Aydin Inci, Villalonga-Planells Ruth, Greune Lilo, Bronnimann Matthew P, Calton Christine M, Becker Miriam, Lai Kun-Yi, Campos Samuel K, Schmidt M Alexander, Schelhaas Mario
Cellular Virology, Institutes of Molecular Virology and Medical Biochemistry, Center for Molecular Biology of Inflammation (ZMBE), University of Münster, Münster, Germany.
Cells in Motion, CiM, Cluster of Excellence EXC, Münster, Germany.
PLoS Pathog. 2017 May 2;13(5):e1006308. doi: 10.1371/journal.ppat.1006308. eCollection 2017 May.
Incoming papillomaviruses (PVs) depend on mitotic nuclear envelope breakdown to gain initial access to the nucleus for viral transcription and replication. In our previous work, we hypothesized that the minor capsid protein L2 of PVs tethers the incoming vDNA to mitotic chromosomes to direct them into the nascent nuclei. To re-evaluate how dynamic L2 recruitment to cellular chromosomes occurs specifically during prometaphase, we developed a quantitative, microscopy-based assay for measuring the degree of chromosome recruitment of L2-EGFP. Analyzing various HPV16 L2 truncation-mutants revealed a central chromosome-binding region (CBR) of 147 amino acids that confers binding to mitotic chromosomes. Specific mutations of conserved motifs (IVAL286AAAA, RR302/5AA, and RTR313EEE) within the CBR interfered with chromosomal binding. Moreover, assembly-competent HPV16 containing the chromosome-binding deficient L2(RTR313EEE) or L2(IVAL286AAAA) were inhibited for infection despite their ability to be transported to intracellular compartments. Since vDNA and L2 were not associated with mitotic chromosomes either, the infectivity was likely impaired by a defect in tethering of the vDNA to mitotic chromosomes. However, L2 mutations that abrogated chromatin association also compromised translocation of L2 across membranes of intracellular organelles. Thus, chromatin recruitment of L2 may in itself be a requirement for successful penetration of the limiting membrane thereby linking both processes mechanistically. Furthermore, we demonstrate that the association of L2 with mitotic chromosomes is conserved among the alpha, beta, gamma, and iota genera of Papillomaviridae. However, different binding patterns point to a certain variance amongst the different genera. Overall, our data suggest a common strategy among various PVs, in which a central region of L2 mediates tethering of vDNA to mitotic chromosomes during cell division thereby coordinating membrane translocation and delivery to daughter nuclei.
入侵的乳头瘤病毒（PVs）依赖有丝分裂时核膜破裂来首次进入细胞核进行病毒转录和复制。在我们之前的研究中，我们推测PVs的次要衣壳蛋白L2将入侵的病毒DNA拴系到有丝分裂染色体上，引导它们进入新生细胞核。为了重新评估在有丝分裂前期L2特异性地招募到细胞染色体上的动态过程，我们开发了一种基于显微镜的定量检测方法，用于测量L2-EGFP在染色体上的招募程度。分析各种HPV16 L2截短突变体发现了一个147个氨基酸的中央染色体结合区域（CBR），该区域赋予与有丝分裂染色体的结合能力。CBR内保守基序（IVAL286AAAA、RR302/5AA和RTR313EEE）的特定突变会干扰染色体结合。此外，含有染色体结合缺陷的L2（RTR313EEE）或L2（IVAL286AAAA）的具有装配能力的HPV16尽管能够转运到细胞内区室，但感染受到抑制。由于病毒DNA和L2也不与有丝分裂染色体相关，感染性可能因病毒DNA与有丝分裂染色体拴系缺陷而受损。然而，消除染色质结合的L2突变也损害了L2跨细胞内细胞器膜的转运。因此，L2的染色质招募本身可能是成功穿透限制膜的必要条件，从而在机制上将这两个过程联系起来。此外，我们证明L2与有丝分裂染色体的结合在乳头瘤病毒科的α、β、γ和ι属中是保守的。然而，不同的结合模式表明不同属之间存在一定差异。总体而言，我们的数据表明各种PVs之间存在一种共同策略，即L2的中央区域在细胞分裂期间介导病毒DNA与有丝分裂染色体的拴系，从而协调膜转运并传递到子细胞核。
