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This dissertation, Identification and Characterization of a Novel Fusion Inhibitor of Influenza A Virus by Kin-kui, Lai, 黎健鉅, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Influenza A virus is the major pathogen contributing to seasonal infections and even severe pandemics. Due to the frequent usage of antiviral drugs like Tamiflu and Amantadine, drug resistance viruses have appeared rapidly. There is an urge to develop effective vaccines or antivirals for influenza A virus associated infections. In the initial step of viral infection, hemagglutinin (HA) plays a critical role in recognizing the host cells and inducing membrane fusion under acidic pH. Using the forward chemical genetic approach, we have selected a small molecule compound named 5831050 from the screening of the chemical library previously done by our lab. 5831050 inhibits H1N1, H5N1, and 2009 swine-origin influenza A viruses effectively at nanomolar median effective concentration (EC_50), To uncover the mechanism of the compound, time-of-addition assay was performed and the early stage of viral life cycle was inhibited. Through subsequent viral passages together with increasing concentration of 5831050, four escape mutant clones that are commonly observed with a methionine to leucine substitution at the amino acid residue 59 of the HA2 protein were isolated by plaque reduction assay, and were further identified by the whole genome sequencing of influenza A virus. Its resistance level to 5831050 was further confirmed by the recombinant virus generated through reverse genetics, suggesting that the molecular target of 5831050 is HA. Molecular docking using available HA crystal structures suggested that the compound may favorably bind to a pocket on HA2 protein in which M59 is located. The viral morphology was found to be intact in the presence of the compound using transmission electron microscopy. This can eliminate the possibility that the compound acts on the viral envelope. It was also found that the presence of 5831050 did not inhibit the receptor binding activity of HA in hemagglutination inhibition assay. The results indicated that the compound might inhibit viral entry by targeting HA via binding to HA_2 〖M59〗_2 (〖M59〗_2) without altering the receptor binding activity of HA. The available three-dimensional structure of HA protein indicates that 〖M59〗_2 is located on the B-loop of HA_2 subunit, which is involved in loop-to-helix transition during membrane fusion. As this transition is critical in the fusion process, compound 5831050 may inhibit viral entry by targeting the membrane fusion. Trypsin protection assay and low-pH inactivation assay were further carried out to confirm its mode of action. Experimental results from both assays supported our postulations that the compound is able to inhibit the membrane fusion by blocking the low-pH induced HA conformational change at the early stage of viral infection. This study may be beneficial to the characterization and development of a novel class of fusion inhibitors for influenza A viruses. Subjects: Virus inhibitorsInfluenza A virus

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