Chen D, Ramig R F
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030.
Virology. 1993 Jun;194(2):743-51. doi: 10.1006/viro.1993.1315.
We recently developed an in vitro transcapsidation system in which infectivity of single-shelled (ss) rotavirus particles was successfully rescued (Chen and Ramig, Virology [1993]). Here, we report the rescue of infectivity of rotavirus core particles using virus strain B223 (G serotype 10) as the core donor and strain SA11-4F (G3) as the capsid donor. Core particles of B223 were obtained by CaCl2 treatment of B223 ss-particles followed by isopycnic CsCl gradient centrifugation. Inner capsid protein VP6 of SA11-4F was prepared by CaCl2 treatment of SA11-4F ss-particles, followed by removal of core particles by two rounds of centrifugation. Outer capsid proteins VP4 and VP7 of SA11-4F were prepared by EDTA treatment of ds-particles, followed by three rounds of centrifugation to remove ss-particles and minimize residual infectivity. No infectivity (< 3 PFU/ml) was detectable in any of the donor preparations. Transcapsidated ss-particles were obtained by mixing B223 core particles and a 5-fold excess of SA11-4F VP6 at neutral pH. The formation of transcapsidated ss-particles was confirmed by electron microscopy, protein composition analysis, and density determination. Along with the formation of ss-particles by in vitro transcapsidation, some infectivity was also detected and transcriptase activity was reconstituted. Semi-purified transcapsidated ss-particles were then mixed with SA11-4F outer capsid proteins VP4 and VP7 at acidic pH to obtain transcapsidated ds-particles, as described previously. The formation of ds-like particles was also confirmed by electron microscopy, protein composition, and density determination. As the result of formation of transcapsidated ds-like particles, viral infectivity increased significantly (80-fold) relative to that of transcapsidated ss-particles. The infectivity of transcapsidated ds-particles was neutralized by polyclonal anti-SA11 serum, but not by polyclonal anti-B223 serum. The transcapsidated particles formed small plaques like B223 (core donor), and all the progeny plaques contained B223 genomes. These results demonstrate that the infectivity of rotavirus core particles can be rescued by sequential addition of inner and outer capsid proteins in vitro.
我们最近开发了一种体外转衣壳化系统，其中成功挽救了单壳（ss）轮状病毒颗粒的感染性（Chen和Ramig，《病毒学》[1993]）。在此，我们报告使用病毒株B223（G血清型10）作为核心供体和株SA11-4F（G3）作为衣壳供体来挽救轮状病毒核心颗粒的感染性。B223的核心颗粒是通过用CaCl2处理B223 ss颗粒，然后进行等密度CsCl梯度离心获得的。SA11-4F的内衣壳蛋白VP6是通过用CaCl2处理SA11-4F ss颗粒，然后通过两轮离心去除核心颗粒来制备的。SA11-4F的外衣壳蛋白VP4和VP7是通过用EDTA处理双链颗粒，然后进行三轮离心以去除ss颗粒并将残余感染性降至最低来制备的。在任何供体制备物中均未检测到感染性（<3 PFU/ml）。通过在中性pH下混合B223核心颗粒和5倍过量的SA11-4F VP6获得转衣壳化的ss颗粒。通过电子显微镜、蛋白质组成分析和密度测定证实了转衣壳化ss颗粒的形成。随着体外转衣壳化形成ss颗粒，还检测到了一些感染性并重建了转录酶活性。然后如前所述，在酸性pH下将半纯化的转衣壳化ss颗粒与SA11-4F外衣壳蛋白VP4和VP7混合以获得转衣壳化的双链颗粒。通过电子显微镜、蛋白质组成和密度测定也证实了双链样颗粒的形成。作为转衣壳化双链样颗粒形成的结果，病毒感染性相对于转衣壳化的ss颗粒显著增加（80倍）。转衣壳化双链颗粒的感染性被多克隆抗SA11血清中和，但不被多克隆抗B223血清中和。转衣壳化颗粒形成了像B223（核心供体）一样的小噬斑，并且所有子代噬斑都含有B223基因组。这些结果表明，通过在体外顺序添加内衣壳蛋白和外衣壳蛋白可以挽救轮状病毒核心颗粒的感染性。
