Mutations at the Alphavirus E2/E1 inter-dimer interface have host-specific phenotypes

Abstract

Alphaviruses are enveloped viruses transmitted by arthropod vectors to vertebrate hosts. The surface of the virion contains 80 glycoprotein spikes embedded in the membrane and these spikes mediate attachment to the host cell and initiate viral fusion. Each spike consists of a trimer of E2-E1 heterodimers. These heterodimers interact at two interfaces: (1) the intra-dimer interactions between E2 and E1 of the same heterodimer, and (2) the inter-dimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (E1). We hypothesized that the inter-dimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus (CPB) where we replaced six inter-dimeric residues in the E2 protein of Sindbis virus (WT SINV) with those from the E2 protein from chikungunya virus, and studied its effect in both mammalian and mosquito cell lines. CPB produced fewer infectious particles in mammalian cells than in mosquito cells, relative to WT SINV. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to capsid protein, and contained defects in particle morphology compared to virus derived from mosquito cells. Using cryo-EM, we determined that the spikes of CPB had a different conformation than WT SINV. Last, we identified two revertants, E2-H333N and E1-S247L, that restored particle growth and assembly to different degrees. We conclude the inter-dimer interface is critical for spike trimerization and is a novel target for potential antiviral drug design. IMPORTANCEAlphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as an emerging pathogen, with infections occurring worldwide. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This is important because it identifies a region in the spike that could be a target for antiviral drug design.