Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes
Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes
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DOI：
10.1128/jvi.00930-16
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发表时间：
2016-11-01
影响因子：
5.4
通讯作者：
Pijlman, G. P.
中科院分区：
文献类型：
--
作者：
Goertz, G. P.;Fros, J. J.;Pijlman, G. P.
黄病毒，如寨卡病毒、黄热病病毒、登革热病毒和西尼罗河病毒（WNV），是人类健康的严重问题。黄病毒在感染细胞中产生丰富的非编码亚基因组黄病毒RNA（sfRNA）。sfRNA由宿主5 '-3'核糖核酸外切酶XRN 1/Pacman在病毒基因组RNA的3'非翻译区（UTR）中的保守RNA结构上的停滞产生。sfRNA的产生在昆虫特异性、蚊媒和蜱媒黄病毒和没有已知载体的黄病毒中是保守的，表明sfRNA在黄病毒生命周期中的关键作用。在这里，我们研究了sfRNA在西尼罗河病毒感染库蚊蚊的功能，并评估其在确定载体能力的作用。产生sfRNA 1缺陷的WNV，其在RNA干扰（RNAi）胜任和受损蚊子细胞系中显示与野生型WNV相似的生长动力学。对WNV感染的蚊子进行的小RNA深度测序表明，野生型和sfRNA 1缺陷型病毒都存在基于小干扰RNA（siRNA）的活性抗病毒反应。此外，我们提供了sfRNA是体内RNAi底物的第一个证据。野生型病毒的3' UTR/sfRNA内的两个可再现的小RNA热点映射到RNA茎环SL-III和3' SL，其突出于三维（3D）sfRNA结构模型。重要的是，我们证明，sfRNA缺陷的西尼罗河病毒显示显着降低感染和传播率在体内时，通过血餐管理。最后，我们表明，传播和感染率不受胸内注射后的sfRNA的影响，从而确定sfRNA作为一个关键的驱动程序，以克服蚊子中肠感染屏障。这是第一份报告描述了一个关键的生物学功能sfRNA的黄病毒感染的节肢动物载体，sfRNA生产的严格保守提供了一个解释。IMPORTANCEUnderstanding the flavivirus transmission cycle is important to identify new target to interfere with disease and to aid development of virus control strategies.黄病毒在节肢动物和哺乳动物细胞中产生大量的非编码病毒RNA，称为sfRNA。为了评估sfRNA在黄病毒传播中的作用，我们用黄病毒西尼罗病毒和sfRNA缺陷突变体西尼罗病毒感染蚊子。我们证明了sfRNA决定了西尼罗河病毒在尖音库蚊中的感染率和传播率。通过血餐与绕过中肠的胸腔内注射的感染的比较揭示了sfRNA对于克服蚊子中肠屏障是重要的。我们还表明，sfRNA是由蚊子中的抗病毒RNA干扰机制处理的。这是第一个报告，描述了一个关键的生物学功能的sfRNA在节肢动物。这些结果解释了为什么sfRNA的产生在进化上是保守的。
Flaviviruses, such as Zika virus, yellow fever virus, dengue virus, and West Nile virus (WNV), are a serious concern for human health. Flaviviruses produce an abundant noncoding subgenomic flavivirus RNA (sfRNA) in infected cells. sfRNA results from stalling of the host 5'-3' exoribonuclease XRN1/Pacman on conserved RNA structures in the 3' untranslated region (UTR) of the viral genomic RNA. sfRNA production is conserved in insect-specific, mosquito-borne, and tick-borne flaviviruses and flaviviruses with no known vector, suggesting a pivotal role for sfRNA in the flavivirus life cycle. Here, we investigated the function of sfRNA during WNV infection of Culex pipiens mosquitoes and evaluated its role in determining vector competence. An sfRNA1-deficient WNV was generated that displayed growth kinetics similar to those of wild-type WNV in both RNA interference (RNAi)-competent and -compromised mosquito cell lines. Small-RNA deep sequencing of WNV-infected mosquitoes indicated an active small interfering RNA (siRNA)-based antiviral response for both the wild-type and sfRNA1-deficient viruses. Additionally, we provide the first evidence that sfRNA is an RNAi substrate in vivo. Two reproducible small-RNA hot spots within the 3' UTR/sfRNA of the wild-type virus mapped to RNA stem-loops SL-III and 3' SL, which stick out of the three-dimensional (3D) sfRNA structure model. Importantly, we demonstrate that sfRNA-deficient WNV displays significantly decreased infection and transmission rates in vivo when administered via the blood meal. Finally, we show that transmission and infection rates are not affected by sfRNA after intrathoracic injection, thereby identifying sfRNA as a key driver to overcome the mosquito midgut infection barrier. This is the first report to describe a key biological function of sfRNA for flavivirus infection of the arthropod vector, providing an explanation for the strict conservation of sfRNA production.IMPORTANCEUnderstanding the flavivirus transmission cycle is important to identify novel targets to interfere with disease and to aid development of virus control strategies. Flaviviruses produce an abundant noncoding viral RNA called sfRNA in both arthropod and mammalian cells. To evaluate the role of sfRNA in flavivirus transmission, we infected mosquitoes with the flavivirus West Nile virus and an sfRNA-deficient mutant West Nile virus. We demonstrate that sfRNA determines the infection and transmission rates of West Nile virus in Culex pipiens mosquitoes. Comparison of infection via the blood meal versus intrathoracic injection, which bypasses the midgut, revealed that sfRNA is important to overcome the mosquito midgut barrier. We also show that sfRNA is processed by the antiviral RNA interference machinery in mosquitoes. This is the first report to describe a pivotal biological function of sfRNA in arthropods. The results explain why sfRNA production is evolutionarily conserved.