Boyle Kathleen A, Greseth Matthew D, Traktman Paula
Department of Microbiology & Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Department of Microbiology & Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
J Virol. 2015 Jun;89(12):6312-27. doi: 10.1128/JVI.00445-15. Epub 2015 Apr 8.
The duplication of the poxvirus double-stranded DNA genome occurs in cytoplasmic membrane-delimited factories. This physical autonomy from the host nucleus suggests that poxvirus genomes encode the full repertoire of proteins committed for genome replication. Biochemical and genetic analyses have confirmed that six viral proteins are required for efficient DNA synthesis; indirect evidence has suggested that the multifunctional H5 protein may also have a role. Here we show that H5 localizes to replication factories, as visualized by immunofluorescence and immunoelectron microscopy, and can be retrieved upon purification of the viral polymerase holoenzyme complex. The temperature-sensitive (ts) mutant Dts57, which was generated by chemical mutagenesis and has a lesion in H5, exhibits defects in DNA replication and morphogenesis under nonpermissive conditions, depending upon the experimental protocol. The H5 variant encoded by the genome of this mutant is ts for function but not stability. For a more precise investigation of how H5 contributes to DNA synthesis, we placed the ts57 H5 allele in an otherwise wild-type viral background and also performed small interfering RNA-mediated depletion of H5. Finally, we generated a complementing cell line, CV-1-H5, which allowed us to generate a viral recombinant in which the H5 open reading frame was deleted and replaced with mCherry (vΔH5). Analysis of vΔH5 allowed us to demonstrate conclusively that viral DNA replication is abrogated in the absence of H5. The loss of H5 does not compromise the accumulation of other early viral replication proteins or the uncoating of the virion core, suggesting that H5 plays a direct and essential role in facilitating DNA synthesis.
Variola virus, the causative agent of smallpox, is the most notorious member of the Poxviridae family. Poxviruses are unique among DNA viruses that infect mammalian cells, in that their replication is restricted to the cytoplasm of the cell. This physical autonomy from the nucleus has both cell biological and genetic ramifications. Poxviruses must establish cytoplasmic niches that support replication, and the genomes must encode the repertoire of proteins necessary for genome synthesis. Here we focus on H5, a multifunctional and abundant viral protein. We confirm that H5 associates with the DNA polymerase holoenzyme and localizes to the sites of DNA synthesis. By generating an H5-expressing cell line, we were able to isolate a deletion virus that lacks the H5 gene and show definitively that genome synthesis does not occur in the absence of H5. These data support the hypothesis that H5 is a crucial participant in cytoplasmic poxvirus genome replication.
痘病毒双链DNA基因组的复制发生在细胞质膜界定的工厂中。这种与宿主细胞核的物理自主性表明，痘病毒基因组编码了用于基因组复制的全部蛋白质。生化和遗传分析已证实高效DNA合成需要六种病毒蛋白；间接证据表明多功能H5蛋白可能也起作用。在这里，我们表明，通过免疫荧光和免疫电子显微镜观察，H5定位于复制工厂，并且在纯化病毒聚合酶全酶复合物时可以回收。通过化学诱变产生的温度敏感（ts）突变体Dts57在H5中有损伤，在非允许条件下，根据实验方案，其在DNA复制和形态发生方面表现出缺陷。该突变体基因组编码的H5变体在功能上是温度敏感的，但稳定性不是。为了更精确地研究H5如何促进DNA合成，我们将ts57 H5等位基因置于其他方面为野生型的病毒背景中，并且还进行了小干扰RNA介导的H5消耗。最后，我们产生了一个互补细胞系CV-1-H5，这使我们能够产生一种病毒重组体，其中H5开放阅读框被删除并用mCherry取代（vΔH5）。对vΔH5的分析使我们能够确凿地证明在没有H5的情况下病毒DNA复制被废除。H5的缺失不会损害其他早期病毒复制蛋白的积累或病毒粒子核心的脱壳，这表明H5在促进DNA合成中起直接和重要作用。
天花的病原体天花病毒是痘病毒科中最臭名昭著的成员。痘病毒在感染哺乳动物细胞的DNA病毒中是独特的，因为它们的复制限于细胞的细胞质。这种与细胞核的物理自主性具有细胞生物学和遗传学后果。痘病毒必须建立支持复制的细胞质生态位，并且基因组必须编码基因组合成所需的蛋白质库。在这里，我们关注H5，一种多功能且丰富的病毒蛋白。我们证实H5与DNA聚合酶全酶相关联并定位于DNA合成位点。通过产生表达H5的细胞系，我们能够分离出一种缺乏H5基因的缺失病毒，并明确表明在没有H5的情况下不会发生基因组合成。这些数据支持H5是细胞质痘病毒基因组复制的关键参与者这一假设。
