Molecular Basis of Receptor Interactions for Venezuelan Equine Encephalitis

Project Summary Title: Molecular basis of receptor interactions for Venezuelan equine encephalitis Venezuelan Equine Encephalitis Virus (VEEV) is a mosquito-transmitted alphavirus that causes severe neurological symptoms in South and Central America and threatens parts of the southern region of the United States. No therapies or licensed vaccines are currently available. Recently, our laboratory identified its major entry receptor, low-density lipoprotein receptor class A domain containing 3 (LDLRAD3). Ldlrad3-/- mice do not develop severe infection when the virus is administered by subcutaneous, intranasal, or even intracranial routes. Nonetheless, the infection still progresses in Ldlrad3-/- mice in peripheral organs and the central nervous system (CNS), albeit it at a lower level. This result highlights the existence of additional, uncharacterized subordinate entry pathways of VEEV. To identify alternative receptors, in this R03 application, we propose to perform a novel CRISPR activation screen targeting the plasma membrane proteins. The top ‘hits’ will be validated in the presence or absence of LDLRAD3 expression. A second component of this application will be to improve the possible therapeutic activity of an LDLRAD3 soluble decoy molecule through a forward genetic screen. Our prior structural and mutagenesis studies show that domain 1 (D1) of LDLRAD3 engages the VEEV in the cleft of the heterodimer of viral envelope proteins E1 and E2. Preliminary structure-guided experiments suggest that amino acid substitutions in D1 can result in variants with enhanced binding affinity for VEEV. We propose to conduct directed protein evolution by mammalian cell display and screening D1 mutants to identify variants with high binding and neutralization capacity to VEEV. Variant LDLRAD3-D1-Fc decoy molecules will be tested for inhibitory activity in cells. Relevance Venezuelan Equine Encephalitis Virus (VEEV) is a mosquito-transmitted alphavirus that causes devastating encephalitis with a high death rate in horses and humans. The presence of its mosquito vectors throughout the Americas makes it a potential threat to public health. No drugs or licensed vaccines are available for humans. Although LDLRAD3 was recently identified as a principal entry receptor of VEEV, additional unknown receptors still sustain infection. Decoy molecules designed based on the receptor LDLRAD3 can neutralize VEEV infection. Here, we will conduct a CRISPR-based screen to find alternative receptors for VEEV and a mutagenesis screen to identify LDLRAD3 D1 variants with greater binding and neutralizing activity. This study will enhance our knowledge of VEEV entry and provide potential avenues for VEEV infection and disease control.