Studies on the structure and function of the influenza virus ribonucleoprotein and polymerase complex.

Klumpp, Klaus (1997). Studies on the structure and function of the influenza virus ribonucleoprotein and polymerase complex. PhD thesis The Open University.

Abstract

Genomic RNPs were isolated from influenza A virus particles and the RNA conformation in the native structure was studied by chemical and enzymatical RNA modification analysis on complete RNPs and on RNPs depleted o f polymerase protein. The results suggest that NP binding to the genomic RNA does not involve the Watson-Crick positions of the bases and the major part of the genomic RNA was found to be in a single-stranded conformation, consistent with the idea that NP functions as a single-stranded RNA binding protein, that facilitates templated RNA synthesis by the polymerase. The polymerase, on the other hand, was found to form a ternary complex with both conserved vRNA ends, but in the absence of the polymerase the RNA ends were completely single-stranded despite a partial, inverted complementarity. The implications of the absence of a stable panhandle RNA secondary structure on possible replicative mechanisms are discussed.

RNP a c tiv ity assays were employed to determ ine basic kinetic constants of the transcription reaction catalyzed by RNPs and to analyse the mechanism of inhibition by 2'-deoxy -2 '-fluororibonucleotides , which were found to be incorporated in to RNA products by the influenza virus polymerase thereby preventing efficient elongation of the transcripts. Transcription reactions in the presence of ATP analogs and ATPase assays revealed a possible requirement for ATP β-ϒ bond hydrolysis during influenza virus transcription.

Finally, a number of different protein expression systems were tested for the production and purification of the PA sub unit of the influenza virus polymerase complex for structural and functional studies. In both procaryotic and eucaryotic systems the PA protein yield was very low and possible reasons for this lack of expression efficiency are discussed. Affinity purified PA preparations from bacterial cultures and semipurified fractions from eucaryotic systems showed that an ATPase activity consistently copurified with PA. Also, preliminary evidence for sequence-specific RNA binding was obtained from in vitro translation experiments.

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