Elucidating the Critical Role of Low-Density Lipoprotein Receptor Class A Domain Containing 3 in Venezuelan Equine Encephalitis Virus Neuron Infection and Pathogenesis

PROJECT SUMMARY Alphaviruses are enveloped positive-stranded RNA viruses of the Togaviridae family whose natural endemic cycles occur between mosquito vectors and nonhuman mammal or avian species. Venezuelan equine encephalitis virus (VEEV) is an encephalitic alphavirus responsible for periodic epidemics in equine and human populations across the Americas. Its potential for use as an aerosolized bioterrorism weapon and the demonstrated risk it poses in research settings highlight the need for countermeasures. VEEV pathogenesis is characterized by central nervous system (CNS) involvement and damage to lymphoid tissues. Our laboratory published the discovery of a novel receptor for VEEV, low-density lipoprotein receptor class A domain containing 3 (LDLRAD3), required for infection and VEEV pathogenesis in vivo. We have demonstrated that a soluble fusion protein with LDLRAD3 linked to an IgG Fc domain (LDLRAD3-Fc) protects mice from lethal infection. Though this shows that identification of an entry receptor can become a target for countermeasures against VEEV, the role of LDLRAD3 in controlling VEEV pathogenesis is still not understood. The first Aim of my proposal will explore LDLRAD3-mediated tropism and neuroinvasion of VEEV. I hypothesize that LDLRAD3 is required for VEEV infection of myeloid cells in peripheral organs and neurons in the central nervous system, which contribute to the development of inflammation and clinical disease. This hypothesis will be tested using in vivo infection studies that analyze viral RNA levels, histopathology, and virus tropism during time courses of infection in Ldlrad3-/- mice. Cell-type specificity of LDLRAD3-mediated VEEV infection will be explored using mixed primary neuron cultures from Ldlrad3-/- mice and tissue-specific conditional Ldlrad3-/- mice. Though the molecular mechanisms of VEEV attachment and entry into host cells during infection are still poorly understood, recently the interaction residues on VEEV have been identified for LDLRAD3 by cryo-electron microscopy. My second Aim will determine which of these contact sites are of functional importance for VEEV infection in vitro and in vivo. Using structure-guided mutagenesis, I will generate VEEV strains encoding mutations at LDLRAD3 interaction sites. These will be tested for altered infection using cell-based assays and differences in pathogenesis using a lethal murine VEEV model. Mutations at LDLRAD3 interaction sites which result in increased VEEV infectivity or binding will be engineered into a LDLRAD3-Fc decoy receptor to optimize its anti- VEEV therapeutic function. Overall, these studies will define how LDLRAD3-dependent tropism of VEEV is critical for the development of clinical disease and what key viral interaction residues required for binding of VEEV to LDLRAD3 might be harnessed for the development of targeted antiviral therapies.