Emerson S U, Wagner R R
J Virol. 1972 Aug;10(2):297-309. doi: 10.1128/JVI.10.2.297-309.1972.
Transcriptase activity was dissociated from vesicular stomatitis virions by highionic-strength buffer containing Triton X-100. Considerable enzyme activity could be restored by recombining inactive sedimentable and nonsedimentable virion fractions. Reconstituted transcriptase activity was dependent on the presence of all four nucleoside triphosphates and the concentration of heat-labile molecules in both supernatant and pellet fractions. Lower NaCl concentrations removed approximately 46% of virion protein, but did not release transcriptase activity from the pellet fraction, nor could incorporation of (3)H-uridine-5'-triphosphate by complete virions be increased by adding soluble transcriptase. Evidence that the virion nucleocapsid is the transcription template was provided by finding that the pellet contained predominantly virion core nucleoprotein, ribonucleic acid, and homogeneous nucleocapsid coils when viewed by electron microscopy. Removal of envelope G and M proteins by Triton and low-salt buffer without decreasing nucleocapsid polymerase activity indicates that neither G nor M protein is necessary for transcription. Additional data are required to determine whether the minor nucleocapsid proteins L or NSl, or both, which are at least partially solubilized in high-salt buffer, are the transcriptase. Preliminary data suggest that the major N nucleoprotein, which was not solubilized by high-salt buffer, is also required for transcription. Defective T virions contained at least as much transcriptase per weight as did B virions, as determined by restoration with T supernatant fluids of transcription function to B nucleocapsid template. However, the T nucleocapsid would not serve as template for B or T transcriptase, a finding which is interpreted as evidence of T template defectiveness. The presence of defective T nucleocapsids did not interfere with B or T transcriptase function reconstituted with B template.
通过含有 Triton X - 100 的高离子强度缓冲液，可将转录酶活性与水泡性口炎病毒粒子分离。通过重新组合无活性的可沉降和不可沉降病毒粒子组分，可恢复相当可观的酶活性。重组后的转录酶活性依赖于所有四种核苷三磷酸的存在以及上清液和沉淀组分中热不稳定分子的浓度。较低的 NaCl 浓度可去除约 46%的病毒粒子蛋白，但不会从沉淀组分中释放转录酶活性，通过添加可溶性转录酶也无法增加完整病毒粒子对（3）H - 尿苷 - 5'-三磷酸的掺入。当通过电子显微镜观察时，发现沉淀主要包含病毒粒子核心核蛋白、核糖核酸和均匀的核衣壳螺旋，这为病毒粒子核衣壳是转录模板提供了证据。用 Triton 和低盐缓冲液去除包膜 G 和 M 蛋白而不降低核衣壳聚合酶活性，表明 G 蛋白和 M 蛋白对于转录都不是必需的。需要更多数据来确定在高盐缓冲液中至少部分溶解的次要核衣壳蛋白 L 或 NSl，或两者是否为转录酶。初步数据表明，未被高盐缓冲液溶解的主要 N 核蛋白对于转录也是必需的。通过用 T 上清液恢复 B 核衣壳模板的转录功能来确定，缺陷型 T 病毒粒子每重量所含的转录酶至少与 B 病毒粒子一样多。然而，T 核衣壳不能作为 B 或 T 转录酶的模板，这一发现被解释为 T 模板缺陷的证据。缺陷型 T 核衣壳的存在并不干扰用 B 模板重组的 B 或 T 转录酶功能。
