Chandrika R, Horikami S M, Smallwood S, Moyer S A
Department of Molecular Genetics, University of Florida College of Medicine, Gainesville 32610-0266, USA.
Virology. 1995 Nov 10;213(2):352-63. doi: 10.1006/viro.1995.0008.
To begin to map functional domains of the Sendai P-L RNA polymerase complex we wanted to characterize the P binding site on the Sendai L protein. Analysis of in vitro and in vivo P-L polymerase complex formation with carboxyl-truncations of the L protein showed that the N-terminal half of the protein was required. Site-directed mutagenesis of the Sendai virus L gene was employed to change amino acids within a highly conserved region of the N-terminal domain I from amino acids (aa) 348-379 singly or in pairs from the Sendai to the corresponding measles L sequence or to alanine. The mutant L proteins coexpressed with the viral P and NP proteins in mammalian cells were assayed for their ability to form the P-L complex and to synthesize RNA in vitro and showed a variety of defective phenotypes. While most of the mutant L proteins still formed the P-L polymerase complex, a change from serine to arginine at aa 368 and a three-amino-acid insertion at aa 379 virtually abolished both complex formation and RNA synthesis. Changes of aas 370 and 376-377 in the L protein gave only small decreases in viral RNA synthesis. Substitutions at either aas 349-350 or aas 354-355 and a three-amino-acid insertion at aa 348 in the L protein yielded enzymes that catalyzed significant transcription, but were defective in DI RNA replication, thus differentially affecting the two processes. Since DI leader RNA, but not genome RNA, was still synthesized by this class of mutants, the defect in replication appears to be in the ability of the mutant enzyme to package newly synthesized nascent RNA. Single changes at aas 362, 363, and 366 in the L protein gave enzymes with severely decreased overall RNA synthesis, although some leader RNA was synthesized, suggesting that they cannot transcribe or replicate past the leader gene. These studies have identified a region in conserved domain I critical for multiple functions of the Sendai virus L protein.
为了开始绘制仙台病毒P-L RNA聚合酶复合物的功能结构域图谱，我们想要确定仙台病毒L蛋白上的P结合位点。对L蛋白进行羧基端截短后，分析其在体外和体内形成P-L聚合酶复合物的情况，结果表明该蛋白的N端一半是必需的。利用定点诱变技术改变仙台病毒L基因N端结构域I高度保守区域内的氨基酸，将单个或成对的氨基酸从仙台病毒的348-379位氨基酸改变为相应的麻疹病毒L序列或丙氨酸。在哺乳动物细胞中与病毒P蛋白和NP蛋白共表达的突变L蛋白，检测其形成P-L复合物以及在体外合成RNA的能力，结果显示出各种缺陷表型。虽然大多数突变L蛋白仍能形成P-L聚合酶复合物，但368位氨基酸由丝氨酸变为精氨酸以及379位氨基酸处插入三个氨基酸，几乎完全消除了复合物的形成和RNA合成。L蛋白中370位和376-377位氨基酸的改变仅使病毒RNA合成略有减少。L蛋白中349-350位氨基酸或354-355位氨基酸的替换以及348位氨基酸处插入三个氨基酸，产生的酶能够催化显著的转录，但在DI RNA复制方面存在缺陷，从而对这两个过程产生不同影响。由于这类突变体仍能合成DI前导RNA，而不能合成基因组RNA，复制缺陷似乎在于突变酶包装新合成的新生RNA的能力。L蛋白中362、363和366位氨基酸的单个改变产生的酶总体RNA合成严重减少，尽管仍能合成一些前导RNA，这表明它们无法转录或复制超过前导基因。这些研究确定了保守结构域I中对仙台病毒L蛋白多种功能至关重要的一个区域。
