Virus attachment and entry is a complex interplay of viral and cellular interaction partners. Employing bovine viral diarrhea virus (BVDV) encoding an mCherry-E2 fusion protein (BVDVE2-mCherry), being the first genetically labelled member of the family Flaviviridae applicable for the analysis of virus particles, the early events of infection-attachment, particle surface transport, and endocytosis-were monitored to better understand the mechanisms underlying virus entry and their dependence on the virus receptor, bovine CD46. The analysis of 801 tracks on the surface of SK6 cells inducibly expressing fluorophore labelled bovine CD46 (CD46fluo) demonstrated the presence of directed, diffusive, and confined motion. 26 entry events could be identified, with the majority being associated with a CD46fluo positive structure during endocytosis and occurring more than 20 min after virus addition. Deletion of the CD46fluo E2 binding domain (CD46fluo∆E2bind) did not affect the types of motions observed on the cell surface but resulted in a decreased number of observable entry events (2 out of 1081 tracks). Mean squared displacement analysis revealed a significantly increased velocity of particle transport for directed motions on CD46fluo∆E2bind expressing cells in comparison to CD46fluo. These results indicate that the presence of bovine CD46 is only affecting the speed of directed transport, but otherwise not influencing BVDV cell surface motility. Instead, bovine CD46 seems to be an important factor during uptake, suggesting the presence of additional cellular proteins interacting with the virus which are able to support its transport on the virus surface.

中文翻译：

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牛病毒性腹泻病毒 (BVDV) 感染及其对牛 CD46 依赖性的实时分析。


病毒附着和进入是病毒和细胞相互作用伙伴之间复杂的相互作用。采用编码 mCherry-E2 融合蛋白 (BVDVE2-mCherry) 的牛病毒性腹泻病毒 (BVDV)，是黄病毒科第一个基因标记成员，适用于病毒颗粒分析、感染附着早期事件、颗粒表面转运、和内吞作用 - 进行监测，以更好地了解病毒进入的机制及其对病毒受体牛 CD46 的依赖性。对诱导表达荧光团标记的牛 CD46 (CD46fluo) 的 SK6 细胞表面上的 801 个轨迹进行分析，证明存在定向、扩散和受限运动。可以识别 26 个进入事件，其中大多数与胞吞过程中的 CD46fluo 阳性结构相关，并在病毒添加后 20 分钟以上发生。 CD46fluo E2 结合域 (CD46fluoΔE2bind) 的删除不会影响在细胞表面观察到的运动类型，但会导致可观察到的进入事件数量减少（1081 个轨道中的 2 个）。均方位移分析显示，与 CD46fluo 相比，CD46fluoΔE2bind 表达细胞上定向运动的颗粒运输速度显着增加。这些结果表明牛 CD46 的存在仅影响定向运输的速度，但不影响 BVDV 细胞表面运动。相反，牛 CD46 似乎是摄取过程中的一个重要因素，这表明存在与病毒相互作用的其他细胞蛋白，这些蛋白能够支持其在病毒表面的运输。