High cooperativity of the SV40 major capsid protein VP1 in virus assembly.
High cooperativity of the SV40 major capsid protein VP1 in virus assembly.
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DOI：
10.1371/journal.pone.0000765
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发表时间：
2007-08-22
期刊：
影响因子：
3.7
通讯作者：
Oppenheim A
中科院分区：
文献类型：
--
作者：
Mukherjee S;Abd-El-Latif M;Bronstein M;Ben-nun-Shaul O;Kler S;Oppenheim A
SV40是一种小的、无包膜的DNA病毒，衣壳二十面体，长度为45 nm。外壳由主要衣壳蛋白VP1的五聚体组成，通过其灵活的羧基末端臂连接。它的形态发生是通过在病毒微染色体周围组装衣壳而发生的。然而，导致成熟病毒形成的步骤却鲜为人知。不能从感染wt SV40的细胞中分离到组装反应的中间产物。在这里，我们使用在昆虫细胞中产生的重组VP1，在携带报告基因的超螺旋异源质粒DNA周围进行体外组装研究。这一策略产生了具有感染性的纳米颗粒，提供了一种简单的定量转导分析。我们证明了VP1在生理条件下组装成与野生型病毒相同的形状、大小和CSCL密度的均匀纳米颗粒。化学计量学是每个衣壳一个DNA分子。在不能组装但可以结合DNA的C-臂上缺失的VP1是无效的，表明真正的组装而不是非特异性的DNA结合。如最近所示，该反应需要宿主的酶活性，与伴侣的参与一致。我们的结果表明Vp1具有戏剧性的协同作用，希尔系数为∼6。这些发现表明组装可能是一种协同反应。我们认为，正如我们最近报道的那样，多个胶囊同时与单个DNA分子结合，从而增加了它们的局部浓度，从而促进了协同组装。SV40组装的新原理可能有助于理解其他复杂系统的组装。此外，这里描述的基于SV40的纳米颗粒是潜在的基因治疗载体，它将有效的基因传递与安全和灵活结合在一起。
SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of ∼6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.
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影响因子：
64.5
作者：
GLUZMAN, Y
通讯作者：
GLUZMAN, Y
影响因子：
11.4
作者：
Chen, XJS;Stehle, T;Harrison, SC
通讯作者：
Harrison, SC
DOI：
10.1073/pnas.85.2.422
发表时间：
1988-01-01
影响因子：
11.1
作者：
BAKER, TS;DRAK, J;BINA, M
通讯作者：
BINA, M
影响因子：
3.4
作者：
Endres, D;Zlotnick, A
通讯作者：
Zlotnick, A
DOI：
10.1073/pnas.1832245100
发表时间：
2003-09-02
影响因子：
11.1
作者：
Chromy, LR;Pipas, JM;Garcea, RL
通讯作者：
Garcea, RL