Alternative amino acids at a single site in the Sendai virus L protein produce multiple defects in RNA synthesis in vitro.
Alternative amino acids at a single site in the Sendai virus L protein produce multiple defects in RNA synthesis in vitro.
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DOI：
10.1006/viro.1995.1440
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发表时间：
1995-08
期刊：
影响因子：
3.7
通讯作者：
S. Horikami;S. Moyer
中科院分区：
文献类型：
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作者：
S. Horikami;S. Moyer
我们的长期目标是确定仙台病毒RNA聚合酶的L蛋白亚基的催化结构域。水泡性口炎病毒tsG 16 L蛋白突变体中的异常聚腺苷酸化表型最近被鉴定为氨基酸1488处的苯丙氨酸至丝氨酸的变化（Hunt和哈钦森，病毒学193，786-793，1993）。为了测试负链RNA病毒L蛋白的功能结构域是否保守，我们试图在仙台病毒L蛋白中产生类似的多聚腺苷酸化缺陷。通过基因的定点诱变构建仙台L蛋白中类似位点处的九个不同氨基酸取代（氨基酸1571处的半胱氨酸）。合成每个突变L蛋白并与仙台P蛋白结合以形成P-L聚合酶复合物。虽然这些L突变体中没有一个表现出多聚腺苷酸化的变化，但单个氨基酸的变化在体外产生了多种活性。在氨基酸1571处含有缬氨酸、亮氨酸或苯丙氨酸（在其它副粘病毒的L蛋白中天然发现的氨基酸）的突变体产生具有等于或优于野生型（WT）聚合酶的生物活性的聚合酶。L蛋白在该位置的丝氨酸或苏氨酸取代也导致具有接近WT合成活性的聚合酶。相比之下，甘氨酸取代显着降低整体聚合酶活性，而酪氨酸取代降低转录，但实际上没有DI基因组体外复制。酪氨酸取代的聚合酶可能无法进行复制的包装步骤，因为DI前导RNA合成在该突变体中是正常的。突变体L蛋白与碱性精氨酸或组氨酸取代是无活性的，在所有的病毒RNA合成在体外，虽然聚合酶复合物可以结合的核衣壳模板。
Our long-term goal is to define the catalytic domains of the L protein subunit of the Sendai virus RNA polymerase. An aberrant polyadenylation phenotype in the vesicular stomatitis virus tsG16 L protein mutant has recently been identified as a phenylalanine to serine change at amino acid 1488 (Hunt and Hutchinson, Virology 193, 786-793, 1993). To test if functional domains are conserved in the L proteins of negative-strand RNA viruses, we attempted to create a similar polyadenylation defect in the Sendai virus L protein. Nine different amino acid substitutions at the analogous site in the Sendai L protein (cysteine at amino acid 1571) were constructed by site-directed mutagenesis of the gene. Each mutant L protein was synthesized and bound to the Sendai P protein to form the P-L polymerase complex. While none of these L mutants exhibited a change in polyadenylation, the single amino acid changes yielded a variety of activities in vitro. Mutants containing valine, leucine, or phenylalanine at amino acid 1571, amino acids found naturally in the L proteins of other paramyxoviruses, yielded polymerases that had biological activity equal to or better than the wild-type (WT) polymerase. Serine or threonine substitutions in the L protein at this position also resulted in polymerases with nearly WT synthetic activity. In contrast, a glycine substitution significantly decreased overall polymerase activity, whereas a tyrosine substitution gave decreased transcription, but virtually no DI genome replication in vitro. The tyrosine-substituted polymerase may be unable to carry out the packaging step of replication, since DI leader RNA synthesis was normal in this mutant. Mutant L proteins with basic arginine or histidine substitutions were inactive in all viral RNA synthesis in vitro, although the polymerase complexes could bind the nucleocapsid template.