Critical regions involved in the regulation and misregulation of the E1 homotrimerization step of the Semliki forest virus membrane fusion mechanism
Liu, Catherine Y.
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Alphaviruses are enveloped viruses that cause severe arthritis and encephalitis. Alphaviruses such as Semliki Forest virus (SFV) use a membrane fusion reaction to infect host cells. Fusion of the virus and cell membranes is triggered by low pH in the endosome and is mediated by the viral membrane protein E1. Formation of the E1 homotrimer is critical to membrane fusion, but the mechanism of trimerization is not well understood. The crystal structure of the postfusion E1 trimer shows that a conserved aspartate residue, D188, is positioned in the central core trimer interface. We tested the contribution of this amino acid to trimerization and fusion. A lysine (D188K) mutation blocked SFV fusion and infection and was trapped as a target membrane-inserted monomer that did not efficiently form the stable core trimer. Thus D188 is involved in the initial steps of core trimer formation.;Selections were carried out for revertants that rescued the fusion and growth defects of D188K. Revertant analysis revealed that acidic or polar neutral residues at E1 position 188 were important for fusion efficiency. A second site revertant containing an E1 K176T mutation rescued the D188K defect, suggesting possible interactions. De novo mutations in K176 to threonine or isoleucine produced similar fusion phenotypes as D188 mutants. Computational analysis using multi-conformation continuum electrostatics (MCCE) revealed an important interaction bridging D188 of one chain with K176 of the adjacent chain in the core trimer. Together our data support a model in which a ring of 3 salt-bridges formed by D188 and K176 stabilize the core trimer, a key intermediate of the alphavirus fusion protein.;Zinc has been shown to block SFV fusion. To study the zinc block mechanism, we developed a robust cell culture system amenable to zinc inhibition of SFV fusion. Selections for zinc escape mutants revealed that E1 H333 is important for mediating the zinc block. While wildtype viruses were blocked in stable trimer formation and fusion, an H333A mutation rescued the trimerization and fusion defect. Insights into the mechanism of zinc inhibition could help with the development of strategies to prevent alphavirus fusion and infection.