Laboratoire de Biocristallographie et Biologie Structurale des Cibles Thérapeutiques, IBCP-BMSSI, UMR 5086 CNRS Université de Lyon, SFR BioSciences Gerland-Lyon Sud, Lyon, France.
PLoS One. 2013;8(2):e56424. doi: 10.1371/journal.pone.0056424. Epub 2013 Feb 15.
The Feline Immunodeficiency Virus (FIV) capsid protein p24 oligomerizes to form a closed capsid that protects the viral genome. Because of its crucial role in the virion, FIV p24 is an interesting target for the development of therapeutic strategies, although little is known about its structure and assembly. We defined and optimized a protocol to overexpress recombinant FIV capsid protein in a bacterial system. Circular dichroism and isothermal titration calorimetry experiments showed that the structure of the purified FIV p24 protein was comprised mainly of α-helices. Dynamic light scattering (DLS) and cross-linking experiments demonstrated that p24 was monomeric at low concentration and dimeric at high concentration. We developed a protocol for the in vitro assembly of the FIV capsid. As with HIV, an increased ionic strength resulted in FIV p24 assembly in vitro. Assembly appeared to be dependent on temperature, salt concentration, and protein concentration. The FIV p24 assembly kinetics was monitored by DLS. A limit end-point diameter suggested assembly into objects of definite shapes. This was confirmed by electron microscopy, where FIV p24 assembled into spherical particles. Comparison of FIV p24 with other retroviral capsid proteins showed that FIV assembly is particular and requires further specific study.
猫免疫缺陷病毒（FIV）衣壳蛋白 p24 寡聚形成一个封闭的衣壳，保护病毒基因组。由于其在病毒粒子中的关键作用，FIV p24 是开发治疗策略的一个有趣的靶点，尽管人们对其结构和组装知之甚少。我们定义并优化了在细菌系统中过表达重组 FIV 衣壳蛋白的方案。圆二色性和等温热滴定实验表明，纯化的 FIV p24 蛋白的结构主要由α-螺旋组成。动态光散射（DLS）和交联实验表明，p24 在低浓度时为单体，在高浓度时为二聚体。我们开发了一种体外组装 FIV 衣壳的方案。与 HIV 一样，增加离子强度会导致 FIV p24 在体外组装。组装似乎依赖于温度、盐浓度和蛋白质浓度。通过 DLS 监测 FIV p24 的组装动力学。一个极限终点直径表明组装成具有确定形状的物体。这通过电子显微镜得到证实，其中 FIV p24 组装成球形颗粒。与其他逆转录病毒衣壳蛋白的比较表明，FIV 组装是特殊的，需要进一步的专门研究。
