Biology Department, Boston College, Chestnut Hill, MA 02467, USA.
Virology. 2012 Jan 20;422(2):413-24. doi: 10.1016/j.virol.2011.11.007. Epub 2011 Dec 6.
In this study we examined the transport signals contributing to HPV16 L2 nucleocytoplasmic traffic using confocal microscopy analysis of enhanced green fluorescent protein-L2 (EGFP-L2) fusions expressed in HeLa cells. We confirmed that both nuclear localization signals (NLSs), the nNLS (1MRHKRSAKRTKR12) and cNLS (456RKRRKR461), previously characterized in vitro (Darshan et al., 2004), function independently in vivo. We discovered that a middle region rich in arginine residues (296SRRTGIRYSRIGNKQTLRTRS316) functions as a nuclear retention sequence (NRS), as mutagenesis of critical arginine residues within this NRS reduced the fraction of L2 in the nucleus despite the presence of both NLSs. Significantly, the infectivity of HPV16 pseudoviruses containing either RR297AA or RR297EE within the L2 NRS was strongly reduced both in HaCaT cells and in a murine challenge model. Experiments using Ratjadone A nuclear export inhibitor and mutation-localization analysis lead to the discovery of a leucine-rich nuclear export signal ((462)LPYFFSDVSL) mediating 16L2 nuclear export. These data indicate that HPV16 L2 nucleocytoplasmic traffic is dependent on multiple functional transport signals.
在这项研究中，我们使用共聚焦显微镜分析，研究了 HPV16 L2 核质转运的信号，方法是在 HeLa 细胞中表达增强型绿色荧光蛋白-L2（EGFP-L2）融合蛋白。我们证实，两个核定位信号（NLS），即先前在体外鉴定的 nNLS（1MRHKRSAKRTKR12）和 cNLS（456RKRRKR461），在体内独立发挥作用。我们发现富含精氨酸残基的中间区域（296SRRTGIRYSRIGNKQTLRTRS316）作为核滞留序列（NRS），因为该 NRS 中关键精氨酸残基的突变尽管存在两个 NLS，但会减少 L2 在核内的比例。重要的是，在 L2 NRS 内含有 RR297AA 或 RR297EE 的 HPV16 假病毒的感染性在 HaCaT 细胞和小鼠挑战模型中均显著降低。使用 Ratjadone A 核输出抑制剂和突变定位分析的实验导致发现了一个富含亮氨酸的核输出信号（（462）LPYFFSDVSL），介导 16L2 的核输出。这些数据表明，HPV16 L2 的核质转运依赖于多个功能转运信号。
