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

Alphaviruses are enveloped viruses that are transmitted by an arthropod vector to vertebrate hosts. Alphaviruses have glycoprotein spikes on their particle surface which are essential for viral entry. Each of the 80 spikes on the surface of an alphavirus particle consists of a trimer of E2-E1 heterodimers. Two types of interactions make up the spikes: ([1][1]) interactions between E2 and E1 of the same heterodimer called intra-dimer contacts, and ([2][2]) inter-dimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (called 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 where we replaced six inter-dimeric residues in Sindbis virus (WT SINV) with those from Chikungunya virus (CHIKV); the mutant is called CPB. CPB grew slower and to lower levels than WT SINV in mammalian cells, but not mosquito cells. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to capsid protein, and defects in particle morphology compared to mosquito cells. CPB transported glycoproteins to the plasma membrane in similar amounts to WT SINV in mammalian cells. Two revertants, E2-H333N and E1-S247L, restored particle assembly to different degrees. The viruses were visualized by cryo-EM. We determined that the spikes of CPB had a different conformation than WT SINV or the revertants. We conclude that the inter-dimer mutant, CPB, has host-dependent defects in spike trimerization and/or particle budding in mammalian cells. IMPORTANCE Alphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as an emerging pathogen with a global distribution. 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. [1]: #ref-1 [2]: #ref-2