Jané-Valbuena J, Nibert M L, Spencer S M, Walker S B, Baker T S, Chen Y, Centonze V E, Schiff L A
Department of Biochemistry, College of Agricultural and Life Sciences, The Graduate School, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
J Virol. 1999 Apr;73(4):2963-73. doi: 10.1128/JVI.73.4.2963-2973.1999.
Structure-function studies with mammalian reoviruses have been limited by the lack of a reverse-genetic system for engineering mutations into the viral genome. To circumvent this limitation in a partial way for the major outer-capsid protein sigma3, we obtained in vitro assembly of large numbers of virion-like particles by binding baculovirus-expressed sigma3 protein to infectious subvirion particles (ISVPs) that lack sigma3. A level of sigma3 binding approaching 100% of that in native virions was routinely achieved. The sigma3 coat in these recoated ISVPs (rcISVPs) appeared very similar to that in virions by electron microscopy and three-dimensional image reconstruction. rcISVPs retained full infectivity in murine L cells, allowing their use to study sigma3 functions in virus entry. Upon infection, rcISVPs behaved identically to virions in showing an extended lag phase prior to exponential growth and in being inhibited from entering cells by either the weak base NH4Cl or the cysteine proteinase inhibitor E-64. rcISVPs also mimicked virions in being incapable of in vitro activation to mediate lysis of erythrocytes and transcription of the viral mRNAs. Last, rcISVPs behaved like virions in showing minor loss of infectivity at 52 degrees C. Since rcISVPs contain virion-like levels of sigma3 but contain outer-capsid protein mu1/mu1C mostly cleaved at the delta-phi junction as in ISVPs, the fact that rcISVPs behaved like virions (and not ISVPs) in all of the assays that we performed suggests that sigma3, and not the delta-phi cleavage of mu1/mu1C, determines the observed differences in behavior between virions and ISVPs. To demonstrate the applicability of rcISVPs for genetic studies of protein functions in reovirus entry (an approach that we call recoating genetics), we used chimeric sigma3 proteins to localize the primary determinants of a strain-dependent difference in sigma3 cleavage rate to a carboxy-terminal region of the ISVP-bound protein.
对哺乳动物呼肠孤病毒的结构-功能研究一直受到缺乏用于在病毒基因组中进行工程突变的反向遗传系统的限制。为了部分克服对主要外衣壳蛋白sigma3的这一限制，我们通过将杆状病毒表达的sigma3蛋白与缺乏sigma3的感染性子病毒颗粒（ISVP）结合，获得了大量病毒样颗粒的体外组装。sigma3的结合水平通常可达到天然病毒粒子中结合水平的近100%。通过电子显微镜和三维图像重建，这些重新包被的ISVP（rcISVP）中的sigma3衣壳与病毒粒子中的非常相似。rcISVP在鼠L细胞中保留了完全的感染性，使其可用于研究sigma3在病毒进入过程中的功能。感染时，rcISVP在指数生长前表现出延长的延迟期，并且在被弱碱NH4Cl或半胱氨酸蛋白酶抑制剂E-64抑制进入细胞方面与病毒粒子表现相同。rcISVP在体外也模仿病毒粒子，无法被激活以介导红细胞裂解和病毒mRNA转录。最后，rcISVP在52℃时表现出与病毒粒子相似的轻微感染性丧失。由于rcISVP含有病毒粒子样水平的sigma3，但含有主要在δ-φ连接处裂解的外衣壳蛋白mu1/mu1C，就像在ISVP中一样，我们进行的所有实验中rcISVP表现得像病毒粒子（而不是ISVP）这一事实表明，是sigma3，而不是mu1/mu1C的δ-φ裂解，决定了病毒粒子和ISVP之间观察到的行为差异。为了证明rcISVP在呼肠孤病毒进入过程中蛋白质功能的遗传研究中的适用性（我们将这种方法称为重新包被遗传学），我们使用嵌合sigma3蛋白将sigma3裂解速率的菌株依赖性差异的主要决定因素定位到与ISVP结合的蛋白的羧基末端区域。
