MOLECULAR DISSECTION OF THE INFLUENZA VIRUS RNA POLYMERASE AND ITS APPLICATION TO ANALYSIS OF VIRAL REPLICATION.

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Infuluenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. In order to study the function of each subunit we performed transcription in vitro using short model RNA templates carrying both termini of 8th genome segment and insect cell nuclear extracts containing subunits at each combination. And we demonstrated that the nuclear extracts containing at least PB1 subunit catalyzed RNA synthesis on model RNA templates with or without dinucleotide ApG.This result directly indicate that PB1 subunit is a catalytic subunit of influenza virus RNA polymerase.By transfection of various combinations of cDNA encoding wild-type and serial deletion mutants of each P protein subunit and co-immunoprecipitation with subunit-specific antibodies, the subunit-subunit contact sites on all the three P proteins were determined. Results indicate that the binary complexes are formed between PB1-PB2 and PB1-PA but not PB2-PA.Therefore, we concluded that PB1 is a core subunit for assembly of the viral RNA polymerase. The C-terminal 158 amino acids of PB1 bound to the N-terminal 249 amino acid stretch of PB2, while the N-terminal 140 amino acids of PB1 bound to the C-terminal two third region of PA.PB2-PA binding was not detected when they were expressed in the absence of PB1 subunit.