Mutagenesis of the central contact interface in the WN E dimer

描述（由申请人提供）：对于许多黄病毒，特别是与神经侵袭和脑炎综合征相关的黄病毒，如西尼罗病毒、日本脑炎病毒或蜱传脑炎病毒，毒力表型的表达至少部分取决于包膜蛋白中的决定簇。一般而言，迄今为止所有已知的减毒突变影响E蛋白的主要功能，其在黄病毒通过融合进入的阶段起主要作用。该应用程序旨在研究以前未知的突变的影响，这些突变影响感染性病毒体表面上存在的E二聚体中单体相互作用的强度。我们假设突变增加单体缔合的强度对病毒组装的危害小于对病毒感染性的危害。此类突变在病毒感染周期结束时对病毒体组装的影响极小或没有影响，但可能由于在下一个繁殖周期的初始阶段的干扰而导致病毒减毒。为了验证这一假设，在本初步研究中定义了以下具体目标：1）研究在所鉴定的接触界面处的突变对蛋白E二聚体的形成和稳定性的影响。我们将分析果蝇表达系统中产生的诱变E400胞外结构域的生物化学性质，如二聚化、二聚体稳定性和解离动力学。预期影响二聚体稳定性的突变已通过使用丰富构象中DEN2和DENS E二聚体的可用坐标对NY99 E400胞外域进行同源建模来选择。2)评价单体接触界面突变对WN病毒生物学特性的影响。将携带所选突变的E蛋白插入NY99感染性DNA构建体中，并研究回收病毒的生物学特性，如组织培养中的生长特性和小鼠中的毒力。E二聚体的形成对于病毒体组装和实现其感染特性都是至关重要的。本初步研究的目的是确定在接触界面的选择性诱变是否会影响二聚体的形成及其稳定性，以及观察到的差异是否会导致对病毒生物学特性的差异效应。拟议的研究结果将表明，如果操纵的E二聚体中发现的感染性黄病毒病毒粒子的包膜单体缔合的强度可以提供新的病毒减毒手段。

DESCRIPTION (provided by applicant): For a number of flaviviruses, in particular for those associated with neuroinvasion and with the encephalitic syndrome such as West Nile, Japanese encephalitis or tick-borne encephalitis viruses, expression of the virulent phenotype at least in part depends on determinants in the envelope protein. In general, all known to date attenuating mutations affect the main functionality of the E protein, which plays the major role at the phase of flavivirus entry by fusion. This application is designed to investigate effects of a previously unknown mutations that influence the strength of monomer interaction in the E dimer existing on the surface of infectious virions. We hypothesize that mutations increasing the strength of monomer association will be less detrimental for virus assembly than for the virus infectivity. Such mutations will have minimal or no effect on virion assembly at the end of the viral infectious cycle, yet may lead to virus attenuation due to interference at the initial phase of the next reproductive cycle. To test this hypothesis, the following specific aims have been defined in this pilot study: 1) To investigate effect of mutations at the identified contact interface on formation and the stability of the protein E dimers. We will analyze biochemical properties, such as dimerization, the dimer stability and dissociation kinetics of mutagenized E400 ectodomains produced in the Drosophila expression system. Mutations that are expected to influence the dimer stability have been selected by homology modeling of the NY99 E400 ectodomain using available coordinates of the DEN2 and DENS E dimers in the profusion conformation. 2) To evaluate effect of mutations at the monomer contact interface on the biological properties of WN virus. E proteins carrying the selected mutations will be inserted into the NY99 infectious DNA construct and biological properties of recovered viruses, such as growth characteristics in tissue culture and virulence in mice, will be investigated. Formation of the E dimer is crucial both for virion assembly and for the realization of its infectious properties. The goal of this pilot study is to determine if selective mutagenesis at the contact interface can influence dimer formation and its stability and whether the observed differences will lead to differential effects on virus biological properties. Results of the proposed study will indicate if manipulation of the strength of monomer association in the E dimer found in the envelope of infectious flavivirus virions can offer new means of virus attenuation.