MECHANICS OF HANTAVIRAL NUCLEOCAPSID PROTEIN MEDIATED TRANSLATION INITIATION OF

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 真核翻译始于在mRNA帽招募eIF4F复合体，在mRNA5‘端与43S预起始复合体结合。另一种被几种病毒充分利用的机制包括IRES翻译启动策略，该策略在内部将核糖体加载到mRNA上，而不依赖于5‘帽。汉坦病毒是布尼亚病毒科(Bunyaviridae)家族的成员，是一种新出现的病毒，它通过一种不同的新机制启动mRNA翻译，利用病毒衣壳蛋白(N)在mRNA帽与核糖体接触，而不依赖于真核细胞中的IF4F复合体。我们将进一步描述N介导的翻译启动机制，并说明这一新策略有利于病毒在感染细胞中复制的可能好处。我们将确定43S预启动复合体的成分，这些组分与N、N特异性地与病毒mRNA5‘UTR高亲和力结合，并在体外促进病毒mRNAs的翻译。我们将鉴定和表征N在病毒mRNA5‘非编码区的结合部位，并确定N是否优先促进病毒RNA在宿主细胞中的翻译。N也是一种RNA伴侣，可以解开RNA双链。然而，这种RNA伴侣活性并不参与N介导的mRNA翻译。在核糖体扫描和AUG密码子鉴定过程中，eIF4A(eIF4F复合体的一个成分)去除了mRNA5‘UTR中的二级结构。由于N在功能上取代了eIF4F复合体，我们假设N将装载的核糖体从5‘端转移到AUG密码子，避免了对5’端前导的常规扫描。已经设计了多方面的实验方法来检验这一假设。我们将使用多种实验方法来检查N介导的翻译策略是否是针对宿主细胞抗病毒反应的病毒对策。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Eukaryotic translation begins with recruitment of eIF4F complex at mRNA cap with the engagement of 43S pre-initiation complex at mRNA 5' terminus. Another well characterized mechanism utilized by several viruses includes IRES translation initiation strategy that internally loads ribosomes on mRNA, independent of 5' cap. Hantaviruses, members of the Bunyaviridae family are emerging viruses that initiate mRNA translation by a different novel mechanism, using viral capsid protein (N) to engage the ribosome at mRNA cap, independent of eukaryotic IF4F complex. We will further characterize N mediated translation initiation mechanism and illustrate possible benefits of this novel strategy that favor virus replication in infected cells. We will identify the components of 43S pre-initiation complex that interact with N. N specifically binds the viral mRNA 5' UTR with high affinity and referentially facilitates the translation of viral mRNAs in vitro. We will identify and characterize the binding site for N on viral mRNA 5' UTR and will determine whether N preferentially facilitates the translation of viral RNAs in host cells. N is also an RNA chaperone that unwinds RNA duplexes. However, this RNA chaperone activity is not involved in N mediated mRNA translation. Secondary structures in mRNA 5' UTR are removed by eIF4A (a component of eIF4F complex) during ribosome scanning and identification of AUG codon. Since N functionally supplants eIF4F complex, we hypothesize that N translocates the loaded ribosomes from 5' cap to the AUG codon, avoiding the regular scanning of 5' leader. Multifaceted experimental approaches have been designed to test this hypothesis. We will use multiple experimental approaches to check whether N mediated translation strategy is a viral counter measure against host cell antiviral response.