Studies of the influenza virus entry mechanism

Infection by enveloped viruses proceeds by fusion of viral and cellular membranes which transfers the genetic material of the virus into the cell. The membrane fusion potential of influenza glycoprotein hemagglutinin is generated by proteolytic cleavage of a biosynthetic precursor into two disulfide-linked polypeptide chains (HA{dollar}sb1{dollar} and HA{dollar}sb2{dollar}) and activated at the low pH of endosomes in a process that involves extensive refolding and conformational rearrangements of the hemagglutinin structure. This thesis is devoted to elucidate the role of hemagglutinin during membrane fusion process at a molecular level. Chapter I, the introduction, reviews current knowledge of the influenza virus and the viral surface glycoprotein hemagglutinin. Chapter II shows that the structure of the low-pH-induced fold of HA{dollar}sb2{dollar} observed crystallographically is the lowest-energy-state of the HA{dollar}sb2{dollar} polypeptide in the absence of HA{dollar}sb1.{dollar} Chapter III describes an engineered HA{dollar}sb2{dollar} chain with a highly polar peptide fused to the amino-terminal of HA{dollar}sb2{dollar} to prevent aggregation of the protein and insertion of the fusion peptide into cell membranes. Chapter IV describes crystallization of a recombinant protein composed of the ectodomain of HA{dollar}sb2{dollar} with the fusion peptide deleted. The structural information of this protein may elucidate the initial step of the membrane fusion mechanism. Chapter V describes determination of the three-dimensional structure of uncleaved precursor hemagglutinin by X-ray crystallography and provides a structural basis to study the proteolytic activation of hemagglutinin and its relation to virus entry.