# Informed mutation of western equine encephalitis virus to heparan sulfate binding: Implications for rational design of alphavirus live attenuated vaccines

## TL;DR

Researchers engineered a safer version of western equine encephalitis virus by altering its protein to bind heparan sulfate, reducing its virulence in mice and offering a new approach for alphavirus vaccine design.

## Contribution

A structure-informed method for creating alphavirus live attenuated vaccines by introducing specific E2 glycoprotein mutations.

## Key findings

- Positively charged E2 mutations increased heparan sulfate binding and reduced virulence in mice.
- Second-site mutations improved infectivity via VLDLR while maintaining attenuation and HS binding.
- Cryo-EM mapping revealed mutation clusters in E2 domains, guiding receptor-specific vaccine design.

## Abstract

Encephalitogenic alphaviruses are mosquito-borne viruses that can cause fatal disease in humans and equines. Currently, there are no licensed vaccines or antiviral treatments for these infections. Western equine encephalitis virus (WEEV) is a member of this group that had not produced a human infection in over a decade. However, an outbreak of WEEV encephalitis in humans and equines was reported recently in South America, indicating a need for additional countermeasures. Blind passage approaches to generation of RNA virus live attenuated vaccines (LAVs) frequently result in acquisition of positively charged amino acid mutations that confer heparan sulfate (HS) binding and that are attenuating factors in resultant LAVs. To develop an informed approach for creation of alphavirus LAVs, we have utilized the WEEV McMillan (McM) strain as an HS weak/non-binding platform into which we have placed positively charged amino acid substitution mutations at positions in the E2 glycoprotein previously shown to confer HS-dependent infection upon other alphaviruses. This approach yielded four mutants with high efficiency HS binding and avirulence in mice, which were further subjected to yield optimization by in vitro selection of second-site mutations. Interestingly, the original mutations concomitantly increased HS interactions and reduced infection promoted by VLDLR and PCDH10 protein receptors, while the second site mutations improved infectivity mediated by VLDLR. Further, we report a newly generated 4.1Å cryo-EM reconstruction of WEEV McM strain into which we have mapped the mutations to provide an E2 glycoprotein domain-based representation of receptor binding site location.

Using the western equine encephalitis virus (WEEV) McMillan strain, we introduced rationally selected, positively charged amino acid substitution mutations into the E2 glycoprotein to promote heparan sulfate (HS)-dependent entry and attenuation. Several mutations resulted in increases in HS binding and mouse attenuation and, surprisingly, reduced engagement of known WEEV protein receptors VLDLR and PCDH10. Additional passage of mutant viruses yielded second-site mutations that preserved HS binding and attenuation, and increased engagement with protein receptors. Structural localization of mutations in a new cryo-EM model of WEEV revealed clustering within E2 functional domains. These findings provide a structure-informed framework for engineering receptor-specific attenuation and optimizing alphavirus LAV candidates for improved safety and production characteristics.