interacts with Influenza Virus Ribonucleoproteins and Is Required for Efficient Virus Multiplication
Human Staufen1 Protein Interacts with Influenza Virus Ribonucleoproteins and Is Required for Efficient Virus Multiplication
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DOI：
10.1128/jvi.00504-10
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发表时间：
2010-08-01
影响因子：
5.4
通讯作者：
Ortin, Juan
中科院分区：
文献类型：
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作者：
de Lucas, Susana;Peredo, Joan;Ortin, Juan
甲型流感病毒基因组由8个负链RNA片段组成。NS 1是一种非结构蛋白，参与病毒感染周期的不同步骤，包括转录，复制和形态发生，并作为毒力因子。人Staufen 1（hStau 1）是一种参与细胞mRNA的转运和调节翻译的蛋白质，以前被鉴定为NS 1相互作用因子。为了研究hStau 1在流感病毒感染中的可能作用，我们表征了从病毒感染的细胞中分离的含有hStau 1的颗粒的组成。通过Western blotting鉴定复合物中病毒NS 1蛋白和核糖核蛋白（RNP），通过逆转录（RT）-PCR检测病毒mRNA和病毒RNA（vRNA）。此外，共定位的hStau 1与NS 1，核蛋白（NP），和PA在病毒感染的细胞的胞质溶胶中的免疫荧光显示。为了分析hStau 1在感染中的作用，我们通过基因沉默下调其表达。使用靶向hStau 1 mRNA中四个独立位点的短发夹RNA（shRNA）或小干扰RNA（siRNA）沉默人HEK 293 T细胞或A549细胞。与对照沉默细胞相比，在各种hStau 1沉默细胞中流感病毒的产量降低了5至10倍。病毒蛋白的表达水平和它们的核质定位不受hStau 1沉默的影响，但病毒颗粒的产生，如通过从上清液中纯化病毒粒子所确定的，减少了。这些结果表明hStau 1在流感病毒感染的晚期事件中的作用，可能在病毒形态发生期间。
The influenza A virus genome consists of 8 negative-stranded RNA segments. NS1 is a nonstructural protein that participates in different steps of the virus infectious cycle, including transcription, replication, and morphogenesis, and acts as a virulence factor. Human Staufen1 (hStau1), a protein involved in the transport and regulated translation of cellular mRNAs, was previously identified as a NS1-interacting factor. To investigate the possible role of hStau1 in the influenza virus infection, we characterized the composition of hStau1-containing granules isolated from virus-infected cells. Viral NS1 protein and ribonucleoproteins (RNPs) were identified in these complexes by Western blotting, and viral mRNAs and viral RNAs (vRNAs) were detected by reverse transcription (RT)-PCR. Also, colocalization of hStau1 with NS1, nucleoprotein (NP), and PA in the cytosol of virus-infected cells was shown by immunofluorescence. To analyze the role of hStau1 in the infection, we downregulated its expression by gene silencing. Human HEK293T cells or A549 cells were silenced using either short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting four independent sites in the hStau1 mRNA. The yield of influenza virus was reduced 5 to 10 times in the various hStau1-silenced cells compared to that in control silenced cells. The expression levels of viral proteins and their nucleocytoplasmic localization were not affected upon hStau1 silencing, but virus particle production, as determined by purification of virions from supernatants, was reduced. These results indicate a role for hStau1 in late events of the influenza virus infection, possibly during virus morphogenesis.