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; 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.

在几种病毒系统中，根据表达病毒蛋白的方式产生不同类型的病毒样颗粒（VLP）。矫正病毒仅需要表达病毒核心前体蛋白（GAG）进行VLP的分泌，但是，在某些情况下，在糖蛋白（ENG）共表达时会观察到增强。泡沫病毒（FV）是逆转录病毒亚科Spumaretrovirae的唯一属，在其复制策略中显示出许多独特的特征，并与肝炎病毒的复制策略具有同源性，但将其与邻术病毒区分开来。 FV粒子导出是特殊的，因为除FV GAG外，该过程严格需要FV ENV的共表达，而FV GAG本身仅形成了细胞质中的非发育的衣壳。这表明插科打g和env都包含对病毒颗粒出口必不可少的决定因素，这一观察结果与乙型肝炎病毒S抗原相似，原本型FV（PFV）ENV的表达本身会导致无辛酸无帽糖糖蛋白含有含有亚病毒亚颗粒（SVPS）。我们最近观察到，经历了高度不寻常的生物合成的PFV Env在其细胞质结构域（CDS）和泛素化中被泛素化调节病毒和SVP释放之间的平衡。在德累斯顿的MPI-CBG和Erlangen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突变体和泛素融合的细胞内分布和运输，以及特定蛋白酶体抑制剂对病毒亚颗粒颗粒释放的影响。此外，我们最近还表征了PFV插入中的经典PSAP晚期组件（L）结构域，将PFV颗粒导出到液泡蛋白排序机制和多囊体物体最近被各种病毒用于颗粒流体。相反，在PFV Env的CD中无法检测到当前已知的L域，并且尚不清楚哪些细胞相互作用伙伴和途径对于释放PFV SVP是必不可少的。在使用高度纯化的PFV SVP和GST融合蛋白的下拉蛋白的蛋白质组学方法中，我们希望与Dresden的MPI-CBG合作识别PFV Env的CD，以及Heidelberg中的MPI-CBG，Heidelberg的病毒学研究所，在Heidelberg中，这些细胞蛋白，这些细胞蛋白，这些细胞蛋白在SVP中专门纳入SVP或与Gredcoprded and decroted and Fudded and dection and budcoproted and budcopred and decropted and budcoproted and budcoprded and cons and budcoproted and cons and unted。此外，我们将通过对泛素化缺陷型PFV糖蛋白变体的诱变分析来表征SVP导出所必需的糖蛋白结构域和序列，该分析分泌了大量的SVP。该项目在一起应导致具有L域或相关活性的未知病毒序列的表征，鉴定进一步参与病毒SVP出口可能涉及的进一步的细胞蛋白或途径，并应进一步了解病毒性糖蛋白的翻译后修饰如何调节这些过程。