Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells.

与其他逆转录病毒不同，目前已知仅少数可通过与病毒结构组分相互作用来辅助泡沫病毒（Foamy Viruses, FVs）复制的宿主细胞因子。本研究以原型泡沫病毒（Prototype Foamy Virus, PFV）的Gag蛋白作为诱饵，通过酵母双杂交（Yeast-two-hybrid, Y2H）筛选，鉴定出人类polo样激酶2（Human Polo-like Kinase 2, hPLK2）——一类细胞周期调控激酶家族成员——为PFV衣壳的新型互作蛋白。进一步的酵母双杂交实验证实，PFV Gag可与多种人类和大鼠来源的PLK发生相互作用。Gag蛋白中存在一段保守的Ser-Thr/Ser-Pro（S-T/S-P）共有基序，该基序在灵长类FVs中保守存在且在PFV病毒粒子中发生磷酸化，是PLK识别所必需的关键序列。就大鼠PLK2而言，其功能性激酶结构域与polo盒（polo-box）结构域均为与PFV Gag发生相互作用所必需。带有荧光标签的PFV Gag可通过其染色质锚定功能，以Gag的STP基序依赖的方式，将异位表达的增强绿色荧光蛋白（enhanced Green Fluorescent Protein, eGFP）标记的PLK蛋白选择性重定位至有丝分裂染色体上，这证实了Gag与PLK之间的相互作用在哺乳动物细胞中具有特异性与主导性。本研究在具有复制能力的FVs以及单轮感染PFV载体体系中，探究了Gag-PLK相互作用的功能相关性。尽管STP基序突变的病毒展现出与野生型（Wild Type, wt）一致的病毒粒子释放、RNA包装以及粒子内逆转录能力，但其复制能力在单周期感染中下降了3倍，在长期扩散感染中更是最高下降达20倍。当用广谱PLK抑制剂处理感染了单轮野生型Gag PFV载体的细胞时，也观察到了极为相似的缺陷表型。对突变病毒的进入动力学分析表明，其存在融合后缺陷，导致整合过程延迟且整合效率降低，同时伴随对异染色质区域整合的偏好性增强。综上，本研究认为PFV Gag与细胞源性PLK蛋白之间的相互作用，对于PFV在宿主细胞内的早期复制步骤至关重要。