Characterization of Viral Capsid Protein Self-Assembly around Short Single-Stranded RNA
Characterization of Viral Capsid Protein Self-Assembly around Short Single-Stranded RNA
复制标题
DOI：
10.1021/jp503050z
复制
发表时间：
2014-07-10
影响因子：
3.3
通讯作者：
Gebart, William M.
中科院分区：
文献类型：
--
作者：
Comas-Garcia, Mauricio;Garmann, Rees F.;Gebart, William M.
对于许多病毒，单链RNA（ss-RNA）基因组的包装是自发的，由衣壳蛋白-衣壳蛋白（CP）和CP-RNA相互作用驱动。此外，对于一些多分裂ss-RNA病毒，两个或更多个RNA分子的共包装是常见的策略。在这里，我们专注于RNA copackaging在体外通过使用豇豆褪绿斑驳病毒（CCMV）CP和RNA分子是短（500个核苷酸（nts））相比，在野生型病毒体包装的长度（约3000 nts）。我们发现，病毒组装的协同性程度不仅取决于CP-CP和CP-RNA相互作用的相对强度，而且还取决于RNA的长度：一个500 nt的RNA分子本身不能形成衣壳，因此其包装需要多个CP-RNA复合物的聚集。通过使用荧光相关光谱（FCS），我们表明，在中性pH值和足够低的浓度RNA和CP形成复合物，小于野生型衣壳和四个500-nt的RNA被包装成病毒样颗粒（VLP），只有在降低pH值。此外，各种散装溶液技术证实，完全有序的VLP只形成酸化。在这些结果的基础上，我们认为，所观察到的高度的协同性涉及多个CP/RNA复合物之间的平衡。
For many viruses, the packaging of a single-stranded RNA (ss-RNA) genome is spontaneous, driven by capsid protein-capsid protein (CP) and CP-RNA interactions. Furthermore, for some multipartite ss-RNA viruses, copackaging of two or more RNA molecules is a common strategy. Here we focus on RNA copackaging in vitro by using cowpea chlorotic mottle virus (CCMV) CP and an RNA molecule that is short (500 nucleotides (nts)) compared to the lengths (approximate to 3000 nts) packaged in wild-type virions. We show that the degree of cooperativity of virus assembly depends not only on the relative strength of the CP-CP and CP-RNA interactions but also on the RNA being short: a 500-nt RNA molecule cannot form a capsid by itself, so its packaging requires the aggregation of multiple CP-RNA complexes. By using fluorescence correlation spectroscopy (FCS), we show that at neutral pH and sufficiently low concentrations RNA and CP form complexes that are smaller than the wild-type capsid and that four 500-nt RNAs are packaged into virus-like particles (VLPs) only upon lowering the pH. Further, a variety of bulk-solution techniques confirm that fully ordered VLPs are formed only upon acidification. On the basis of these results, we argue that the observed high degree of cooperativity involves equilibrium between multiple CP/RNA complexes.