Host Genetic Requirements for Viral Infection: A Systems Virology Approach

描述(申请人提供)：先前的研究表明，与野生型大肠杆菌相比，tRNA2-硫代尿苷合成蛋白(TU)途径的失活会导致噬菌体lambda感染的受损，而铁硫簇(ISC)途径的失活则会促进lambda感染。这项拟议研究的主要目标是了解这两条途径如何影响其宿主E中的lambda复制，这两条途径共享半胱氨酸脱氢酶ISCS的共同节点。Coli.ISCS通过IscU和TusA的直接结合将硫转移到这些途径下。TU途径是tRNAGlu、tRNAGln和tRNAALys中U34(S2U34)2-硫基化所必需的。这一修改在生活的所有分支中都是保守的，并已被证明可以改善翻译动态并减少不必要的移帧。有趣的是，在lambda噬菌体中，尾区蛋白gpg和gpGT的产物比例受核糖体移码控制。编码二肽-甘氨酸-赖氨酸-的特定“滑动序列”约有3.5%的时间沿-1方向滑动，产生更长的融合蛋白gpGT。虽然不是最终尾巴结构的一部分，但准确的gpg-gpgt比率对于正确的波长尾巴形成是必要的。我推测，当硫磺沿着TUS途径传递被破坏时，低调的tRNALys增加了在GPG和gpGT之间的“滑动序列”上的移码频率，影响了lambda噬菌体尾巴的合成。我将通过(1)检测TusA和iscU之间对lambda复制的潜在上位效应，(2)确定s2U34对gpg/gpGT比率和所产生的活性噬菌体的影响，以及(3)确定s2U34突变体中Lambda噬菌体复制受损是否部分是一般翻译缺陷的结果来研究这一点。这项研究应该阐明ISC和TU通路在Lambda噬菌体感染中的相互作用，并应该与其他尾部噬菌体和哺乳动物病毒(如HIV)相关，这些病毒使用“滑序列”来控制基因表达比率。最终，这项工作应该会加深对病毒复制的理解，并在翻译水平上创新抗病毒策略。 与公共卫生相关：拟议研究的首要目标是调查一种扰乱病毒复制的新技术。我的研究可能适用于所有使用一种名为程序移码技术来控制蛋白质比例的病毒。我希望这项工作将对我们对病毒复制的理解产生重大影响，并可能导致新的抗病毒策略。好了！

DESCRIPTION (provided by applicant): Previous research has shown that inactivation of the tRNA 2-thiouridine synthesizing protein (tus) pathway leads to impaired bacteriophage lambda infection, while inactivation of the iron-sulfur clusters (isc) pathway facilitates lambda infection relative to wild-type E. coli. The overarching objective of the proposed research is to understand how these two pathways, which share a common node at cysteine desulferase IscS, affect lambda replication in its host-E. coli. IscS transfers sulfur down these pathways through direct binding of IscU and TusA. The tus pathway is required for 2-thiolation of U34 (s2U34) in tRNAGlu, tRNAGln, and tRNALys. This modification is conserved in all branches of life and has been shown to improve translation dynamics and reduce unwanted frameshifting. Interestingly, in lambda phage the product ratio of tail region proteins gpG and gpGT is controlled by ribosomal frameshifting. A specific "slippery sequence" encoding the dipeptide -Gly-Lys- slips in the -1 direction about 3.5% of the time, creating the longer fusion protein gpGT. While not part of the final tail structure, the precise gpG-gpGT ratio is necessary for proper lambda tail formation. I hypothesize that when sulfur relay down the tus pathway is disrupted, the hypomodified tRNALys increases the frequency of frameshifting at the "slippery sequence" between gpG and gpGT, affecting lambda phage tail synthesis. I will investigate this by (1) examining potential epistatic effects between tusA and iscU on lambda replication, (2) determining the effect of s2U34 on the gpG to gpGT ratio and the resulting viable phage, and (3) determining if impaired lambda phage replication in mutants for s2U34 is, in part, a result of general translational defects. This research should elucidate of the interplay between the isc and tus pathways in lambda phage infection and should be relevant to other tailed bacteriophages and mammalian viruses (like HIV) which use "slippery sequences" to control gene expression ratios. In the end, this work should lead to a deeper understanding of viral replication and innovative antiviral strategies at the translational level. PUBLIC HEALTH RELEVANCE: The overarching objective of the proposed research is to investigate a new technique for disrupting virus replication. My research is potentially applicable to all viruses that use a technique called programmed frameshifting to control its protein ratios. I am hopeful that this work will have a dramatic impact on our understanding of virus replication and could lead to new antiviral strategies. !