Hendra virus trafficking and assembly

性状（由申请方提供）：亨德拉病毒（HeV）和尼帕病毒是副粘病毒科亨尼帕病毒属的成员，是在人类中具有高死亡率的人畜共患病毒。亨尼帕病毒颗粒装配由病毒基质（M）蛋白协调，所述病毒基质（M）蛋白将病毒糖蛋白和病毒核糖核蛋白连接在一起。荷兰实验室最近定义了亨尼帕病毒融合（F）蛋白的蛋白水解切割的新机制。与其他副粘病毒F蛋白相反，亨尼帕病毒F蛋白以无活性、未切割的形式到达细胞表面，从质膜（PM）内吞，并在早期内体（EE）内遇到组织蛋白酶。在组织蛋白酶切割后，F通过含有Rab 11的再循环内体（RE），然后返回PM以掺入出芽病毒颗粒中。施密特实验室最近鉴定了亨尼帕病毒基质（M）蛋白和AP-3衔接子复合物的β亚基之间的相互作用，所述AP-3衔接子复合物参与内体运输。M蛋白定位于Rab 11区室，并且这种定位在M结合AP 3B 1衍生的抑制性多肽表达后被破坏。为了探索Rab 11阳性RE中M和F蛋白会聚的意义，荷兰和施密特实验室现在已经联合他们的努力，发现内吞运输缺陷的F突变体不能参与VLP组装，并且引入显性阴性Rab 11蛋白显著抑制Hendra VLP的产生。因此，我们假设含有Rab-11的内体区室作为病毒萌芽之前关键病毒蛋白的关键组装位点。在目的1中，我们将通过使用一组全面的运输改变的F蛋白突变体来检查HeV病毒F蛋白运输在病毒组装中的作用，并将使用灵敏的邻近连接测定来分析Rab 11 RE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定义之间的相互作用HeV M蛋白和AP-3适配器复合物的β亚基，检查这种相互作用的作用，在M贩运，描绘的贩运途径，并检查这种相互作用的作用，在apial靶向的HeV M蛋白。最后，在目标3中，我们将讨论HeV G蛋白与M或F的相互作用对G组装的作用，测试G蛋白组装成VLP是由与M和F蛋白的冗余相互作用介导的假设。这些实验将为HeV组装提供关键的新信息，并测试Rab 11 RE可能作为病毒组装位点的新假设。如果这一假设是正确的，Rab 11 RE组件参与膜弯曲可能作为新的主机因子非ESCRT介导的出芽。

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.