Tracking the virus-like particles of Macrobrachium rosenbergii nodavirus in insect cells.
Tracking the virus-like particles of Macrobrachium rosenbergii nodavirus in insect cells.
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作者：
Hanapi UF;Yong CY;Goh ZH;Alitheen NB;Yeap SK;Tan WS
罗氏沼虾诺达病毒 (MrNv) 对虾业构成重大威胁。目前，尚无有效的疫苗和治疗方法可以预防 MrNv 的传播。它的感染机制和在宿主细胞中的定位也没有得到很好的表征。大肠杆菌中产生的 MrNv 衣壳蛋白 (MrNvc) 自组装成类似于天然病毒的病毒样颗粒 (VLP)。因此，荧光素标记的 MrNvc VLP 被用作模型来研究病毒在草地贪夜蛾 Sf9 细胞中的进入和定位。通过荧光显微镜和亚细胞分级分离，显示MrNvc进入Sf9细胞，并最终到达细胞核。 Z-stack 成像进一步证实了 MrNvc 在 Sf9 细胞的细胞质和细胞核中的存在。氯化铵 (NH4Cl)、金雀异黄素、甲基-β-环糊精或氯丙嗪 (CPZ) 的存在抑制 MrNvc 进入 Sf9 细胞，但细胞松弛素 D 不抑制这一过程。这表明，小窝和网格蛋白介导的内吞作用促进了 MrNvc VLP 的内化。通过活细胞成像记录了 MrNvc VLP 进入 Sf9 细胞的整个内化过程。我们还通过缺失诱变鉴定了 MrNvc 的潜在核定位信号（NLS），并通过经典 NLS 作图进行验证。总的来说，这项研究提供了对 MrNvc VLP 在昆虫细胞中的旅程的深入了解。
Macrobrachium rosenbergii nodavirus (MrNv) poses a major threat to the prawn industry. Currently, no effective vaccine and treatment are available to prevent the spread of MrNv. Its infection mechanism and localisation in a host cell are also not well characterised. The MrNv capsid protein (MrNvc) produced in Escherichia coli self-assembled into virus-like particles (VLPs) resembling the native virus. Thus, fluorescein labelled MrNvc VLPs were employed as a model to study the virus entry and localisation in Spodoptera frugiperda, Sf9 cells. Through fluorescence microscopy and sub-cellular fractionation, the MrNvc was shown to enter Sf9 cells, and eventually arrived at the nucleus. The presence of MrNvc within the cytoplasm and nucleus of Sf9 cells was further confirmed by the Z-stack imaging. The presence of ammonium chloride (NH4Cl), genistein, methyl-β-cyclodextrin or chlorpromazine (CPZ) inhibited the entry of MrNvc into Sf9 cells, but cytochalasin D did not inhibit this process. This suggests that the internalisation of MrNvc VLPs is facilitated by caveolae- and clathrin-mediated endocytosis. The whole internalisation process of MrNvc VLPs into a Sf9 cell was recorded with live cell imaging. We have also identified a potential nuclear localisation signal (NLS) of MrNvc through deletion mutagenesis and verified by classical-NLS mapping. Overall, this study provides an insight into the journey of MrNvc VLPs in insect cells.