Anthony P. Hanson, PhD

Research Title: Characterization of Stabilizing Mutations in the H5N1 Hemagglutinin Influenza Protein

Research Summary: To identify mutations in an influenza virus H5 hemagglutinin (HA) protein that affect protein stability, Anthony Hanson and members the Kawaoka laboratory created an H5 HA virus library with random mutations in the extracellular domain of HA. They used PCR mutagenesis to introduce random mutations into the extracellular region of a modified A/Vietnam/1203/2004 (H5N1) HA gene, which also possesses the N224K/Q226L mutations that confer humantype receptor-binding preference (Imai et al., Nature 486:420-428, 2012). These randomly mutated HA plasmids were then combined with the other seven influenza gene plasmids from the 2009 H1N1 pandemic virus A/California/04/2009 virus to generate a mutant virus library via a plasmid-driven reverse genetics system. This mutant virus library was subjected to multiple rounds of heat treatment at 50°C followed by subsequent amplification. This selection process revealed 9 mutations in an H5 HA that increased heat stability; of those, six mutants that exhibited the most significant increase in heat stability were pursued. The influenza virus databases were queried for the identified mutations.  They found that the identified amino acid positions were highly conserved and that these mutations were rare to nonexistent in sequenced H5N1 isolates. Heat-treatment at 50°C indicates the stability of the HA protein, but is not biologically pertinent. It serves as a surrogate assay for stability under varying pH values. It was previously demonstrated that a mutant H5 HA which binds human-type receptors will undergo the conformational change in late endosomes at a more basic pH value than the wild-type protein (Imai et al., Nature, 2012). The addition of compensatory stabilizing mutations restored the pH of fusion to wild-type levels and facilitated respiratory droplet transmission in the ferret model (Imai et al., Nature, 2012). A polykaryon formation assay was performed in which cells are transfected to express the HA proteins and then briefly exposed to a range of acidic pH buffers initiating cell fusion. Anthony and his colleagues tested wildtype H5 HA, H5 HA possessing the N224K/Q226L mutations which confer binding to human-type receptors, and H5 HA possessing the N224K/Q226L and the selected stability-enhancing mutations. Indeed, the stability-enhancing mutations in HA resulted in fusion at a more acidic pH than the control viruses. Anthony was interested in isolating stabilizing mutations in an H5 HA that is able to bind to human-type receptors. To determine whether any of the new stabilizing mutations affected receptor-binding preference, he tested HAs possessing the N224K/Q226L and stabilizing mutations in an agglutination assay with turkey red blood cells, a solid-phase binding assay, and glycan arrays. The stability-enhancing mutations did not interfere with the ability to bind to human-type receptors (conferred by the N224K/Q226L mutations). In summary, novel HA stabilizing mutations that affect the pH of fusion of HA and may affect virus transmissibility were identified.

Currently, Dr. Hanson is a Regional Associate for WiSys Technology Foundation.  WiSys Technology Foundation is a non-profit organization of the University of Wisconsin System, serving as the dedicated technology transfer office for the 11 four-year Comprehensive Campuses, 13 two-year Colleges and the statewide UW-Extension.