Activity of polymerase proteins of vaccine and wild-type measles virus strains in a minigenome replication assay
Activity of polymerase proteins of vaccine and wild-type measles virus strains in a minigenome replication assay
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DOI：
10.1128/jvi.76.14.7073-7081.2002
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发表时间：
2002-07-01
影响因子：
5.4
通讯作者：
Rota, PA
中科院分区：
文献类型：
--
作者：
Bankamp, B;Kearney, SP;Rota, PA
在细胞内基于质粒的复制测定中比较了五种麻疹病毒（W）聚合酶（L）蛋白的相对活性。当与减毒株的N和P蛋白共表达时，来自两种减毒病毒的L蛋白指导从W微型基因组产生比三种野生型L蛋白多八倍的报告蛋白。北方印迹分析表明，报告蛋白的生产与mRNA转录水平的差异。来自减毒病毒的聚合酶活性增加同样影响mRNA转录和微型基因组复制。较高水平的转录可能是模板可用性增加的结果，或者可能是减弱的聚合酶的活性升高的独立效应。野生型L蛋白与同源N和P蛋白的共表达不影响野生型聚合酶的活性，表明差异活性是L蛋白单独的功能。在三种野生型病毒的基因组前导序列中发现了两个核苷酸变化，使用这种微基因组并没有提高野生型L蛋白的活性。这些数据表明，增加的聚合酶活性区分从野生型病毒减毒，并建议。参与RNA合成的功能有助于W疫苗株的减毒表型。
The relative activities of five measles virus (W) polymerase (L) proteins were compared in an intracellular, plasmid-based replication assay. When coexpressed with N and P proteins from an attenuated strain, L proteins from two attenuated viruses directed the production of up to eight times more reporter protein from an W minigenome than the three wild-type L proteins. Northern blot analysis demonstrated that the differences in reporter protein production correlated with mRNA transcription levels. Increased activity of polymerases from attenuated viruses equally affected mRNA transcription and minigenome replication. The higher level of transcription may be a consequence of increased template availability or may be an independent effect of the elevated activity of the attenuated polymerases. Coexpression of wild-type L proteins with homologous N and P proteins did not affect the activity of the wild-type polymerases, indicating that the differential activity was a function of the L proteins alone. Use of a minigenome that incorporated two nucleotide changes found in the genomic leader of the three wild-type viruses did not raise the activity of the wild-type L proteins. These data demonstrate that increased polymerase activity differentiates attenuated from wild-type viruses and suggest. that functions involved in RNA synthesis contribute to the attenuated phenotype of W vaccine strains.