Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
J Gen Virol. 2010 Jun;91(Pt 6):1428-38. doi: 10.1099/vir.0.016717-0. Epub 2010 Feb 10.
For many viruses, endocytosis and exposure to the low pH within acidic endosomes is essential for infection. It has previously been reported that feline calicivirus uses clathrin-mediated endocytosis for entry into mammalian cells. Here, we report that infection of RAW264.7 macrophages by the closely related murine norovirus-1 (MNV-1) does not require the clathrin pathway, as infection was not inhibited by expression of dominant-negative Eps15 or by knockdown of the adaptin-2 complex. Further, infection was not inhibited by reagents that raise endosomal pH. RAW264.7 macrophages were shown not to express caveolin, and flotillin depletion did not inhibit infection, suggesting that caveolae and the flotillin pathway are not required for cell entry. However, MNV-1 infection was inhibited by methyl-beta-cyclodextrin and the dynamin inhibitor, dynasore. Addition of these drugs to the cells after a period of virus internalization did not inhibit infection, suggesting the involvement of cholesterol-sensitive lipid rafts and dynamin in the entry mechanism. Macropinocytosis (MPC) was shown to be active in RAW264.7 macrophages (as indicated by uptake of dextran) and could be blocked by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), which is reported to inhibit this pathway. However, infection was enhanced in the presence of EIPA. Similarly, actin disruption, which also inhibits MPC, resulted in enhanced infection. These results suggest that MPC could contribute to virus degradation or that inhibition of MPC could lead to the upregulation of other endocytic pathways of virus uptake.
对于许多病毒来说，内吞作用和暴露于酸性内体中的低 pH 值对于感染是必不可少的。先前已经报道，猫杯状病毒使用网格蛋白介导的内吞作用进入哺乳动物细胞。在这里，我们报告称，与猫杯状病毒密切相关的鼠诺如病毒-1 (MNV-1) 感染 RAW264.7 巨噬细胞不需要网格蛋白途径，因为表达显性失活的 Eps15 或敲低衔接蛋白-2 复合物不能抑制感染。此外，感染也不受提高内体 pH 值的试剂的抑制。RAW264.7 巨噬细胞不表达窖蛋白，并且 flotillin 耗竭也不能抑制感染，这表明 caveolae 和 flotillin 途径不是细胞进入所必需的。然而，MNV-1 感染被甲基-β-环糊精和 dynamin 抑制剂 dynasore 抑制。在病毒内化一段时间后将这些药物添加到细胞中不会抑制感染，这表明胆固醇敏感的脂筏和 dynamin 参与了进入机制。巨胞饮作用（MPC）在 RAW264.7 巨噬细胞中显示为活性（如葡聚糖摄取所表明），并且可以被 5-(N-乙基-N-异丙基)amiloride (EIPA) 阻断，据报道该药物抑制这种途径。然而，在存在 EIPA 的情况下感染增强。同样，肌动蛋白的破坏，也抑制 MPC，导致感染增强。这些结果表明，MPC 可能有助于病毒降解，或者 MPC 的抑制可能导致病毒摄取的其他内吞途径的上调。
