Global effects of flavivirus sfRNA on translation determined by ribosome profiling

黄病毒包括许多严重的病原体，如寨卡病毒、登革热病毒、西尼罗河病毒和黄热病病毒。在受感染的细胞中，这些病毒产生大量的非编码短黄病毒rna (sfRNAs)，这些rna包含病毒基因组约500个核苷酸3 '非翻译区域的大部分。这些新型病毒rna最近被证明与许多宿主蛋白相互作用，并抑制先天免疫系统某些基因的翻译（蛋白质合成）。因此，产生高水平sfRNA的病毒株更具致病性，而不产生sfRNA的突变体非常温和，因此有望成为候选疫苗。然而，对于sfRNA如何影响细胞mrna的翻译，目前还缺乏一个全面的了解。2014- 2016年寨卡病毒（ZIKV）流行导致因在子宫内接触寨卡病毒而导致的小头畸形和其他发育和神经疾病病例频发，令人震惊。除了抑制免疫应答外，ZIKV sfRNA还抑制脆性X智力迟钝蛋白（FMRP）， FMRP是神经发育的关键翻译调节因子。因此，了解寨卡病毒sfRNA对宿主翻译的全球影响可能有助于未来研究了解该病毒如何操纵宿主并引起疾病。方法：我们将采用核糖体分析的转化方法（RiboSeq）对细胞中整个mrna群体的翻译水平进行取样，以鉴定在sfRNA存在下（无论是单独存在还是在复制ZIKV的情况下）翻译上调或下调的基因。RiboSeq是一种高通量mRNA测序（RNAseq）的修饰，它只显示受翻译核糖体保护的mRNA片段。核糖体保护的片段从给定mRNA中读取的次数与mRNA翻译的活跃程度成正比。这种信息丰富的方法仅少量应用于黄病毒，据我们所知，从未明确确定sfRNA（一种已知的翻译调节因子）的作用。我们还将开发新的统计方法，以改进目前不完善的计算mrna在治疗后翻译效率变化意义的方法。最后，我们将使用多种生物信息学工具来确定mrna的共同结构特征，这些mrna的翻译效率同样受到sfRNA的影响。预期的结果。鉴定翻译受sfRNAs影响的基因将揭示促进或防御寨卡病毒感染的潜在基因和遗传途径。这些可能是我们和其他人未来研究的重点，以确定它们在病毒感染、免疫反应或可能的神经发育障碍中的作用和调节。此外，这些结果可能揭示调控翻译的新途径。这项研究可以为合理设计针对寨卡病毒和其他黄病毒的疫苗或抗病毒药物提供信息，并更好地了解RNA对蛋白质合成的控制，从而有益于人类健康。

Flaviviruses include many serious pathogens such as Zika, dengue, West Nile, and yellow fever viruses. In infected cells these viruses produce abundant, noncoding, short flavivirus RNAs (sfRNAs) that comprise most of the ~500 nucleotide 3’ untranslated region of the viral genome. These novel viral RNAs have been shown recently to interact with numerous host proteins and inhibit translation (protein synthesis) of certain genes of the innate immune system. Thus, virus strains that produce high levels of sfRNA are more pathogenic, and mutants that produce no sfRNA are so mild as to be promising vaccine candidates. However, a picture of how sfRNA globally affects translation of cellular mRNAs is lacking. The Zika virus (ZIKV) epidemic of 2014-16 resulted in frighteningly frequent cases of microcephaly and other developmental and neurological disorders caused by exposure to ZIKV in utero. In addition to inhibiting the immune response, ZIKV sfRNA inhibits Fragile X Mental Retardation Protein (FMRP), a key translational regulator of neurological development. Thus, this knowledge of the global effects of ZIKV sfRNA on host translation could contribute to future research on understanding how this virus manipulates the host and causes disease. Method: We will employ the transformative method of ribosome profiling (RiboSeq) to sample the level of translation across the entire population of mRNAs in the cell in order to identify genes that are translationally up- or down-regulated in the presence of sfRNA, either alone, or in the context of replicating ZIKV. RiboSeq - a modification of high-throughput mRNA sequencing (RNAseq) - reveals only the fragments of mRNAs that are protected by translating ribosomes. The number of reads of ribosome-protected fragments from a given mRNA is proportional to how actively that mRNA is translated. This highly informative method has been applied only sparingly to flaviviruses and, to our knowledge, never to determine specifically the effects of sfRNA, a known translational regulator. We will also develop new statistical methods to improve on current imperfect approaches for calculating significance of changes in translational efficiency of mRNAs in response to a treatment. Finally, we will use a variety of bioinformatics tools to identify common structural features of mRNAs whose translation efficiencies are similarly affected by sfRNA. Expected Outcomes. Identification of genes whose translation is affected by sfRNAs will reveal potential genes and genetic pathways that facilitate - or defend against – ZIKV infection. These can be the focus of future studies by us and others to determine their role and regulation in virus infection, the immune response, or possibly neurodevelopmental disorders. Moreover, these results may reveal new ways of regulating translation. This research can benefit human health by informing rational design of vaccines or antivirals targeting ZIKV and other flaviviruses, and by providing better understanding of control of protein synthesis by RNA.