Hendra virus trafficking and assembly

描述(申请人提供)：亨德拉病毒(HEV)和尼帕病毒是副粘病毒家族亨尼帕病毒属的成员，是人畜共患病病毒，在人类中死亡率很高。汉尼帕病毒颗粒组装由病毒基质(M)蛋白协调，M蛋白连接病毒糖蛋白和病毒核糖蛋白。荷兰实验室最近定义了一种新的蛋白水解性切割HenipaVirus Fusion(F)蛋白的机制。与其他副粘病毒F蛋白不同的是，汉尼帕病毒F蛋白以不活跃的、未被切割的形式到达细胞表面，从质膜(PM)内吞，并在早期内小体(EES)中遇到组织蛋白酶。组织蛋白酶裂解后，F通过含有Rab11的循环内小体(RES)进行传递，然后返回PM并入萌芽中的病毒颗粒。施密特实验室最近发现了汉尼帕病毒基质(M)蛋白与AP-3接头复合体的β亚基之间的相互作用，这与内体运输有关。M蛋白定位于Rab11区段，这种定位被与M结合的AP3B1衍生抑制多肽的表达干扰。为了探索在Rab11阳性的RES中M和F蛋白会聚的意义，荷兰和施密特实验室现在联合努力发现，内吞转运缺陷的F突变体无法参与VLP组装，并且引入显性的负Rab11蛋白显著抑制Hendra VLP的产生。因此，我们假设Rab-11含有内体隔间，在病毒萌发之前是关键病毒蛋白的关键组装部位。在目标1中，我们将通过使用一组全面的运输改变的F蛋白突变体来研究HEV病毒F蛋白运输在VUS组装中的作用，并将使用敏感的邻近连接试验分析Rab11 RES中的F-M相互作用。我们的第二个目标将建立在令人兴奋的初步数据的基础上，定义HEV M蛋白和AP-3接头复合体的β亚基之间的相互作用，检查这种相互作用在M运输中的作用，描绘运输途径，并研究这种相互作用在HEV M蛋白的心尖靶向中的作用。最后，在目标3中，我们将讨论HEV G蛋白与M或F蛋白相互作用在G组装中的作用，验证G蛋白组装成VLP是通过与M和F蛋白的冗余相互作用介导的假设。这些实验将提供有关HEV组装的关键新信息，并验证Rab11 Res可能作为病毒组装位点的新假设。如果这一假设是正确的，参与膜弯曲的Rab11 RE组分可能成为非ESCRT介导的出芽的新的宿主因子。

DESCRIPTION (provided by applicant): Hendra virus (HeV) and Nipah virus, members of the Henipavirus genus of the paramyxovirus family, are zoonotic viruses with high mortality rates in humans. Henipavirus particle assembly is coordinated by the viral matrix (M) proteins, which link together the viral glycoproteins and the viral ribonucleoproteins. The Dutch lab recently defined a novel mechanism for proteolytic cleavage of the Henipavirus fusion (F) proteins. In contrast to other paramyxovirus F proteins, Henipavirus F proteins reach the cell surface in the inactive, uncleaved form, are endocytosed from the plasma membrane (PM), and encounter cathepsin proteases within early endosomes (EEs). Following cathepsin cleavage, F is routed through Rab11-containing recycling endosomes (REs) before returning to the PM for incorporation into budding virus particles. The Schmitt lab recently identified interactions between Henipavirus matrix (M) proteins and the beta subunit of AP-3 adapter complexes which are involved in endosomal trafficking. M protein localized in Rab11 compartments, and this localization was disrupted upon expression of M- binding AP3B1-derived inhibitory polypeptides. To explore the significance of M and F protein convergence in Rab11-positive REs, the Dutch and Schmitt labs have now combined their efforts and discovered that an F mutant defective in endocytic trafficking fails to participate in VLP assembly, and that introduction of a dominant negative Rab11 protein dramatically inhibits Hendra VLP production. We therefore hypothesize that Rab-11 containing endosomal compartments serve as a critical assembly site for key viral proteins prior to viral budding. In Aim 1, we will examine the role of HeV virus F protein trafficking in vius assembly, through use of a comprehensive set of trafficking-altered F protein mutants, and will analyze F- M interactions in Rab11 REs using a sensitive proximity ligation assay. Our second aim will build on the exciting preliminary data defining an interaction between HeV M protein and the beta subunit of the AP-3 adapter complex, examining the role of this interaction in M trafficking, delineating the trafficking pathway, and examining the role of this interaction in apial targeting of the HeV M protein. Finally, in Aim 3 we will address the role of HeV G protein interactions with M or F on G assembly, testing the hypothesis that G protein assembly into VLPs is mediated by redundant interactions with M and F proteins. These experiments will provide critical new information on HeV assembly, and test the novel hypothesis that Rab11 REs may serve as viral assembly sites. If this hypothesis is correct, Rab11 RE components involved in membrane bending may serve as novel host factors for non-ESCRT mediated budding.