Inhibition of the interferon-mediated antiviral response is a major determinant of virulence in influenza A virus. The NS1 protein of influenza A virus has been identified as the IFN antagonist. However, the specific mechanisms for IFN antagonism are not known. NS1 binds both the PKR as well as single and double stranded RNA to inhibit activation of PKR. Adaptation of human influenza virus to the mouse lung may likewise involve mutations that affect IFN antagonism. The prototype human influenza A virus A/HK/1/68 (H3N2 subtype) was utilized for this project. Six mouse-adapted variants possessing four different point mutations on NS gene were obtained from independently derived mouse-adapted variants. The mutations were located in the RNA binding domain and several sites in an 8 amino acid region from as 98 to 106. Mutant HKMA20c was of interest because it possessed a mutation in common with highly pathogenic avian influenza virus H5N1 (Leucine on AA 103 on NS1 protein). Base on these phenomena, a hypothesis was brought out that this region may encode a site of interaction with a host or viral factor and that mutation(s) on it may enhance the ability of NS1 protein to function as an IFN antagonist. The approach was to first characterize the IFN resistance and IFN induction properties of HK mouse-adapted mutants as well as pathology in mice lung, followed by the generation of defined recombinant viruses possessing desired mutation(s), then analysis of these recombinant viruses for IFN induction and IFN resistance. Rescuing of the HK wild type NS gene and those of HK mouse-adapted NS mutant genes into the backbone of parental HK genome discovered that NS20, NS20c and NS411 produced attenuating phenotypes on their own. Finally, all these mutant NS genes were inserted into backbone of WSN (lab adapted strain, A/WSN/33, H1N1) genome to construct recombinant viruses. Using recombinant viruses that differ due to individual NS1 gene showed that all of the NS1 mutations increased resistance to IFN in mouse cells. The extent of IFN resistance due to individual mutation was influenced by cell types: epithelium versus fibroblast, as well as host type: mouse versus human. Interestingly most of the NS1 mutations attenuated growth of virus which suggests that resistance to IFN involves changes in host interaction that are not optimal for growth in the absence of IFN. Infection assay showed mouse-adapted mutant NS genes conferred resistance to mouse IFN and vulnerability to human IFN. And mutant NS1 protein NSMA20c had the most potent ability to resist human and mouse IFN. IFN assay showed that mutant NS1 protein NSMA20c had the highest ability to induce mouse IFN. Furthermore, all synthetic recombinant viruses induced low amounts of IFN in human cells. Immunopathology of infected lungs showed that mouse adapted progeny virus HKMA20C had acquired a crucial ability to spread to and infected alveoli which may be influenced by IFN resistance. Mouse-adapted variants possess mutations that increase IFN resistance that in some instances leads to higher IFN induction. One of the key discussions was that most of these mutations cluster in a small region that has been previously mapped to involve a region of host protein (eIF4GI) interaction.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/27266 |
| Date | January 2006 |
| Creators | Li, Yishan |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 171 p. |
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