Bina M, Ng S C, Blasquez V
Purdue University, Department of Chemistry, West Lafayette, Indiana 47907.
J Biomol Struct Dyn. 1983 Dec;1(3):689-704. doi: 10.1080/07391102.1983.10507475.
It has been established that both in virions and in infected cells, the cellular core histones fold the SV40 DNA into nucleosomes to form the SV40 chromosome or chromatin. We and others have begun to examine how the capsid proteins assemble the SV40 chromatin into virions and to investigate whether these proteins interact with the encapsidated chromatin. To follow the pathway of virus assembly, we have analyzed the nucleoproteins which accumulate in cells infected with the SV40 mutants temperature-sensitive in assembly: tsC, tsBC, and tsB. (The temperature-sensitivity of these mutants result from alterations in the amino acid sequence of the major capsid protein VP1). We have found that mutants belonging to the same class accumulate similar types of nucleoproteins at the nonpermissive temperature (40 degrees C) and thus, share characteristics in common. For example, the tsC mutants accumulate only the 75 S chromatin. Both tsBC and tsB mutants produce in addition to chromatin, nucleoprotein complexes which sediment broadly from 100-160 S and contain all the three capsid proteins VP1, VP2, and VP3. These nucleoproteins can be distinguished morphologically, however. Under the electron microscope, the tsBC 100-160 S nucleoproteins appear as chromatin to which a small cluster of the capsid proteins is attached; the tsB nucleoproteins appear as partially assembled virions. In addition, we find that the 220 S virions are assembled in cells coinfected with tsB and tsC mutants at 40 degrees C, in agreement with genetic analysis. Our observations favor the hypothesis that the VP1 protein contains three discrete domains. We speculate that each domain may play a specific function in SV40 assembly. To gain more insight into VP1-VP1 interactions, we have examined the nucleoproteins which result from treatment of the mature wild-type virions with increasing concentrations of the reducing agent DTT. In the presence of as low a concentration of DTT as 0.1 mM, the virion shell can be penetrated by micrococcal nuclease, which then cleaves the viral DNA. This result indicates that some of the disulfide bonds bridging the VP1 proteins are on the virion surface.
现已确定，在病毒粒子和受感染细胞中，细胞核心组蛋白会将SV40 DNA折叠成核小体，以形成SV40染色体或染色质。我们和其他人已开始研究衣壳蛋白如何将SV40染色质组装成病毒粒子，并研究这些蛋白是否与被包裹的染色质相互作用。为了追踪病毒组装途径，我们分析了在感染了组装温度敏感型SV40突变体（tsC、tsBC和tsB）的细胞中积累的核蛋白。（这些突变体的温度敏感性是由主要衣壳蛋白VP1的氨基酸序列改变引起的）。我们发现，属于同一类别的突变体在非允许温度（40摄氏度）下积累相似类型的核蛋白，因此具有共同特征。例如，tsC突变体仅积累75S染色质。tsBC和tsB突变体除了产生染色质外，还产生核蛋白复合物，这些复合物在100 - 160S范围内广泛沉降，并包含所有三种衣壳蛋白VP1、VP2和VP3。然而，这些核蛋白在形态上可以区分。在电子显微镜下，tsBC 100 - 160S核蛋白表现为附着有一小簇衣壳蛋白的染色质；tsB核蛋白表现为部分组装的病毒粒子。此外，我们发现220S病毒粒子在40摄氏度下与tsB和tsC突变体共感染的细胞中组装，这与遗传分析一致。我们的观察结果支持VP1蛋白包含三个离散结构域的假设。我们推测每个结构域可能在SV40组装中发挥特定功能。为了更深入了解VP1 - VP1相互作用，我们检查了用浓度不断增加的还原剂DTT处理成熟野生型病毒粒子后产生的核蛋白。在低至0.1 mM的DTT存在下，微球菌核酸酶可以穿透病毒粒子外壳，然后切割病毒DNA。这一结果表明，连接VP1蛋白的一些二硫键位于病毒粒子表面。
