Whitlow Zackary W, Connor John H, Lyles Douglas S
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
J Virol. 2006 Dec;80(23):11733-42. doi: 10.1128/JVI.00971-06. Epub 2006 Sep 27.
Host protein synthesis is inhibited in cells infected with vesicular stomatitis virus (VSV). It has been proposed that viral mRNAs are subjected to the same inhibition but are predominantly translated because of their abundance. To compare translation efficiencies of viral and host mRNAs during infection, we used an enhanced green fluorescent protein (EGFP) reporter expressed from a recombinant virus or from the host nucleus in stably transfected cells. Translation efficiency of host-derived EGFP mRNA was reduced more than threefold at eight hours postinfection, while viral-derived mRNA was translated around sevenfold more efficiently than host-derived EGFP mRNA in VSV-infected cells. To test whether mRNAs transcribed in the cytoplasm are resistant to shutoff of translation during VSV infection, HeLa cells were infected with a recombinant simian virus 5 (rSV5) that expressed GFP. Cells were then superinfected with VSV or mock superinfected. GFP mRNA transcribed by rSV5 was not resistant to translation inhibition during superinfection with VSV, indicating that transcription in the cytoplasm is not sufficient for preventing translation inhibition. To determine if cis-acting sequences in untranslated regions (UTRs) were involved in preferential translation of VSV mRNAs, we constructed EGFP reporters with VSV or control UTRs and measured the translation efficiency in mock-infected and VSV-infected cells. The presence of VSV UTRs did not affect mRNA translation efficiency in mock- or VSV-infected cells, indicating that VSV mRNAs do not contain cis-acting sequences that influence translation. However, we found that when EGFP mRNAs transcribed by VSV or by the host were translated in vitro, VSV-derived EGFP mRNA was translated 22 times more efficiently than host-derived EGFP mRNA. This indicated that VSV mRNAs do contain cis-acting structural elements (that are not sequence based), which enhance translation efficiency of viral mRNAs.
在感染水疱性口炎病毒（VSV）的细胞中，宿主蛋白合成受到抑制。有人提出，病毒mRNA也会受到同样的抑制，但由于其丰度高，所以主要进行翻译。为了比较感染期间病毒mRNA和宿主mRNA的翻译效率，我们使用了一种增强型绿色荧光蛋白（EGFP）报告基因，该基因由重组病毒或稳定转染细胞中的宿主细胞核表达。感染后8小时，宿主来源的EGFP mRNA的翻译效率降低了三倍多，而在VSV感染的细胞中，病毒来源的mRNA的翻译效率比宿主来源的EGFP mRNA高约七倍。为了测试在VSV感染期间细胞质中转录的mRNA是否对翻译关闭具有抗性，用表达GFP的重组猿猴病毒5（rSV5）感染HeLa细胞。然后用VSV对细胞进行超感染或进行模拟超感染。在VSV超感染期间，rSV5转录的GFP mRNA对翻译抑制没有抗性，这表明细胞质中的转录不足以防止翻译抑制。为了确定非翻译区（UTR）中的顺式作用序列是否参与VSV mRNA的优先翻译，我们构建了带有VSV或对照UTR的EGFP报告基因，并测量了模拟感染和VSV感染细胞中的翻译效率。VSV UTR的存在不影响模拟感染或VSV感染细胞中的mRNA翻译效率，这表明VSV mRNA不包含影响翻译的顺式作用序列。然而，我们发现，当VSV或宿主转录的EGFP mRNA在体外进行翻译时，VSV来源的EGFP mRNA的翻译效率比宿主来源的EGFP mRNA高22倍。这表明VSV mRNA确实包含顺式作用结构元件（不是基于序列的），这些元件可提高病毒mRNA的翻译效率。
