School of Cellular and Molecular Medicine, Faculty of Medical and Veterinary Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.
J Biol Chem. 2013 Aug 2;288(31):22621-35. doi: 10.1074/jbc.M113.481382. Epub 2013 Jun 13.
The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-β did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes.
登革病毒（DENV-1 至 -4）的四个血清型引起了最重要的虫媒病毒性人类疾病。DENV 非结构蛋白 5（NS5）包含在宿主细胞质中发生的病毒 RNA 基因组加帽和复制所需的酶活性。然而，先前的研究表明，DENV-2 NS5 在感染过程中积累在核内。在这项研究中，我们检查了所有四种 DENV 血清型的 NS5 的核定位。我们首次证明 NS5 的核定位存在血清型差异。虽然 DENV-2 和 -3 蛋白积累在核内，但 DENV-1 和 -4 NS5 主要（如果不是唯一）定位于细胞质。对 DENV-2 和 -4 NS5 蛋白的比较研究表明，DENV-4 NS5 核定位的差异不是由于核快速输出，而是缺乏功能性核定位序列。使用 DENV-2 和 -4 NS5 和人 importin-α 同种型进行的相互作用研究未能鉴定出支持 NS5 差异核定位的相互作用。siRNA 敲低人 importin-α 同种型 KPNA2（与先前显示与 DENV-2 NS5 结合的鼠 importin-α 同种型相对应）并未显著影响 DENV-2 NS5 的核定位，而 importin-β 的敲低则影响。NS5 的核定位的血清型差异与 IL-8 基因表达的差异无关。结果表明，NS5 的核定位并非严格需要病毒复制，但更可能在特定 DENV 血清型的生命周期中具有辅助功能。
