Hendra virus trafficking and assembly

亨德拉病毒贩运和装配

• 批准号: 8722208
• 负责人: Rebecca E. Dutch
• 金额: $ 23.4万
• 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 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8722208
• 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; 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; public health relevance; 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）：Henipavirus家族的Henipavirus属的成员Hendra病毒（HEV）和Nipah病毒是人类死亡率高死亡率的人畜共患病病毒。 HENIPAVIRUS颗粒组件由病毒基质（M）蛋白协调，该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一种新型的HENIPAVIRUS融合蛋白蛋白水解裂解的机制。与其他帕托粘病毒F蛋白相比，HENIPAVIRUS F蛋白从质膜（PM）（PM）内胞吞以无效，未溶解形式到达细胞表面，并在早期内体（EES）内遇到calter蛋白蛋白酶。在组织蛋白酶裂解后，F通过含RAB11的回收内体（RES）进行路由，然后返回PM以掺入萌芽的病毒颗粒中。施密特实验室最近确定了与内体贩运有关的HENIPAVIRUS基质（M）蛋白与AP-3适配器复合物的β亚基之间的相互作用。 M蛋白位于Rab11室中，并且在表达M-结合AP3B1衍生的抑制性多肽时，该定位被破坏。为了探索M和F蛋白收敛在RAB11阳性RES中的重要性，荷兰语和施密特实验室现在已经结合了他们的努力，发现内吞交易中的F突变体有缺陷，无法参与VLP组装，并且引入了显着的负面RAB11蛋白质极大地抑制Hendra VLP VLP的生产。因此，我们假设含有内体隔室的RAB-11充当关键病毒蛋白在病毒萌芽之前的关键组装位点。在AIM 1中，我们将通过使用一组全面的移植F蛋白突变体来检查HEV病毒F蛋白运输在VIUS组装中的作用，并将使用敏感的接近连接测定法分析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，该数据定义了HEV M蛋白与AP-3适配器综合体的β亚基之间的相互作用，研究了这种相互作用在M运输中的作用，描述运输途径，并检查了这种相互作用在HEV M蛋白的APIAL靶向中的作用。最后，在AIM 3中，我们将解决HEV G蛋白与M或F在G组装中的作用，从而检验了G蛋白组装到VLP中的假设是通过与M和F蛋白的冗余相互作用介导的。这些实验将提供有关HEV组装的关键新信息，并测试RAB11 RES可以用作病毒组装位点的新假设。如果该假设是正确的，则与膜弯曲有关的RAB11 RE组件可能是非ESCRT介导的出芽的新型宿主因子。