Ravkov E V, Nichol S T, Peters C J, Compans R W
Department of Microbiology and Immunology, School of Medicine, Emory University, Atlanta, Georgia 30322, USA.
J Virol. 1998 Apr;72(4):2865-70. doi: 10.1128/JVI.72.4.2865-2870.1998.
We have investigated the involvement of cytoskeletal proteins in the morphogenesis of Black Creek Canal virus (BCCV), a New World hantavirus. Immunofluorescent staining of BCCV-infected cells revealed a filamentous pattern of virus antigen, the appearance of which was sensitive to treatment with cytochalasin D, an actin microfilament-depolymerizing drug. Double immunofluorescence staining of BCCV-infected Vero cells with anti-BCCV nucleocapsid (N) monoclonal antibody and phalloidin revealed a colocalization of the BCCV N protein with actin microfilaments. A similar, though less prominent, filamentous pattern was observed in BHK21 cells transiently expressing the BCCV N protein alone but not in cells expressing the BCCV G1 and G2 glycoproteins. Moreover, the association of the N protein with actin microfilaments was confirmed by coimmunoprecipitation with beta-actin-specific antibody. Treatment of the BCCV-infected Vero cells at 3 days postinfection with cytochalasin D decreased the yield of released BCCV by 94% relative to the yield from untreated cells. Pretreatment of Vero cells with cytochalasin D prior to and during BCCV adsorption and entry had no effect on the outcome of virus production. These results indicate that actin filaments may play an important role in hantavirus assembly and/or release.
我们研究了细胞骨架蛋白在新世界汉坦病毒——黑溪运河病毒（BCCV）形态发生过程中的作用。对感染BCCV的细胞进行免疫荧光染色，结果显示病毒抗原呈丝状分布，其形态对肌动蛋白微丝解聚药物细胞松弛素D的处理敏感。用抗BCCV核衣壳（N）单克隆抗体和鬼笔环肽对感染BCCV的Vero细胞进行双重免疫荧光染色，结果显示BCCV N蛋白与肌动蛋白微丝共定位。在单独瞬时表达BCCV N蛋白的BHK21细胞中观察到类似的丝状分布模式，不过不太明显，而在表达BCCV G1和G2糖蛋白的细胞中未观察到这种模式。此外，通过与β-肌动蛋白特异性抗体进行共免疫沉淀，证实了N蛋白与肌动蛋白微丝的结合。在感染后3天用细胞松弛素D处理感染BCCV的Vero细胞，相对于未处理细胞，释放的BCCV产量降低了94%。在BCCV吸附和进入之前及过程中用细胞松弛素D预处理Vero细胞，对病毒产生的结果没有影响。这些结果表明，肌动蛋白丝可能在汉坦病毒的组装和/或释放中起重要作用。
