Chandran K, Walker S B, Chen Y, Contreras C M, Schiff L A, Baker T S, Nibert M L
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
J Virol. 1999 May;73(5):3941-50. doi: 10.1128/JVI.73.5.3941-3950.1999.
Reovirus outer-capsid proteins mu1, sigma3, and sigma1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed mu1 and sigma3 onto purified cores that lack mu1, sigma3, and sigma1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein sigma1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of mu1 and sigma3 onto cores had induced rearrangement of the pentameric lambda2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH4Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the sigma1 protein. To examine the utility of r-cores for genetic dissections of mu1 functions in reovirus entry, we generated r-cores containing a mutant form of mu1 that had been engineered to resist cleavage at the delta:phi junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in delta:phi cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH4Cl, providing new evidence that this cleavage is dispensable for productive infection.
呼肠孤病毒外衣壳蛋白μ1、σ3和σ1被认为是在受感染细胞内组装到新生的类核心颗粒上，从而产生子代病毒粒子。与该模型一致，我们报道了杆状病毒表达的μ1和σ3在体外组装到缺乏μ1、σ3和σ1的纯化核心上。通过扫描冷冻电子显微镜观察，所得颗粒（重新包被的核心，或r-核心）在蛋白质组成（除了缺乏细胞附着蛋白σ1）、浮力密度和颗粒形态方面与天然病毒粒子非常相似。透射冷冻电子显微镜和r-核心的图像重建证实，它们在外衣壳结构上与病毒粒子非常相似，并揭示了μ1和σ3在核心上的组装诱导了五聚体λ2炮塔重排为接近病毒粒子中的构象。r-核心与病毒粒子一样，在蛋白质组成、颗粒形态和体外透化膜的能力方面经历蛋白水解转化为类似于天然感染性子病毒粒子（ISVP）的颗粒。r-核心在小鼠L细胞中的感染性比核心高250至500倍，并且与病毒粒子一样，但与ISVP或核心不同，NH4Cl或E-64的存在会抑制它们有效感染这些细胞。后一结果表明，r-核心和病毒粒子使用相似的进入L细胞的途径，包括通过溶酶体半胱氨酸蛋白酶进行加工，尽管前者颗粒缺乏σ1蛋白。为了研究r-核心在呼肠孤病毒进入过程中对μ1功能进行基因剖析的效用，我们生成了含有突变形式μ1的r-核心，该突变形式经过工程改造，在体外被胰凝乳蛋白酶转化为类ISVP颗粒的过程中，能抵抗在δ:φ连接处的切割。尽管它们在δ:φ切割方面存在缺陷，但这些类ISVP颗粒在体外完全有能力透化膜并在NH4Cl存在的情况下感染L细胞，这提供了新的证据表明这种切割对于有效感染是可有可无的。
