Generation of recombinant influenza A virus without M2 ion channel protein by introducing a point mutation at the 5' end of viral intron

by Cheung, Kai-wing

(Uncorrected OCR) Abstract of thesis entitled ?eneration of recombinant influenza A virus without M2 ion channel protein by introducing a point mutation at the 5?end of viral intron?submitted by Cheung Kai Wing for the Degree of Master of Philosophy at The University of Hong Kong in December 2004 Influenza viruses are medically important viral pathogens causing significant mortality and morbidity throughout the entire world for centuries. The continuous outbreaks of influenza A virus in the last few decades highlighted the fact that the current vaccine approach has limited success as this pathogen will undergo unpredictable genetic reassortments naturally. This issue poses serious threats to the health of human being. The recent advance of molecular techniques allows novel approaches to provide better understanding and alternative measures to control this particular infectious agent. In this study, we aimed at knocking down the influenza M2 protein expression by mutating the splicing signal of M gene. Mutations were introduced into the GU dinucleotide sequence at the 5?proximal splicing site of the M gene (corresponds to nt 52-53 of M cRNA). Transfected cells expressing mutated M viral ribonucleoproteins failed to generate M2 mRNA. Interestingly, recombinant viruses with mutations at the dinucleotide sequence were viable though attenuated in cell culture. These recombinants failed to express M2 mRNA and M2 proteins. In addition, these mutants were less susceptible to pH perturbants. These observations demonstrate that the GU invariant dinucleotide sequence at the 5?proximal splicing site of M gene is essential for M2 mRNA synthesis. Our results also indicate the M2 ion channel protein is critical, but not absolutely essential for virus to replicate in cell culture. Besides, this approach might provide a new way in attenuating influenza A virus. (236 words)