SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release.
SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release.
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SV40 晚期蛋白 VP4 形成环形孔，破坏膜以释放病毒。
DOI：
10.1021/bi400036z
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发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
Hebert,DanielN
中科院分区：
文献类型：
--
作者：
Raghava,Smita;Giorda,KristinaM;Romano,FabianB;Heuck,AlejandroP;Hebert,DanielN
无包膜病毒通常通过及时裂解宿主细胞膜而从细胞中释放。SV 40已被用作研究裂解性无包膜病毒生命周期的模型病毒。SV 40 VP 4在感染后期的表达伴随着细胞裂解。为了研究VP 4在病毒释放中的作用及其作用机制，从细菌中表达并纯化VP 4作为用于膜破坏测定的融合蛋白。纯化的VP 4穿孔膜，如通过包封在大单层囊泡或脂质体内的荧光标记物的释放所证明的。动态光散射结果显示，VP 4处理不引起膜溶解或改变脂质体的大小。使用用4，4-二氟-5，7-二甲基-4-硼杂-3a，4a-二氮杂-3-引达省标记的链霉亲和素包封的脂质体来显示VP 4在膜中形成稳定的孔。这些VP 4孔具有1-5 nm的内径。在外层单层中含有芘标记的脂质的不对称脂质体用于监测跨双层脂质扩散。与VP 4在膜中形成环形孔结构一致，VP 4诱导跨双层脂质扩散或脂质翻转。总之，这些研究支持VP 4作为病毒孔蛋白在破坏细胞膜中的核心作用，通过形成将膜双层的外小叶和内小叶结合在一起的环形孔来触发SV 40病毒释放。
Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results revealed that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of 1–5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers.
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