Cellular proteins and pathways involved in subviral particle release of foamy viruses

参与泡沫病毒亚病毒颗粒释放的细胞蛋白和途径

• 批准号: 13043321
• 负责人: Professor Dr. Dirk Lindemann
• 金额: --
• 依托单位: Institut für Medizinische Mikrobiologie und Virolgie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2005
• 资助国家: 德国
• 起止时间: 2004-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/13043321?language=en
• 关键词: Cellular; proteins; pathways; involved; subviral

In several viral systems, different types of virus-like particles (VLPs) are produced depending on which viral proteins are expressed. Orthoretroviruses require only expression of the viral core precursor protein (Gag) for secretion of VLPs, however, in some cases an enhancement is observed upon glycoprotein (Env) coexpression. Foamy viruses (FV) are the only genus of the retrovirus subfamily spumaretrovirinae that show many unique features in their replication strategy and bearing homology to those of hepadnaviruses but setting them apart from orthoretroviruses. FV particle export is special because coexpression of FV Env is strictly required for this process in addition to FV Gag, which by itself only forms non-enveloped capsids in the cytoplasm. This indicates that both Gag and Env contain determinants essential for viral particle egress which is supported by the observation that, similar to hepatitis B virus S-antigen, prototype FV (PFV) Env expression by itself leads to release of capsid-less glycoprotein containing subviral particles (SVPs). We have observed recently that PFV Env, which undergoes a highly unusual biosynthesis, is ubiquitylated at its cytoplasmic domains (CDs) and ubiquitylation regulates the balance between viral and SVP release. In collaboration with the MPI-CBG in Dresden and the Institute of Virology in Erlangen we want to determine the mechanism underlying this regulation by posttranslational modification of the glycoprotein. Therefore the intracellular distribution and trafficking of various PFV Env mutants and ubiquitin fusions thereof as well as the influence of specific proteasome inhibitors on viraland subviral particle release will be studied. In addition we recently characterized a classical PSAP late-assembly (L) domain in PFV Gag linking PFV particle export to the vacuolar protein sorting machinery and multivesicular body that were demonstrated recently to be used by a variety of viruses for particle egress. In contrast, none of the currently known L-domains can be detected in the CDs of PFV Env and it is unclear which cellular interaction partners and pathways are essential for the release of PFV SVPs. In a proteomic approach using highly purified PFV SVPs and pull-downs of GST fusion proteins containing the CDs of PFV Env we want to identify, in collaboration with the MPI-CBG in Dresden and the Institute of Virology in Heidelberg, cellular proteins that are specifically incorporated into SVPs or interact with the glycoprotein CDs during budding and particle release. In addition we will characterize glycoprotein domains and sequences essential for SVP export by mutagenesis analysis of a ubiquitylation-deficient PFV glycoprotein variant that secretes high amounts of SVPs. Taken together the project should lead to the characterization of yet unknown viral sequences with L-domain or related activity, the identification of further cellular proteins or pathways potentially involved in viraland SVP egress and should further the understanding on how posttranslational modifications of viral glycoproteins regulate these processes.

在一些病毒系统中，根据表达的病毒蛋白产生不同类型的病毒样颗粒（VLP）。正逆转录病毒仅需要表达病毒核心前体蛋白（Gag）来分泌VLP，然而，在某些情况下，在糖蛋白（Env）共表达时观察到增强。泡沫病毒 (FV) 是逆转录病毒亚科 spumaretrovirinae 中唯一的属，其复制策略显示出许多独特的特征，与嗜肝DNA病毒具有同源性，但又与正逆转录病毒不同。 FV 颗粒输出很特殊，因为除了 FV Gag 之外，此过程还严格需要 FV Env 的共表达，而 FV Gag 本身仅在细胞质中形成无包膜衣壳。这表明 Gag 和 Env 都含有病毒颗粒排出所必需的决定因素，这一点得到了观察的支持，即与乙型肝炎病毒 S 抗原类似，原型 FV (PFV) Env 表达本身会导致含有亚病毒的无衣壳糖蛋白的释放。粒子（SVP）。我们最近观察到，PFV Env 经历了一种非常不寻常的生物合成，其细胞质结构域 (CD) 被泛素化，泛素化调节病毒和 SVP 释放之间的平衡。通过与德累斯顿的 MPI-CBG 和埃尔兰根的病毒学研究所合作，我们希望通过糖蛋白的翻译后修饰来确定这种调节的机制。因此，将研究各种PFV Env突变体及其泛素融合物的细胞内分布和运输以及特定蛋白酶体抑制剂对病毒和亚病毒颗粒释放的影响。此外，我们最近在 PFV Gag 中表征了一个经典的 PSAP 后期组装 (L) 结构域，该结构域将 PFV 颗粒输出连接到液泡蛋白分选机器和多泡体，最近证明这些结构可被多种病毒用于颗粒出口。相比之下，在 PFV Env 的 CD 中无法检测到目前已知的 L 结构域，并且尚不清楚哪些细胞相互作用伙伴和途径对于 PFV SVP 的释放至关重要。在使用高度纯化的 PFV SVP 和含有 PFV Env CD 的 GST 融合蛋白下拉的蛋白质组学方法中，我们希望与德累斯顿的 MPI-CBG 和海德堡病毒学研究所合作，鉴定出特异性的细胞蛋白在出芽和颗粒释放过程中掺入 SVP 或与糖蛋白 CD 相互作用。此外，我们将通过对分泌大量 SVP 的泛素化缺陷 PFV 糖蛋白变体进行诱变分析，来表征 SVP 输出所必需的糖蛋白结构域和序列。总而言之，该项目应能够鉴定具有 L 结构域或相关活性的未知病毒序列，识别可能参与病毒和 SVP 出口的进一步细胞蛋白或途径，并应进一步了解病毒糖蛋白的翻译后修饰如何调节这些蛋白。流程。