Investigating a hemagglutinin mediated molecular mechanism of avian influenza A virus host tropism

Project Summary Zoonotic transmission of avian Influenza A virus (IAV) to humans poses a pandemic threat. Recent transmission of avian IAV to dairy cattle, and to dairy farm employees emphasizes the importance of identifying molecular mechanisms that regulate zoonotic events. It is thought that the IAV envelope glycoprotein, hemagglutinin (HA), must adapt its receptor specificity to bind α2,6-linked SA that predominates the human upper airway to initiate infection. However, recognition of α2,6-linked SA by HA is insufficient for avian H5N1 IAVs to enter human cells, indicating that other adaptations are necessary for zoonotic transmission to occur. Although evidence suggests that the pH and temperature sensitivity of HA are important factors that govern host tropism of IAV, a molecular mechanism that explains the temperature and pH dependence is missing. To define the pH and temperature dependence of HAs pre- to post-fusion conformational change, we first focused on visualizing conformational dynamics of HA with single-molecule FRET (smFRET). Preliminary data reveal that under neutral pH and room temperature conditions, the head domains of A/Vietnam/1194/2004 (H5N1) (VN04) HA, HA1, undergo a breathing motion where the heads are either fully caged, or partially uncaged. At pH8.0 HA spends 50% of the time in the fully caged conformation and 50% of the time in the partially uncaged conformation. Decreasing the pH to 6.5 shifts the equilibrium to where HA spends 30% of the time in the fully caged conformation and 80% of the time in the partially uncaged conformation. These data allow us to establish a conformational phenotype for HA where can determine the pH and temperature dependence of a given conformational phenotype. Thus, the central hypotheses of this proposal are that (1) the regulation of the pre- to post-fusion conformational change of avian HA is differentially regulated by pH and temperature compared to human adapted HA and (2) this difference in regulation directly affects the fusogenic function of HA. Experiments performed in Aim 1 will characterize the pH and temperature dependence of HAs conformational phenotype by monitoring conformational dynamics of HA at pH 8.0, pH 6.5, and pH 5.5 as well as at room temperature and 37°C. I will implement the smFRET for three HA serotypes: A/Vietnam/1194/2004 (H5N1) (VN04), A/California/07/2009 (H1N1) (CA09) and A/Hong Kong/1/1968 (H3N2) (HK68). Furthermore, mutations that alter pH and temperature stability will be characterized to evaluate a molecular mechanism that can explain the differences in the conformational phenotype between the different HA serotypes. Aim 2 will characterize the pH and temperature dependence on HA-mediated membrane fusion by monitoring the extent and rates of fusion of HA pseudo-typed virus with the three previously mentioned HA serotypes. Additionally, the same pH and temperature stability mutations will be used to assess their impact membrane fusion. Collectively, these data will reveal a mechanism that explains the zoonotic transmission potential of IAV and help improve genetic surveillance efforts to identify variants of concern.