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.

在几种病毒系统中，不同类型的病毒样颗粒（VLP）的产生取决于表达的病毒蛋白。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（foamy viruses，FV）是逆转录病毒亚科中唯一一个在复制策略上与嗜肝DNA病毒具有同源性但又与正逆转录病毒不同的病毒属。FV颗粒输出是特殊的，因为除了FV Gag之外，该过程严格需要FV Env的共表达，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簇，这得到以下观察结果的支持：与B型肝炎病毒S-抗原类似，原型FV（PFV）Env表达本身导致释放含有亚病毒颗粒（SVP）的无衣壳糖蛋白。我们最近观察到，PFV Env，这经历了一个非常不寻常的生物合成，是泛素化的细胞质结构域（CD）和泛素化调节病毒和SVP释放之间的平衡。我们与德累斯顿的MPI-CBG和埃尔兰根的病毒学研究所合作，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体的细胞内分布和运输，以及特异性蛋白酶体抑制剂对病毒和亚病毒颗粒释放的影响。此外，我们最近的特点是一个经典的PSAP后期组装（L）结构域PFV Gag连接PFV颗粒出口的液泡蛋白分选机器和多泡体，最近被证明是由各种病毒的颗粒出口。相比之下，目前已知的L-结构域中没有一个可以检测到的PFV Env的CD，目前还不清楚哪些细胞相互作用伴侣和途径是必不可少的PFV SVP的释放。在使用高度纯化的PFV SVP和含有PFV Env CD的GST融合蛋白的下拉的蛋白质组学方法中，我们希望与德累斯顿的MPI-CBG和海德堡的病毒学研究所合作，鉴定特异性掺入SVP或在出芽和颗粒释放期间与糖蛋白CD相互作用的细胞蛋白。此外，我们将通过对分泌大量SVP的泛素化缺陷型PFV糖蛋白变体进行诱变分析，来表征SVP出口所必需的糖蛋白结构域和序列。总之，该项目应导致尚未未知的病毒序列与L-结构域或相关活动的特征，进一步的细胞蛋白或途径的鉴定可能涉及病毒和SVP的出口，并应进一步了解病毒糖蛋白的翻译后修饰如何调节这些过程。