RACK1-mediated control of mRNA selectivity by poxviruses

摘要 病毒已经进化出使用5‘非翻译区(UTRs)来控制病毒蛋白质合成的机制 感染。例如，许多RNA病毒在5‘UTRs中使用内部核糖体进入位点。相反， 痘病毒，如痘苗病毒，编码多聚腺苷(PolyA)重复序列在复制后的5‘非编码区 增强痘病毒转录本翻译的mRNA。中的Polya增强器效果的功能 人类细胞依赖于痘病毒介导的小亚基蛋白延伸环的磷酸化 活化C激酶受体1(RACK1)。尽管我们知道核糖体的特性在 选择性地调节痘病毒蛋白质的合成，目前还不清楚环中的负电荷如何调节mrna。 痘病毒定制的核糖体的选择性以及这种选择性如何受到细胞内全球变化的影响 在感染过程中的转录和翻译活动。使用结构建模和生化方法， 我们先前已经证明，RACK1环中的电荷增加了与RACK1环的排斥静电相互作用 18S rRNA在mRNA出口通道附近的带负电荷的主干。因此，我们假设 负电荷的引入重塑了环或信使核糖核酸的出口通道，从而通过以下方式更广泛地调节翻译 改变对特定5‘UTR元素的识别，例如Polya Leader。我们将通过以下方式解决这一假设 发展两个互补的细胞系统，我们将使用它们来研究RACK1环介导的mRNA选择性 在不同的条件下模拟痘病毒感染的各个方面。我们将使用多聚体图谱和RNA-Seq 分析翻译效率的全球变化，以更广泛地了解特定于文字记录的内容 由RACK1环路调制的翻译。拟议中的研究结果将奠定坚实的基础 以了解痘病毒如何控制翻译，以及未来对核糖体为中心的模式的研究 在不同的生物和病理背景下的翻译调控。

ABSTRACT Viruses have evolved mechanisms to use 5’ untranslated regions (UTRs) to control viral protein synthesis during infection. For example, many RNA viruses use internal ribosome entry sites in the 5’ UTRs. By contrast, poxviruses, such as Vaccinia virus, encode polyadenosine (polyA) repeats in the 5’ UTR of post-replicative mRNA that enhance the translation of poxviral transcripts. The functionality of the polyA enhancer effect in human cells is dependent on poxvirus-mediated phosphorylation of an extended loop of the small subunit protein receptor of activated C kinase 1 (RACK1). Although we know ribosome specification plays a key role in selectively regulating poxviral protein synthesis, it is unclear how negative charge in the loop regulates mRNA selectivity of poxvirus-customized ribosomes and how this selectivity is influenced by global changes in cellular transcriptional and translational activity during infection. Using structure modeling and biochemical approaches, we have previously shown that charge in the RACK1 loop increases repulsive electrostatic interactions with the negatively charged backbone of 18S rRNA near the mRNA exit channel. Therefore, we hypothesize that introduction of negative charge remodels the loop or mRNA exit channel to more broadly regulate translation by altering recognition of specific 5’ UTR elements, such as polyA leaders. We will address this hypothesis by developing two complementary cell systems that we will use to study RACK1-loop mediated mRNA selectivity under different conditions that mimic aspects of poxvirus infection. We will use polysome profiling and RNA-Seq to analyze global changes in translational efficiency to gain a broader understanding of transcript-specific translation modulated by the RACK1 loop. The findings from the proposed research will lay a solid foundation for our understanding of how poxviruses control translation, as well as future studies of ribosome-centric modes of translation regulation in diverse biological and pathological contexts.