Alphavirus Minus Strand RNA Synthesis and RNase L

描述（由申请人提供）：我们正在提交一份新申请的修订，该申请利用了我们认为将为该病毒家族开启新的研究方向的发现所建议的可能性。甲病毒引起普遍关注，因为它们在动物和人类中产生疾病，并在无脊椎动物和脊椎动物中复制。感染性子代的合成始于将感染基因组翻译和复制成负链RNA模板。值得注意的是，甲病毒负链的合成在早期阶段结束时，即感染后约4小时（pi），以一种未知的机制停止。以前的模型认为停止是病毒的特性，宿主因子或组装位点饱和，或者可能是过早和不适当的复制酶处理起了作用。我们现在知道宿主细胞激活潜伏的抗病毒反应，导致进入后4小时内停止。为了支持宿主功能的作用，在自然缺乏RNase L并形成持续感染的C7/10蚊子细胞或RNase L敲除（ko）小鼠胚胎成纤维细胞（MEF）中没有发生负链停止，这些细胞缺乏dsRNA-2‘-5’寡oa合成酶途径的这种潜在内切酶介质；RNaseL在ko细胞中的重建恢复了停止。此外，与野生型nsP2复制子不同，持续感染突变型Sindbis nsP2复制子的BHK细胞也继续负链合成。出乎意料的是，在缺乏RNase L或表达突变的nsP2蛋白的细胞中，Sindbis复制复合体（RC）现在的转录寿命很短。因此，RNase L和nsP2在停止和RC稳定性中起作用。停止在PKR ko和wt MEF中是正常的，消除了Mx1和PKR的作用。提出了四个目标。Aim 1将绘制停止所需的RNase L区域。目的2将使用基因微阵列来鉴定感染和停止期间宿主mrna的变化。Aim 3将分析突变的nsP2蛋白，该蛋白在BHK细胞中的存在导致失去与RNase L-/- MEF和伊蚊细胞相同的病毒复制特征。目的4将探讨Sindbis RC在RNase L缺失和突变的nsP2细胞环境中丢失（翻转）的分子基础。总之，这些结果将阐明对甲病毒感染的先天反应以及宿主RNase L和病毒nsP2蛋白在其结果中的作用。

DESCRIPTION (provided by applicant): We are submitting a revision of a new application that took advantage of possibilities suggested by findings that we believe will begin a new research direction for this family of viruses. Alphaviruses are of general interest as they produce disease in animals and humans, and replicate in invertebrates and vertebrates. Synthesis of infectious progeny starts with translation and copying of the infecting genome into a minus strand RNA template. Of note, alphavirus minus strand synthesis ceases by an as yet unknown mechanism at the end of the early phase, ~4 h post-infection (pi). Previous models argued cessation was a virus property, with saturation of host factors or assembly sites, or possibly premature and improper replicase processing playing a role. We now know the host cell activates a latent, antivirus response leading to cessation during the 4 h period after entry. In support of a role for host functions, minus strand cessation did not occur in C7/10 mosquito cells that naturally lack RNase L and form persistent infections or in RNase L knockout (ko) mouse embryo fibroblasts (MEF) that are deficient in this latent endonuclease mediator of the dsRNA-2'-5' oligoA synthetase pathway; reconstitution of RNaseL in ko cells restored cessation. Moreover, BHK cells persistently infected with mutant Sindbis nsP2 replicons also continued minus strand synthesis, unlike wildtype nsP2 replicons. Unexpectedly, in cells lacking RNase L or expressing mutant nsP2 proteins, the Sindbis replication complex (RC) was now transcriptionally shortlived. Thus, RNase L and nsP2 play role(s) in cessation and RC stability. Cessation was normal in PKR ko and wt MEF, eliminating roles for Mx1 and PKR. Four aims are proposed. Aim 1 will map the region of RNase L required for cessation. Aim 2 will use gene microarrays to identify changes in host mRNAs during infection and with cessation. Aim 3 will analyze mutant nsP2 proteins, whose presence in BHK cells led to loss of the same virus replication features as seen in RNase L-/- MEF and Aedes mosquito cells. Aim 4 will probe the molecular basis of the loss (turnover) of Sindbis RC in RNase L deficient and mutant nsP2 cell environments. Together, the results will elucidate the innate response to alphavirus infection and the role of the host RNase L and the viral nsP2 protein in its outcome.