“Determining the Functional Characteristics of Precursors to Drug-Resistant Influenza”
Viral membrane fusion to the host endosomal membrane is an essential step to successful infection. The rate and efficiency at which fusion occurs is determined by multiple independent variables and is mediated by the surface protein hemagglutinin (HA) in influenza. Membrane fusion inhibitors, such as Tert-butylhydroquinone (TBHQ), inhibit viral infectivity by binding to and stabilizing the native HA structure.
This research project aims to investigate the mechanism of resistance emergence against cell-entry inhibitors for influenza virus. Our fusion model reveals that 3-5 HAs combine forces while pulling on viral and host cell membranes to induce membrane fusion. This active HA cluster is formed by random chance within the virus-cell contact interface containing more than 100 HAs. While a large fraction of HAs become inactivated and cannot contribute to the cluster formation, more than 70% of HAs need to become inactivated for any decrease in fusion yield. Based on this model, we predict that membrane fusion inhibitors act by increasing the apparent fraction of inactivated HAs and in this way primarily serve to slow fusion rate rather than decrease fusion efficiency. As such, we predict that resistance mutations are more likely to emerge in viruses with faster fusion kinetics. We further hypothesize that the crippling effects of resistance mutations will be compensated by the genetic context of fast fusing viruses.
Small scale infections were performed under varying concentrations of TBHQ to determine the inhibitory effects of TBHQ on viral strains with differing fusion kinetics. Stochastic simulations were performed to predict the effects of changes in independent fusion variables on fusion kinetics. Higher sensitivity of the slower fusing mutants to TBHQ inhibition supports our hypothesis that fusion inhibitors act by slowing fusion and that fast fusing viruses are thus the more likely population from which resistance might emerge.
In the future, we plan passage virus strains in presence of TBHQ to see from which fusion kinetics background resistance will emerge first, and any resistant viruses will be sequenced and analyzed for further functional studies.
SMURF (Summer MRSEC Undergrad Research Fellowship)