The residue 86 of the Getah virus E2 glycoprotein mediates both glycosaminoglycan- and LDLR-dependent infection

Getah virus (GETV), a mosquito-borne alphavirus, poses an emerging threat to public health with its increasingly broad host spectrum. While glycosaminoglycans (GAGs) serve as critical attachment factors for many alphaviruses and the low-density lipoprotein receptor (LDLR) facilitates the cellular entry of several members, the precise viral determinants governing these interactions and their implications for viral virulence remain poorly defined. Here, we introduced an H86Y substitution, a potential adaptive mutation site, within the E2 glycoprotein of GETV using reverse genetics. The H86Y mutant replicated more efficiently in mosquito C6/36 cells but was consistently attenuated across several mammalian cell lines. In susceptible mouse models, H86Y infection led to reduced viral loads, milder histopathology, and lower inflammatory responses compared with the parental virus, yet still elicited robust protective immunity in adult mice. Mechanistically, a series of functional assays, including infection in GAG-deficient cells, decoy inhibition, co-immunoprecipitation, receptor overexpression and knockdown, and biolayer interferometry, demonstrated that the residue 86 in E2 glycoprotein is a critical determinant for GETV binding to both GAGs and LDLR. The H86Y mutation concurrently reduces these interactions, contributing the impairment of virus attachment and entry into mammalian cells. Furthermore, the GAG-binding site functionally overlaps with the LDLR interaction interface. In LDLR-deficient suckling mice, the impaired replication of H86Y persisted in examined tissues. However, pre-treatment with heparinase nearly completely eliminated this attenuation phenotype, further confirming that LDLR and GAG are the key host factors mediating attenuation phenotype for H86Y. In summary, the residue 86 of the GETV E2 glycoprotein represents a determinant of viral virulence, and an H86Y mutation attenuates GAGs and LDLR-dependent infection, providing mechanistic insights into alphavirus-host interactions and a potential target for antiviral and vaccine development. Getah virus is an emerging mosquito-borne alphavirus. We investigated a naturally occurring substitution in the viral E2 glycoprotein, where histidine at position 86 is replaced by tyrosine. This single change, which we call H86Y, weakened the virus in mammalian cells and mice but unexpectedly enhanced its ability to infect mosquito cells. This host-specific switch may explain why the mutation persists in nature despite reducing disease severity in animals. Mechanistically, the attenuation was linked to impaired viral attachment and entry, resulting from a specific reduction in binding affinity for two critical host molecules, the attachment factor GAGs and the cellular entry receptor LDLR. Also, the GAG-binding site functionally overlaps with the LDLR interaction interface. Multiple mouse models supported that LDLR and GAGs are the key host factors mediating attenuation phenotype for H86Y. Despite being weakened, the mutant virus still triggered strong protective immunity, making it a promising vaccine candidate. Together, our findings identify E2 residue 86 as a key virulence determinant that modulates receptor usage, offering a precise target for antiviral and vaccine development against alphaviruses.