Host Genetic Requirements for Viral Infection: A Systems Virology Approach

描述（由申请人提供）：先前的研究表明，tRNA 2-硫尿苷合成蛋白（tus）途径的失活导致λ噬菌体感染受损，而铁硫簇（isc）途径的失活相对于野生型大肠杆菌促进λ噬菌体感染。杆菌拟议研究的总体目标是了解这两种途径如何在半胱氨酸转移酶IscS上共享一个共同的节点，影响其宿主E中的lambda复制。杆菌IscS通过IscU和杜莎的直接结合将硫转移到这些途径。tus途径是tRNAGlu、tRNAGln和tRNALys中U34（s2 U34）的2-巯基化所必需的。这种修饰在生命的所有分支中都是保守的，并且已被证明可以改善翻译动力学并减少不必要的移码。有趣的是，在λ噬菌体中，尾区蛋白gpG和gpGT的产物比率由核糖体移码控制。编码二肽-Gly-Lys-的特定“滑动序列”在-1方向上滑动约3.5%的时间，产生较长的融合蛋白gpGT。虽然不是最终尾翼结构的一部分，但精确的gpG-gpGT比率对于适当的λ尾翼形成是必要的。我推测，当硫中继下的tus途径被破坏，低修饰tRNALys增加的频率移码在“滑序列”之间的gpG和gpGT，影响λ噬菌体尾合成。我将通过（1）检查杜莎和iscU对λ复制的潜在上位效应，（2）确定s2 U34对gpG与gpGT比率和所得活噬菌体的影响，以及（3）确定s2 U34突变体中λ噬菌体复制受损是否部分是一般翻译缺陷的结果。这项研究应该阐明λ噬菌体感染中isc和tus途径之间的相互作用，并且应该与其他尾噬菌体和哺乳动物病毒（如HIV）相关，这些病毒使用“滑动序列”来控制基因表达比例。最后，这项工作应该导致更深入地了解病毒复制和创新的抗病毒策略在翻译水平。 公共卫生相关性：拟议研究的总体目标是研究一种破坏病毒复制的新技术。我的研究可能适用于所有使用程序移码技术来控制其蛋白质比例的病毒。我希望这项工作将对我们对病毒复制的理解产生巨大影响，并可能导致新的抗病毒策略。!

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. !