Hendra virus trafficking and assembly

亨德拉病毒贩运和组装

• 批准号: 8803767
• 负责人: Rebecca E. Dutch
• 金额: $ 18.42万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-10 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8803767
• 关键词: Address; Antiviral Agents; Apical; Binding; Biological Assay; Cathepsins; Cell membrane; Cell surface; Cells; Complex; Cytoplasmic Tail; Data; Defect; Development; Dominant-Negative Mutation; Early Endosome; Endocytosis; Endosomes; Family; GTP-Binding Proteins; Glycoproteins; Health; Hendra Virus; Henipavirus; Henipavirus Infections; Human; Integration Host Factors; Lead; Ligation; Link; Measures; Mediating; Membrane; Nipah Virus; Paramyxovirus; Pathway interactions; Peptide Hydrolases; Process; Production; Proteins; Reagent; Recycling; Ribonucleoproteins; Role; Series; Site; Surface; Testing; Ubiquitination; Vaccines; Viral; Viral Proteins; Virion; Virus; Virus Assembly; member; mortality; multiple myeloma M Protein; mutant; novel; particle; pathogen; polarized cell; polypeptide; protein transport; rab11 protein; research study; trafficking

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.

描述（由申请人提供）：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，是人类高死亡率的人畜共患病毒。亨尼帕病毒的粒子组装是由病毒基质(M)蛋白协调的，它将病毒糖蛋白和病毒核糖核蛋白连接在一起。荷兰实验室最近确定了亨尼帕病毒融合(F)蛋白的蛋白水解裂解新机制。与其他副粘病毒F蛋白不同，亨尼帕病毒F蛋白以无活性、未裂解的形式到达细胞表面，从质膜（PM）内吞，并在早期内体（EEs）内遇到组织蛋白酶蛋白酶。在组织蛋白酶裂解后，F穿过含有rab11的再循环核内体（REs），然后返回PM并入出芽病毒颗粒。Schmitt实验室最近发现了亨尼帕病毒基质(M)蛋白与参与内体运输的AP-3适配器复合物的β亚基之间的相互作用。M蛋白定位于Rab11区室，当M结合ap3b1衍生的抑制多肽表达时，这种定位被破坏。为了探索Rab11阳性REs中M和F蛋白趋同的意义，Dutch和Schmitt实验室现在联合他们的努力，发现在内噬运输中有缺陷的F突变体不能参与VLP组装，并且引入显性阴性Rab11蛋白显著抑制亨德拉VLP的产生。因此，我们假设含有Rab-11的内体室在病毒出芽之前作为关键病毒蛋白的关键组装位点。在Aim 1中，我们将通过使用一套全面的运输改变的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，并将使用敏感的近距离连接试验分析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基础上，确定HeV M蛋白与AP-3适配器复合体的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，并检查这种相互作用在HeV M蛋白的资本靶向中的作用。最后，在Aim 3中，我们将讨论HeV G蛋白与M或F的相互作用在G组装中的作用，验证G蛋白组装成VLPs是通过与M和F蛋白的冗余相互作用介导的假设。这些实验将为HeV组装提供重要的新信息，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成分可能是非escrt介导的出芽的新宿主因子。