Lombardo E, Maraver A, Espinosa I, Fernández-Arias A, Rodriguez J F
Department of Biología Molecular y Celular, Centro Nacional de Biotecnología, Cantoblanco, 28049 Madrid, Spain.
Virology. 2000 Nov 25;277(2):345-57. doi: 10.1006/viro.2000.0595.
Infectious bursal disease virus (IBDV) encodes a 17-kDa nonstructural polypeptide known as VP5. This polypeptide is not essential for virus replication in vitro but it plays an important role in in vivo dissemination and pathogenesis. We have characterized the expression of VP5 in three eukaryotic systems: (i) IBDV-infected chicken embryo fibroblasts; (ii) BSC-1 cells infected with a recombinant vaccinia virus vector; and (iii) Cos-1 cells transiently transfected with a plasmid vector. Immunofluorescence analyses showed that upon expression VP5 accumulates within the plasma membrane. This finding was consistent with sequence-based topology predictions, indicating that VP5 is a class II membrane protein with a cytoplasmic N-terminus and an extracellular C-terminal domain. Brefeldin A treatment of VP5-expressing cells prevented the accumulation of this polypeptide in the plasma membrane, thus showing the requirement of an active exocytic pathway to reach that compartment. Expression of VP5 was shown to be highly cytotoxic. Induction of VP5 expression resulted in the alteration of cell morphology, the disruption of the plasma membrane, and a drastic reduction of cell viability. VP5-induced cytotoxicity was prevented by blocking its transport to the membrane with Brefeldin A. Our findings suggest that VP5 plays an important role in the release of the IBDV progeny.
传染性法氏囊病病毒（IBDV）编码一种名为VP5的17 kDa非结构多肽。这种多肽对于病毒在体外的复制并非必需，但在体内传播和发病机制中发挥重要作用。我们已对VP5在三种真核系统中的表达进行了表征：（i）IBDV感染的鸡胚成纤维细胞；（ii）用重组痘苗病毒载体感染的BSC-1细胞；以及（iii）用质粒载体瞬时转染的Cos-1细胞。免疫荧光分析表明，VP5表达后会在质膜内积累。这一发现与基于序列的拓扑预测一致，表明VP5是一种II类膜蛋白，其N端位于细胞质中，C端结构域位于细胞外。用布雷菲德菌素A处理表达VP5的细胞可阻止该多肽在质膜中的积累，从而表明需要一条活跃的胞吐途径才能到达该区室。已证明VP5的表达具有高度细胞毒性。VP5表达的诱导导致细胞形态改变、质膜破裂以及细胞活力急剧下降。用布雷菲德菌素A阻断VP5向膜的转运可防止其诱导的细胞毒性。我们的研究结果表明，VP5在IBDV子代的释放中起重要作用。
