The splicing factor proline-glutamine rich (SFPQ/PSF) is involved in influenza virus transcription.
The splicing factor proline-glutamine rich (SFPQ/PSF) is involved in influenza virus transcription.
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DOI：
10.1371/journal.ppat.1002397
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发表时间：
2011-11
期刊：
影响因子：
6.7
通讯作者：
Ortín J
中科院分区：
文献类型：
--
作者：
Landeras-Bueno S;Jorba N;Pérez-Cidoncha M;Ortín J
甲型流感病毒RNA聚合酶是一种异源三聚体复合物，在感染细胞的细胞核中负责病毒基因组转录和复制。我们最近对人类细胞中表达的纯化聚合酶进行了蛋白质组学分析，并鉴定了许多与聚合酶相关的细胞蛋白。在这里，我们描述了一种这样的宿主因子SFPQ/PSF在病毒感染期间的作用。通过沉默两个独立的sirna来下调SFPQ/PSF，在低多重感染中使病毒产量降低2-5倍，而不相关的病毒如VSV或腺病毒的复制几乎不受影响。由于SFPQ/PSF蛋白经常与NonO/p54相关，我们测试了后者在流感病毒复制中的潜在意义。然而，用两个独立的sirna沉默来下调NonO/p54并不影响病毒产量。通过siRNA沉默下调SFPQ/PSF导致流感病毒基因表达的减少和延迟。免疫荧光分析显示感染细胞中SFPQ/PSF与NP水平有良好的相关性。对沉默细胞中病毒RNA积累的分析表明，mRNA、cRNA和vRNA的产生减少了5倍以上，但剪接不受影响。同样，在环己胺处理的细胞中，病毒mRNA的积累减少了3倍。相比之下，在重组病毒复制子系统中，SFPQ/PSF的下调表明，虽然病毒mRNA的积累减少了5倍，但vRNA水平略有增加。在SFPQ/ psf沉默细胞中产生的重组RNPs的体外转录表明，聚腺苷酸化减少了4 - 5倍，但帽抢夺没有改变。这些结果表明，SFPQ/PSF是流感病毒转录所必需的宿主因子，可提高病毒mRNA聚腺苷化的效率，并为开发针对初级转录物积累的新抗病毒药物提供了可能，初级转录物是感染的早期步骤。甲型流感病毒每年都会引起流行病，偶尔也会引起可能危及生命的呼吸道感染大流行。病毒基因组包含8个形成核糖核蛋白的RNA分子，这些核糖核蛋白在感染细胞的细胞核中复制和转录。流感病毒是细胞内寄生虫，需要宿主细胞机制进行复制。为了更好地理解这种病毒与细胞的相互作用，我们纯化了在人细胞中表达的病毒RNA聚合酶，并鉴定了几种特异性相关的细胞蛋白。在这里，我们描述了其中一个，脯氨酸-谷氨酰胺富剪接因子（SFPQ/PSF）的作用。SFPQ/PSF的下调表明它是病毒增殖所必需的。具体来说，感染细胞中的SFPQ/PSF沉默减少了信使rna和基因组病毒特异性rna的积累。此外，SFPQ/PSF下调会影响亲本核糖核蛋白的转录。沉默SFPQ/PSF对病毒重组复制子转录和复制的影响表明，它是病毒转录所必需的，而不是病毒RNA复制所必需的。体外转录实验表明，SFPQ/PSF可提高病毒mRNA多聚腺苷化的效率。这是对流感病毒转录必不可少的细胞因子的首次描述，并为鉴定针对宿主-病毒相互作用并阻断病毒基因表达的抑制剂提供了可能性。
The influenza A virus RNA polymerase is a heterotrimeric complex responsible for viral genome transcription and replication in the nucleus of infected cells. We recently carried out a proteomic analysis of purified polymerase expressed in human cells and identified a number of polymerase-associated cellular proteins. Here we characterise the role of one such host factors, SFPQ/PSF, during virus infection. Down-regulation of SFPQ/PSF by silencing with two independent siRNAs reduced the virus yield by 2–5 log in low-multiplicity infections, while the replication of unrelated viruses as VSV or Adenovirus was almost unaffected. As the SFPQ/PSF protein is frequently associated to NonO/p54, we tested the potential implication of the latter in influenza virus replication. However, down-regulation of NonO/p54 by silencing with two independent siRNAs did not affect virus yields. Down-regulation of SFPQ/PSF by siRNA silencing led to a reduction and delay of influenza virus gene expression. Immunofluorescence analyses showed a good correlation between SFPQ/PSF and NP levels in infected cells. Analysis of virus RNA accumulation in silenced cells showed that production of mRNA, cRNA and vRNA is reduced by more than 5-fold but splicing is not affected. Likewise, the accumulation of viral mRNA in cicloheximide-treated cells was reduced by 3-fold. In contrast, down-regulation of SFPQ/PSF in a recombinant virus replicon system indicated that, while the accumulation of viral mRNA is reduced by 5-fold, vRNA levels are slightly increased. In vitro transcription of recombinant RNPs generated in SFPQ/PSF-silenced cells indicated a 4–5-fold reduction in polyadenylation but no alteration in cap snatching. These results indicate that SFPQ/PSF is a host factor essential for influenza virus transcription that increases the efficiency of viral mRNA polyadenylation and open the possibility to develop new antivirals targeting the accumulation of primary transcripts, a very early step during infection. The influenza A viruses cause annual epidemics and occasional pandemics of respiratory infections that may be life threatening. The viral genome contains 8 RNA molecules forming ribonucleoproteins that replicate and transcribe in the nucleus of infected cells. Influenza viruses are intracellular parasites that need the host cell machinery to replicate. To better understand this virus-cell interplay we purified the viral RNA polymerase expressed in human cells and identified several specifically associated cellular proteins. Here we characterise the role of one of them, the proline-glutamine rich splicing factor (SFPQ/PSF). Down-regulation of SFPQ/PSF indicated that it is essential for virus multiplication. Specifically, the accumulation of messenger and genomic virus-specific RNAs was reduced by SFPQ/PSF silencing in infected cells. Furthermore, transcription of parental ribonucleoproteins was affected by SFPQ/PSF down-regulation. The consequences of silencing SFPQ/PSF on the transcription and replication of a viral recombinant replicon indicated that it is required for virus transcription but not for virus RNA replication. In vitro transcription experiments indicated that SFPQ/PSF increases the efficiency of virus mRNA polyadenylation. This is the first description of a cellular factor essential for influenza virus transcription and opens the possibility to identify inhibitors that target this host-virus interaction and block virus gene expression.
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