Institute of Virology, Vetmeduni Vienna, 1210 Vienna, Austria.
VetCore Facility for Research, Vetmeduni Vienna, 1210 Vienna, Austria.
Viruses. 2020 Jan 17;12(1):116. doi: 10.3390/v12010116.
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 (BVDV), being the first genetically labelled member of the family 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 (CD46) demonstrated the presence of directed, diffusive, and confined motion. 26 entry events could be identified, with the majority being associated with a CD46 positive structure during endocytosis and occurring more than 20 min after virus addition. Deletion of the CD46 E2 binding domain (CD46∆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 CD46∆E2bind expressing cells in comparison to CD46. 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.
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。本研究使用牛病毒性腹泻病毒（BVDV）编码的 mCherry-E2 融合蛋白（BVDV），这是该家族中第一个可用于分析病毒颗粒的遗传标记成员，监测感染早期事件-附着、颗粒表面转运和内吞作用，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。在可诱导表达荧光标记牛 CD46（CD46）的 SK6 细胞表面分析了 801 条轨迹，结果表明存在定向、扩散和受限运动。可以识别 26 个进入事件，其中大多数与内吞作用过程中的 CD46 阳性结构相关，并且发生在病毒添加后 20 分钟以上。CD46 E2 结合域（CD46∆E2bind）的缺失不影响细胞表面观察到的运动类型，但导致可观察到的进入事件数量减少（1081 条轨迹中有 2 个）。均方根位移分析表明，与 CD46 相比，CD46∆E2bind 表达细胞上定向运动的颗粒转运速度显著增加。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 细胞表面的迁移。相反，牛 CD46 似乎是摄取过程中的一个重要因素，表明存在与病毒相互作用的其他细胞蛋白，能够支持其在病毒表面的运输。
