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

在几种病毒系统中，根据表达的病毒蛋白的不同，会产生不同类型的病毒样颗粒(VLP)。直肠病毒只需要表达病毒核心前体蛋白(GAG)就可以分泌VLP，然而在某些情况下，糖蛋白(Env)的共同表达被观察到增强。泡沫病毒(Foamy Virus，FV)是逆转录病毒亚科Spumaretrovirinae中唯一一个在复制策略上表现出许多独特特征的属，它与Hepadna病毒具有同源性，但与正反转录病毒不同。FV颗粒输出是特殊的，因为FV Env在这一过程中严格需要共表达FV Env，而Fv Gag本身只在细胞质中形成无包膜的衣壳。这表明Gag和Env都含有病毒颗粒外出所必需的决定因素，这一点得到了如下观察的支持：与乙肝病毒S抗原类似，FV(PFV)原型env自身表达会导致含有亚病毒颗粒(SVP)的无衣壳糖蛋白(SVP)的释放。我们最近观察到，PFV env经历了一种非常不寻常的生物合成，在其细胞质结构域(CDs)上泛素化，泛素化调节病毒和SVP之间的平衡。与德累斯顿的MPI-CBG和Erlangen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控的机制。因此，我们将研究各种PFV Env突变体及其泛素融合蛋白在细胞内的分布和转运，以及特定的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中鉴定了一个经典的PSAP晚期组装(L)结构域，将PFV颗粒输出连接到空泡蛋白分选机械和多囊泡小体，最近证明这些结构域被多种病毒用于颗粒输出。相比之下，目前已知的所有L结构域都不能在PFV Env的CD中检测到，目前还不清楚哪些细胞相互作用伙伴和途径是PFV SVP释放的关键。在使用高纯度的PFV SVP和包含PFV Env的CDS的GST融合蛋白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，鉴定在萌发和颗粒释放过程中特定地被结合到SVP中或与糖蛋白CDS相互作用的细胞蛋白。此外，我们将通过对泛素化缺陷的PFV糖蛋白变体的突变分析来表征SVP出口所必需的糖蛋白结构域和序列，这种糖蛋白变体分泌大量的SVP。综上所述，该项目将导致表征具有L结构域或相关活性的未知病毒序列，鉴定可能参与病毒SVP和SVP出口的更多细胞蛋白或途径，并应进一步理解病毒糖蛋白的翻译后修饰如何调控这些过程。

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.