Dual Role of the pestiviral non-structural protein NS4A in RNA Replication and virion morphogenesis

对于黄病毒科的所有病毒，除结构蛋白外，非结构蛋白（NSPs）是形成感染性病毒粒子所必需的。因此，NSPs在基因组复制和病毒粒子形态发生中具有双重功能。本提案的目的是阐明这种多功能性的分子基础。对于鼠疫病毒来说，NS2-3的切割决定了它的功能：未切割的NS2-3是产生感染性颗粒所必需的，而游离的NS3是RNA复制所必需的。基于一个能够产生病毒粒子的病毒突变体，尽管NS2-3被完全切割，我们可以证明NS3/NS4A表面相互作用是决定该蛋白复合物是否在病毒粒子形态发生或RNA复制中起作用的决定性因素。此外，在详细的功能研究中，我们在BVDV NS4A的c端区域研究了其作为(I) NS3蛋白酶辅助因子，（II）复制酶模块或（III）病毒粒子形态发生组分的功能所必需的氨基酸。这些分析将最后定稿。该应用的核心方面是鉴定复制酶和包装复合物的组成，特别关注鉴定两个单价NS3/NS4A复合物的病毒相互作用伙伴（在复制酶或病毒粒子形态发生复合物中活性）和参与这种相互作用的蛋白质表面。使用的技术是通过“正交蛋白标记”和“生物素-连接酶蛋白融合”的蛋白质-“交联”。目前的研究表明，在NS2和NS3中发现的未切割的NS2-3缺失时，病毒粒子形态发生的功能获得突变增强了NS2/NS3的相互作用。此外，我们观察到NS4A的c端截断导致NS2/NS3相互作用的大量增加。总之，这些发现表明NS2和NS4A的c端部分竞争结合到NS3相同或重叠的表面积上。因此，与NS3表面结合的蛋白伴侣决定了RNA复制或病毒粒子形态发生的复合物的功能化。NS2/NS3的相互作用需要通过相关NS3和NS4A表面的诱变来进一步表征。将使用正交蛋白标记/交联测试关键氨基酸在蛋白质结合中的功能。了解病毒蛋白多功能性的分子基础具有很高的科学意义，因为通过选择性地将相互作用伙伴招募到多功能相互作用表面来实现蛋白质复合物功能化的基本原理很可能也适用于其他正链RNA病毒和逆转录病毒的多蛋白。

For all viruses of the Flaviviridae family the non-structural proteins (NSPs) are in addition to the structural proteins essential for the formation of infectious virions. Accordingly, the NSPs have a dual function in genome replication as well as in virion morphogenesis. Aim of this proposal is the elucidation of the molecular basis of this multifunctionality. For pestiviruses the NS2-3 cleavage decides on its function: while uncleaved NS2-3 is required for the production of infectious particles, free NS3 is essential for RNA replication. Based on a virus mutant which is capable of virion production despite of complete NS2-3 cleavage, we could demonstrate that NS3/NS4A surface interactions are decisive for the decision whether this protein complex functions in virion morphogenesis or RNA replication. Moreover, in a detailed functional study we studied in the C-terminal region of BVDV NS4A which amino acids are essential for its function as (I) NS3 protease cofactor, (II) replicase modul, or (III) component in virion morphogenesis. These analyses are to be finalized. The central aspect of the application is the identification of the composition of the replicase- and packaging-complexes, with special focus on the identification of the viral interaction partners of the two monovalent NS3/NS4A complexes (active either in the replicase or in virion morphogenesis complexes) and the protein surfaces involved in this interaction. Techniques to be used are protein-„crosslinking“ via „orthogonal protein labeling“ und „biotin-ligase protein fusions“. Current studies have shown that gain-of-function mutations for virion morphogenesis in the absence of uncleaved NS2-3, identified in NS2 and NS3, enhance the NS2/NS3 interaction. Furthermore, it was observed that a C-terminal truncation of NS4A induced a massive increase in NS2/NS3 interaction. Together, these findings indicate that NS2 and the C-terminal part of NS4A compete for binding to the same or an over-lapping surface area of NS3. Accordingly, the protein partner binding to this NS3 surface area decides on the functionalization of the complexes for either RNA replication or virion morphogenesis. The NS2/NS3 interaction shall be further characterized by mutagenesis of the relevant NS3 and NS4A surfaces. Critical amino acids will be tested for their functionality in protein binding using orthogonal protein labeling/crosslinking. Understanding the molecular basis for the multifunctionality of viral proteins is of high scientific relevance since the underlying principle of the functionalization of protein complexes by selective recruitment of interaction partners to multifunctional interaction surfaces will most likely be also applied in the polyproteins of other positive-strand RNA viruses and retroviruses.