Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration.
Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration.
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DOI：
10.1371/journal.ppat.1005860
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发表时间：
2016-08
期刊：
影响因子：
6.7
通讯作者：
Lindemann D
中科院分区：
文献类型：
--
作者：
Zurnic I;Hütter S;Rzeha U;Stanke N;Reh J;Müllers E;Hamann MV;Kern T;Gerresheim GK;Lindel F;Serrao E;Lesbats P;Engelman AN;Cherepanov P;Lindemann D
与其他逆转录病毒不同，只有少数宿主细胞因子通过与病毒结构组分相互作用来帮助泡沫病毒（FV）复制。以FV（PFV）Gag蛋白为诱饵进行酵母双杂交（Y2 H）筛选，我们鉴定了一种新的PFV衣壳相互作用蛋白--人polo样激酶2（hPLK 2）。进一步的Y2 H研究证实了PFV Gag与人和大鼠来源的几种PLK的相互作用。Gag中的共有Ser-Thr/Ser-Pro（S-T/S-P）基序在灵长类FV中是保守的，在PFV病毒粒子中是磷酸化的，对于PLK的识别是必需的。在大鼠PLK 2的情况下，功能性激酶和polo盒结构域需要与PFV Gag相互作用。标记的PFV Gag通过其染色质拴系功能，以Gag STP基序依赖的方式选择性地将异位表达的EGFP标记的PLK蛋白重新定位到有丝分裂染色体，证实了哺乳动物细胞中Gag-PLK相互作用的特异性和显性性质。在复制能力的FV和单轮PFV载体的背景下检查了Gag-PLK相互作用的功能相关性。尽管STP基序突变的病毒显示野生型（wt）颗粒释放、RNA包装和颗粒内逆转录，但它们的复制能力在单循环感染中降低3倍，并且在延长的时间段内在扩散感染中降低高达20倍。当用pan PLK抑制剂处理用单轮wt Gag PFV载体感染的细胞时，观察到惊人相似的缺陷。突变病毒的进入动力学分析表明融合后缺陷导致延迟和减少的整合，这是伴随着增强的偏好整合到异染色质。我们的结论是，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。病毒是利用宿主细胞机制进行复制的高手。对于人类免疫缺陷病毒1型（HIV-1），最好的研究代表正逆转录病毒亚科慢病毒属，许多重要的病毒-宿主相互作用已被描述。相比之下，只有少数细胞蛋白质已知影响泡沫病毒（FV，也称为泡沫病毒）的复制，泡沫病毒是一种有趣类型的复杂逆转录病毒，其复制策略结合了逆转录病毒和嗜肝DNA病毒的特征。考虑到FV作为基因转移工具的兴趣越来越大，以及它们在逆转录病毒家族中的独特地位，这种差异促使鉴定FV结构组分的新宿主细胞相互作用伴侣。本研究集中在原型FV（PFV），最具特征的成员的FV，其衣壳蛋白，Gag，作为病毒复制的中心球员。有丝分裂调节，polo样激酶（PLK）家族的成员被确定为新的Gag结合伴侣。Gag与PLK 1（可能还有PLK 2）的相互作用促进了PFV基因组有效整合到宿主染色质中，确保了在感染的靶细胞培养物中成功复制和病毒传播。总的来说，我们的研究结果阐明了细胞周期调控网络和有丝分裂依赖的PFV整合过程之间的第一个联系。
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. Viruses are masters at exploiting host cell machineries for their replication. For human immunodeficiency virus type 1 (HIV-1), the best-studied representative of the Orthoretrovirinae subfamily from the genus lentiviruses, numerous important virus-host interactions have been described. In contrast, only a few cellular proteins are known to influence the replication of foamy viruses (FVs, also known as spumaviruses), an intriguing type of complex retrovirus of the Spumaretrovirinae subfamily that combines features of both retroviruses and hepadnaviruses in its replication strategy. Given the increasing interest in FVs as gene transfer tools and their unique status within the retrovirus family, this discrepancy urged the identification of novel host cell interaction partners of FV structural components. This study focused on prototype FV (PFV), the best-characterized member of FVs, and its capsid protein, Gag, as the central player of viral replication. Members of the mitosis-regulatory, polo-like kinase (PLK) family were identified as novel Gag binding partners. The Gag interaction with PLK1 (and possibly also PLK2) facilitated efficient PFV genome integration into host chromatin, ensuring successful replication and viral spread in infected target cell cultures. Collectively, our results elucidate the first link between cell cycle regulatory networks and the mitosis-dependent PFV integration process.
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DOI：
10.1038/nrmicro3503
发表时间：
2015-08
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
Campbell EM;Hope TJ
通讯作者：
Hope TJ
影响因子：
5.4
作者：
Giron, ML;Colas, S;EmanoilRavier, R
通讯作者：
EmanoilRavier, R
影响因子：
5.4
作者：
Chen, Yung-Chia;Su, Wen-Chi;Lai, Michael M. C.
通讯作者：
Lai, Michael M. C.
影响因子：
5.3
作者：
DUBRIDGE, RB;TANG, P;CALOS, MP
通讯作者：
CALOS, MP
影响因子：
5.4
作者：
Huetter, Sylvia;Muellers, Erik;Lindemann, Dirk
通讯作者：
Lindemann, Dirk