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）：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，是人畜共患病毒，在人类中具有高死亡率。亨尼帕病毒颗粒组装由病毒基质 (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的亨尼帕病毒融合 (F) 蛋白的蛋白水解裂解机制。与其他副粘病毒 F 蛋白相比，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。组织蛋白酶裂解后，F 穿过含有 Rab11 的回收内体 (RE)，然后返回 PM 并入出芽的病毒颗粒中。 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 接头复合物的 β 亚基之间的相互作用。 M 蛋白定位于 Rab11 区室中，并且这种定位在 M 结合 AP3B1 衍生的抑制性多肽的表达时被破坏。为了探索 Rab11 阳性 RE 中 M 和 F 蛋白趋同的重要性，Dutch 和 Schmitt 实验室现已联合努力，发现内吞运输缺陷的 F 突变体无法参与 VLP 组装，并且显性失活 Rab11 蛋白的引入会显着抑制 Hendra VLP 的产生。因此，我们假设含有 Rab-11 的内体区室是病毒出芽前关键病毒蛋白的关键组装位点。在目标 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 vius 组装中的作用，并将使用敏感的邻近连接测定分析 Rab11 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的基础上，这些数据定义了 HeV M 蛋白和 AP-3 接头复合物的 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 蛋白的顶端靶向中的作用。最后，在目标 3 中，我们将探讨 HeV G 蛋白与 M 或 F 相互作用对 G 组装的作用，检验 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。这些实验将提供有关 HeV 组装的重要新信息，并测试 Rab11 RE 可能作为病毒组装位点的新假设。如果这一假设正确，那么参与膜弯曲的 Rab11 RE 成分可能作为非 ESCRT 介导的出芽的新宿主因子。