Becker Katrin A, Florin Luise, Sapp Cornelia, Maul Gerd G, Sapp Martin
Institute of Medical Microbiology and Hygiene, University of Mainz, 55101 Mainz, Germany.
J Virol. 2004 Feb;78(3):1121-8. doi: 10.1128/jvi.78.3.1121-1128.2004.
Recent reports suggest that nuclear domain(s) 10 (ND10) is the site of papillomavirus morphogenesis. The viral genome replicates in or close to ND10. In addition, the minor capsid protein, L2, accumulates in these subnuclear structures and recruits the major capsid protein, L1. We have now used cell lines deficient for promyelocytic leukemia (PML) protein, the main structural component of ND10, to study the role of this nuclear protein for L2 incorporation into virus-like particles (VLPs). L2 expressed in PML protein knockout (PML(-/-)) cells accumulated in nuclear dots, which resemble L2 aggregates forming at ND10 in PML protein-containing cells. These L2 assemblies also attracted L1 and the transcriptional repressor Daxx, suggesting that they are functional in the absence of PML protein. In addition, L2-containing VLPs assembled in PML(-/-) cells. In order to analyze whether incorporation of L2 into VLPs requires any specific subcellular localization, an L1 mutant defective for nuclear transport and L2 mutants deficient in nuclear translocation and/or ND10 localization were constructed. Using this approach, we identified two independent L2 domains interacting with L1. Mutant L2 proteins not accumulating in ND10 were incorporated into VLPs. Mutant L1 protein, which assembled into VLPs in the cytoplasm, did not incorporate L2 defective for nuclear translocation. The same mutant L2 protein, which passively diffuses into the nucleus, is incorporated into wild-type L1-VLPs in the nucleus. Our data demonstrate that the incorporation of L2 into VLPs requires nuclear but not ND10 localization.
最近的报告表明，核区10（ND10）是乳头瘤病毒形态发生的场所。病毒基因组在ND10内或其附近复制。此外，次要衣壳蛋白L2在这些核亚结构中积累，并募集主要衣壳蛋白L1。我们现在利用缺乏早幼粒细胞白血病（PML）蛋白（ND10的主要结构成分）的细胞系，来研究这种核蛋白在L2掺入病毒样颗粒（VLP）中的作用。在PML蛋白敲除（PML(-/-)）细胞中表达的L2积聚在核点中，这些核点类似于在含PML蛋白的细胞中于ND10形成的L2聚集体。这些L2组装体还吸引了L1和转录抑制因子Daxx，这表明它们在没有PML蛋白的情况下仍具有功能。此外，在PML(-/-)细胞中组装了含L2的VLP。为了分析L2掺入VLP是否需要任何特定的亚细胞定位，构建了一种核转运缺陷的L1突变体以及核易位和/或ND10定位缺陷的L2突变体。通过这种方法，我们鉴定出了与L1相互作用的两个独立的L2结构域。未在ND10中积累的突变L2蛋白被掺入VLP。在细胞质中组装成VLP的突变L1蛋白，没有掺入核易位缺陷的L2。被动扩散到细胞核中的相同突变L2蛋白，在细胞核中被掺入野生型L1-VLP。我们的数据表明，L2掺入VLP需要细胞核定位，但不需要ND10定位。
