RNA-POLYMERASE OF INFLUENZA-VIRUS .12. RECONSTITUTION OF INFLUENZA-VIRUS RNA-POLYMERASE FROM 3 SUBUNITS EXPRESSED USING RECOMBINANT BACULOVIRUS SYSTEM

被引:44
作者
KOBAYASHI, M
TUCHIYA, K
NAGATA, K
ISHIHAMA, A
机构
[1] NATL INST GENET, DEPT MOLEC GENET, MISHIMA, SHIZUOKA 411, JAPAN
[2] GRAD UNIV ADV STUDIES, SCH LIFE SCI, SHIZUOKA, JAPAN
[3] NIPPON INST BIOL SCI, TOKYO, JAPAN
关键词
RNA POLYMERASE; INFLUENZA VIRUS; ENZYME RECONSTITUTION; SUBUNIT ASSEMBLY; BACULOVIRUS VECTOR;
D O I
10.1016/0168-1702(92)90055-E
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Influenza virus RNA polymerase catalyzes multiple step reactions in transcription and replication of the genome RNA. The core enzyme is composed of each one of the three P proteins, PB1, PB2 and PA (Honda et al. (1990) J. Biochem. 107, 624-628). For detailed analysis of the role of each P protein and of the functional domains on each P polypeptide, we expressed individual P proteins in cultured insect cells after infection with recombinant baculoviruses. PB1 and PB2 accumulated in cell nuclei whereas PA stayed in cytoplasm. Both the PB1 and PB2 proteins were purified from aggregates in the respective nuclear extract, and the PA was partially purified from the cytoplasm. RNA polymerase was reconstituted by mixing the three P proteins in a urea solution and then dialyzing against a reconstitution buffer. The reconstituted enzyme was able to transcribe model RNA templates. Minus-sense RNA was a better template than plus-sense RNA.
引用
收藏
页码:235 / 245
页数:11
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