Mechanisms of selective host gene translation regulation in picornavirus infection

小核糖核酸病毒感染导致宿主基因翻译的全球关闭，但仍维持支持病毒复制的某些宿主基因的必要翻译。在寻找这样的宿主细胞mRNAs的过程中，5‘端(末端寡嘧啶束)mRNAs是感兴趣的候选者。5‘顶端基序由7-甲基-G帽和C组成，然后在mRNA的5’端延伸4-15CU。5‘端的mRNAs(约占细胞总mRNAs的30%)编码翻译机制的蛋白质，如核糖体蛋白、翻译起始和延伸因子等。先前研究表明，在饥饿、辐射和热休克等细胞应激条件下，这些mRNAs中5‘顶端基序的存在会导致其翻译抑制。令人惊讶的是，我们最近发现，由微小核糖核酸病毒家族成员柯萨奇病毒B3(CVB3)感染引起的细胞应激诱导真核翻译延伸因子1A(EEF1A)在RNA和蛋白质水平上显著增强。我们还发现，这种上调与病毒蛋白质合成的增加是平行的。然而，潜在的机制尚不清楚。 在这个项目中，我们的目标是了解5‘端顶端mRNAs的选择性翻译与增强的病毒感染之间的关系，以揭示CVB3进化的潜在机制，从而利用宿主基因表达机制为自己谋利。我们推测，CVB3感染通过基因启动子的表观遗传修饰、mTORC1生存信号通路的激活和5‘TOP基序的募集反式作用因子来上调eEF1a的表达，eEF1a通过与病毒RNA和蛋白质的特异性相互作用促进CVB3的复制。在本研究中，我们将使用CVB3/eEF1A作为交互作用对： 目的1：探讨CVB3感染上调eEF1A1转录的机制。 1.1确定eEF1A1启动子可能的甲基化变化，以促进转录。 1.2评估eEF1A1转录是否通过mTORC1-SP1信号通路上调。 目的2：阐明CVB3上调eEF1A1翻译的机制。 2.1测试5‘端帽位点的第一个C残基是否使5’端顶端mRNA与帽结合蛋白eEF4E结合。 2.2确定启动eEF1A1 5‘TOP mRNA翻译的反式作用因素。 目的3：确定eEF1A1促进CVB3复制的机制。 3.1评估eEF1A1与CVB3 3‘非编码区和/或3D RNA聚合酶之间的潜在相互作用，以支持CVB3 RNA转录。 3.2确定eEF1A1与CVB3 5‘端非编码区启动CVB3翻译的潜在相互作用。 这个项目将揭示微小核糖核酸病毒CVB3颠覆宿主机制以促进病毒复制的新机制。这一结果将对病毒学和基因调控领域产生广泛的影响。

Picornaviral infection causes global shutdown of host gene translation but still maintains essential translation of certain host genes supporting viral replication. In the search for such host cellular mRNAs, the 5'TOP (terminal oligopyrimidine tract) mRNAs are candidates of interest. The 5'TOP motif is defined by a 7-methyl-G cap followed by a C and then a stretch of 4-15 CU at the 5' end of mRNA. The 5'TOP mRNAs (~30% of total cellular mRNAs) encode proteins of translational machinery such as ribosomal proteins and translation initiation and elongation factors. The presence of the 5'TOP motif within these mRNAs has previously been shown to cause its translation repression in conditions of cellular stress, such as starvation, radiation, and heat shock. Surprisingly, we recently found that cellular stress caused by infection of coxsackievirus B3 (CVB3), a member of picornavirus family, induced significantly enhanced expression of eukaryotic translation elongation factor 1A (eEF1A), a 5'TOP mRNA, at both the RNA and protein levels. We also found that this upregulation is parallel with increased viral protein synthesis. However, the underlying mechanism is unknown. In this program, our Goal is to understand the relationship of selective translation of 5'TOP mRNAs and the enhanced viral infection to unravel the underlying mechanism by which CVB3 evolves to exploit the host gene expression machinery to its own benefit. We hypothesize that CVB3 infection upregulates the eEF1A expression, a 5'TOP mRNA, by epigenetic modification of gene promoter, activation of mTORC1 prosurvival signal pathway and recruiting trans-acting factor of 5'TOP motif; the upregulated eEF1A promotes CVB3 replication by specific interactions with viral RNA and proteins. We will use CVB3/eEF1A as an interaction pair for this study: Aim 1: To elucidate the mechanism by which CVB3 infection upregulates eEF1A1 transcription. 1.1 To identify potential methylation changes of the eEF1A1 promoter which may enhance transcription. 1.2 To evaluate if upregulation of eEF1A1 transcription occurs via the mTORC1-SP1 signaling pathway. Aim 2: To elucidate the mechanism by which CVB3 upregulates eEF1A1 translation. 2.1 To test if the first C residue at the 5'cap site enables 5'TOP mRNA binding to the cap-binding protein eEF4E. 2.2 To identify the trans-acting factors that initiate eEF1A1 5'TOP mRNA translation. Aim 3: To determine the mechanism by which eEF1A1 promotes CVB3 replication. 3.1 To evaluate the potential interaction of eEF1A1 with CVB3 3'UTR and/or 3D RNA polymerase to support CVB3 RNA transcription. 3.2 To determine the potential interaction of eEF1A1 with CVB3 5'UTR to initiate CVB3 translation. This program will reveal novel mechanisms by which picornavirus CVB3 subverts host machinery to promote viral replication. The results will have wide ranging impact on the fields of virology and gene regulation.