CHARACTERIZATION OF THE ACTIVE SITE OF THE INFLUENZA VIRUS NEURAMINIDASE PROTEIN (MUTAGENESIS)

The influenza virus NA protein possesses enzyme activity which is highly conserved in all strains despite 50-60% variation in amino acid sequences of this protein. The enzyme facilitates movement of the virus to and from the site of infection in the host by cleaving terminal sialic acid residues from glycoproteins. The conserved nature of this enzyme activity makes the active site of the NA a possible target for antiviral therapy. This dissertation describes experiments designed to further our understanding of the active site of this enzyme.; A full-length cDNA clone of the NA gene from the A/Tokyo/3/67 strain of influenza was synthesized from viral RNA and sequenced. The cDNA was then inserted into an SV40 virus recombinant expression vector designed and constructed to facilitate expression of the cloned NA gene. The vector expressed NA which was indistinguishable from NA from influenza virions; the protein was transported efficiently to the infected cell surface and was enzymatically active.; The NA gene was subsequently cloned into an M13 phage vector and used as template for site-specific oligonucleotide-directed mutagenesis. Amino acids which are conserved and located in the substrate binding pocket were chosen as targets for a total of twelve mutations, ranging from very conservative substitutions to more dramatic changes. The mutated NA genes were cloned back into the SV40 expression vector, and synthesis, transport, and enzyme activity of the NA proteins in infected cells was assayed.; All but two of the mutant proteins were indistinguishable from wild-type in their expression, transport and folding. The enzyme activity of these mutants ranged from fully active in the case of one active site mutant and one control, to completely inactive in others. Several mutants had intermediate activity and the effect of pH on these enzymes was determined. The results obtained suggest a possible mechanism for neuraminidase catalytic activity which has features in common with the mechanism of lysozyme. It is likely that histidine 274 elevates the pK(,a) of nearby glutamic acid 276 which is then able to donate a proton to the glycosidic bond. Other conserved residues in the active site pocket are likely to be involved in binding the substrate or stabilizing a charged intermediate. (Abstract shortened with permission of author.)...