Chen D, Ramig R F
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030.
Virology. 1993 Feb;192(2):422-9. doi: 10.1006/viro.1993.1057.
We investigated the possibility of rescuing the infectivity of noninfectious single-shelled rotavirus particles by in vitro transcapsidation. The soluble outer capsid proteins VP4 and VP7 were prepared by EDTA treatment of double-shelled (ds) particles of SA11-4F (G serotype 3), followed by removal of single-shelled (ss) particles by three sequential rounds of centrifugation. Ss-particles of B223 (G serotype 10) were prepared by two cycles of EDTA treatment of ds-particles followed by iospycnic CsCl gradient purification. A trace of infectivity (< 1000 PFU/ml) was always detected in the preparations of ss-particles, while no detectable infectivity (< 5 PFU/ml) was present in the preparations of outer capsid proteins. By mixing soluble outer capsid proteins VP4 and VP7 purified from SA11-4F and ss-particles of B223 at acidic pH (5.4), ds-like, transcapsidated particles were obtained. The transcapsidated particles were indistinguishable from genuine ds-particles by negative stain electron microscopy. However, the particles had a density intermediate between that of ds- and ss-particles. Protease-resistance studies revealed that VP7 was assembled onto transcapsidated particles in a resistant (native) form, but VP4 associated with the particles was completely protease sensitive. Viral infectivity was rescued by in vitro transcapsidation as indicated by a 500- to 1000-fold increase over background. The increased infectivity was neutralized by antiserum against SA11 (outer capsid donor), but not by antiserum against B223 (ss-particle donor). The transcapsidated particles formed small plaques like the B223 parent, and all the infectious progeny viruses contained the B223 genome. These results strongly indicate that the observed increase of infectivity was the result of in vitro transcapsidation.
我们研究了通过体外转衣壳来挽救无感染性的单壳轮状病毒颗粒感染性的可能性。通过用乙二胺四乙酸（EDTA）处理SA11 - 4F（G血清型3）的双壳（ds）颗粒来制备可溶性外衣壳蛋白VP4和VP7，随后通过三轮连续离心去除单壳（ss）颗粒。通过对ds颗粒进行两轮EDTA处理，然后进行等密度氯化铯梯度纯化来制备B223（G血清型10）的ss颗粒。在ss颗粒制剂中总能检测到微量感染性（<1000 PFU/ml），而在外衣壳蛋白制剂中未检测到可检测到的感染性（<5 PFU/ml）。通过在酸性pH（5.4）下混合从SA11 - 4F纯化的可溶性外衣壳蛋白VP4和VP7以及B223的ss颗粒，获得了类似ds的转衣壳颗粒。通过负染色电子显微镜观察，转衣壳颗粒与真正的ds颗粒无法区分。然而，这些颗粒的密度介于ds颗粒和ss颗粒之间。蛋白酶抗性研究表明，VP7以抗性（天然）形式组装到转衣壳颗粒上，但与颗粒相关的VP4对蛋白酶完全敏感。如背景之上增加500至1000倍所示，体外转衣壳挽救了病毒感染性。增加的感染性被抗SA11（外衣壳供体）抗血清中和，但不被抗B223（ss颗粒供体）抗血清中和。转衣壳颗粒形成了像B223亲本一样的小噬斑，并且所有感染性子代病毒都含有B223基因组。这些结果有力地表明，观察到的感染性增加是体外转衣壳的结果。
