Studies on Conformational Stability of the Ectodomain of Influenza Virus Hemagglutinin

Enveloped viruses enter host cells by a process that involves fusion of the viral envelope with a membrane of the host cell. The process of membrane fusion which is typical for various cellular processes has been best studied in viral infections, especially in influenza virus. Hemagglutinin (HA), a major envelope glycoprotein is responsible for fusing viral and endosomal membranes during influenza virus entry. This membrane fusion is mediated by a conformational change of HA at low pH conditions in the endosome. The analysis of 3D crystal structure of the bromelain cleaved HA ectodomain shows that the stability of protein is maintained by both non-covalent and covalent interactions. The irreversible conformational change of HA at low pH indicates a role for protonation effects of the ionisable amino acids. While many models explain the various intermediates of the membrane fusion process, the early steps leading to conformational changes have not been consider. In addition, the presence of extensive salt networks in the 3D crystal structure of HA has not been contended. The present research work focuses on these queries in order to bring out the importance of electrostatic interactions for the stability of the spike-like ectodomain in its non-fusogenic structure. Structural investigations were done using “site directed mutagenesis” in order to conceive the importance of charged amino acids and more emphatically the involvement of salt bridges. The mutagenesis was carried out either at the interface of HA1 and HA2 monomers, or between the HA1 or HA2 monomers. Thus, mutations are designed so as to investigate electrostatic interactions both at inter-monomer and intra-monomer interfaces. The approach was to make new salt bridge or break the existing salt bridge, so as to increase or decrease the interactions within or between the monomers. Fourteen mutants were constructed based on careful analysis of the 3D crystal structure of the X-31 influenza A virus. The selected amino acids were analysed for their evolutionary significance by sequence homology. All the mutant and HA-wt constructs were expressed in CV-1 cells by transient T7-RNA-polymerase vaccinia virus system. The surface expression of the expressed HA proteins were characterised. The effect of mutations on the conformational change and fusion activity was probed by proteinase K assay and fluorescence microscopy respectively. An effort was made to correlate the results from proteinase K assay with those from fusion assays. It was observed that HA-wt and all the mutants except R109E showed comparable surface expression. The difference in pH threshold between the HA-wt and the mutants showed that breakage of salt bridge and