Structural and functional basis of VLDLR receptor usage by Eastern equine encephalitis virus

The very low-density lipoprotein receptor (VLDLR) is comprised of eight LDLR type A (LA) domains and supports entry of distantly related Eastern equine encephalitis (EEEV) and Semliki Forest (SFV) alphaviruses. Here, by resolving multiple cryo-electron microscopy structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we show that EEEV uses multiple sites (E1/E2 cleft and E2 A domain) to engage different LA domains simultaneously. However, no single LA domain is necessary or sufficient to support efficient EEEV infection, highlighting complexity in domain usage. Whereas all EEEV strains show conservation of two VLDLR binding sites, the EEEV PE–6 strain and other EEE complex members feature a single amino acid substitution that mediates binding of LA domains to an additional site on the E2 B domain. These structural and functional analyses informed the design of a minimal VLDLR decoy receptor that neutralizes EEEV infection and protects mice from lethal challenge. ### Competing Interest Statement M.S.D. is a consultant for Inbios, Ocugen, Vir Biotechnology, Senda Biosciences, Moderna, and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Emergent BioSolutions, Moderna, Generate Biomedicines, Vir Biotechnology, and Immunome. D.H.F. is a founder of Courier Therapeutics and has received unrelated funding support from Emergent BioSolutions and Mallinckrodt Pharmaceuticals.