Zurnic Irena, Hütter Sylvia, Rzeha Ute, Stanke Nicole, Reh Juliane, Müllers Erik, Hamann Martin V, Kern Tobias, Gerresheim Gesche K, Lindel Fabian, Serrao Erik, Lesbats Paul, Engelman Alan N, Cherepanov Peter, Lindemann Dirk
Institute of Virology, Medical Faculty "Carl Gustav Carus", Technische Universität Dresden, Dresden, Germany.
CRTD/DFG-Center for Regenerative Therapies Dresden, Technische Universität Dresden, Dresden, Germany.
PLoS Pathog. 2016 Aug 31;12(8):e1005860. doi: 10.1371/journal.ppat.1005860. eCollection 2016 Aug.
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，目前已知仅有少数宿主细胞因子通过与病毒结构成分相互作用来辅助泡沫病毒（FV）的复制。我们以原型FV（PFV）的Gag蛋白为诱饵进行酵母双杂交（Y2H）筛选，鉴定出细胞周期调节激酶家族成员人类polo样激酶2（hPLK2）为PFV衣壳的新相互作用蛋白。进一步的Y2H研究证实了PFV Gag与人和大鼠来源的几种PLK之间存在相互作用。Gag中一个在灵长类FV中保守且在PFV病毒粒子中被磷酸化的共有丝氨酸 - 苏氨酸/丝氨酸 - 脯氨酸（S - T/S - P）基序，对于PLK的识别至关重要。就大鼠PLK2而言，与PFV Gag相互作用需要功能性的激酶结构域和polo框结构域。通过其染色质 tethering功能，荧光标记的PFV Gag以依赖于Gag STP基序的方式将异位表达的eGFP标记的PLK蛋白选择性地重新定位到有丝分裂染色体上，证实了Gag - PLK相互作用在哺乳动物细胞中的特异性和主导性。在具有复制能力的FV和单轮PFV载体的背景下研究了Gag - PLK相互作用的功能相关性。尽管STP基序突变的病毒表现出野生型（wt）的粒子释放、RNA包装和粒子内逆转录，但其在单周期感染中的复制能力降低了3倍，在长时间的传播感染中最多降低了20倍。当用泛PLK抑制剂处理感染单轮wt Gag PFV载体的细胞时，观察到了惊人相似的缺陷。对突变病毒进入动力学的分析表明存在融合后缺陷，导致整合延迟和减少，同时伴随着整合到异染色质的偏好增强。我们得出结论，PFV Gag与细胞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很重要。
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