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

在一些病毒系统中，不同类型的病毒样颗粒（vlp）的产生取决于所表达的病毒蛋白。正转录逆转录病毒仅需要表达病毒核心前体蛋白（Gag）来分泌VLPs，然而，在某些情况下，观察到糖蛋白（Env）共表达增强。泡沫病毒（foam virus， FV）是逆转录病毒（spumaretrovirinae）亚科中唯一一种在复制策略上表现出许多独特特征的病毒属，它们与肝病毒具有同源性，但又与正逆转录病毒不同。FV颗粒输出是特殊的，因为除了FV Gag本身只在细胞质中形成非包膜衣壳外，FV Env的共表达也是这个过程严格要求的。这表明Gag和Env都含有病毒颗粒出口所必需的决定因素，这一点得到了观察结果的支持，类似于乙型肝炎病毒s抗原，原型FV (PFV) Env表达本身导致释放含有亚病毒颗粒（SVPs）的无衣壳糖蛋白。我们最近观察到，经过高度不寻常的生物合成，PFV Env在其细胞质结构域（CDs）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MPI-CBG和埃尔兰根病毒学研究所的合作，我们希望通过对糖蛋白的翻译后修饰来确定这种调控的机制。因此，将研究各种PFV环境突变体的细胞内分布和运输及其泛素融合，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，我们最近在PFV Gag中发现了一个经典的PSAP后期组装(L)结构域，该结构域将PFV颗粒出口连接到液泡蛋白分选机制和多泡体，最近被证明被多种病毒用于颗粒出口。相比之下，目前已知的l结构域在PFV Env的cd中均未检测到，并且尚不清楚哪些细胞相互作用伙伴和途径对PFV svp的释放至关重要。在蛋白质组学方法中，我们与德累斯顿MPI-CBG和海德堡病毒学研究所合作，使用高度纯化的PFV svp和含有PFV Env cd的GST融合蛋白的拉下，我们希望鉴定出在出芽和颗粒释放期间特异性结合到svp或与糖蛋白cd相互作用的细胞蛋白。此外，我们将通过诱变分析一种分泌大量SVP的泛素化缺陷PFV糖蛋白变体来表征SVP输出所需的糖蛋白结构域和序列。总之，该项目将导致具有l结构域或相关活性的未知病毒序列的表征，进一步鉴定可能参与病毒和SVP输出的细胞蛋白或途径，并进一步了解病毒糖蛋白的翻译后修饰如何调节这些过程。

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.