Date of Award
Doctor of Philosophy (PhD)
Alphaviruses are arthropod-transmitted positive sense, single-stranded RNA viruses of the Togaviridae family that cause explosive, global outbreaks with significant morbidity. These viruses are classified into two groups based on genetic relatedness and clinical manifestations. Arthritogenic alphaviruses include chikungunya (CHIKV), Mayaro (MAYV), Ross River (RRV), O’nyong’nyong (ONNV) and generally cause acute and chronic musculoskeletal disease. To date, CHIKV has caused >1.8 million cases in over 40 countries. Encephalitic alphaviruses, including Eastern (EEEV), Venezuelan (VEEV), and Western (WEEV) equine encephalitis viruses, infect the central nervous system and result in encephalitic sequelae. In 2019, outbreaks of EEEV in the United States have resulted in 38 confirmed cases, including 15 deaths, with many more infections suspected but not diagnosed. Despite their epidemic potential, there exist no approved vaccines or treatments for these alphaviruses. Further understanding of alphavirus-host interactions can provide a strategy to mitigate alphavirus infection and disease. These studies will investigate the molecular basis of alphavirus host-engagement, and upon infection, the features required for antibody-mediated neutralization in vitro and in vivo.
A recent CRISPR-Cas9 genome-wide screen identified the matrix remodeling-associated protein 8 (Mxra8), a poorly characterized cell adhesion molecule, as a host receptor required for infection by multiple arthritogenic alphaviruses. Deletion of Mxra8 resulted in markedly reduced levels of infection by arthritogenic alphaviruses. Infection was restored upon re-introduction of mouse or human Mxra8 gene in cells. In vivo genetic-editing of Mxra8 or Mxra8 blockade using antibodies reduced CHIKV infection and clinical disease. Mxra8 directly interacts with CHIKV, but not with the encephalitic alphavirus EEEV. Competition binding studies with previously mapped anti-CHIKV antibodies and alanine-scanning mutagenesis revealed that Mxra8 recognizes domains A and B on the CHIKV E2 envelope protein. To determine the structural basis of Mxra8 engagement with CHIKV, we obtained a 2.2 Å crystal structure of Mxra8 and a 4 to 5 Å cryo-electron microscopy reconstruction of Mxra8 bound to CHIKV. Mxra8 contains an unusual Ig-like fold topology and recognizes a quaternary binding site that spans between the CHIKV E2 and E1 proteins on the virion. Evolutionary analysis of the Mxra8 receptor revealed a unique mechanism of evasion of virus-host receptor interactions. Mxra8 of several lineage within the Bovinae family (e.g., cattle, bison, buffalo, and antelope) contained a unique three quasi-identical 5-residue repeats in the virus binding interface, which conferred resistance to alphavirus binding and infection. Removal of this 15-residue insertion from cattle Mxra8 restored alphavirus infection and reciprocally, addition of the 15-residue insertion into mouse Mxra8 conferred resistance to alphavirus infection. A 2.3 Å crystal structure of cattle Mxra8 and cryo-EM docking revealed that the Mxra8 insertion hinders CHIKV engagement and binding. Analysis of Mxra8 gene sequences from animals in the Saint Louis Zoo dated the insertion to at least 5 million years ago. To determine significance of the 15-residue Mxra8 insertion in vivo, we genetically engineered mice with the 15-residue insertion in Mxra8 gene using CRISPR-Cas9. These gene-edited mice exhibited decreased clinical disease and exhibited reduced viral replication and pathogenesis, essentially behaving like Mxra8 knockout mice with respect to CHIKV infection.
Understanding the molecular determinants of in vitro neutralization and in vivo protection by antibodies upon alphavirus infection can be informative towards therapeutic and vaccine strategies. We identified a panel of potently neutralizing murine monoclonal antibodies against EEEV. Mapping of these antibodies through alanine-scanning mutagenesis, virus escape, and cryo-EM revealed that the antibodies recognize epitopes in distinct regions of domains A or B of the E2 envelope protein. In vivo administration of EEEV antibodies protected mice from a lethal subcutaneous or aerosol challenge and significantly decreased viral replication in the brain. In addition, we identified human monoclonal antibodies that are cross-reactive and protective against both arthritogenic and encephalitic alphaviruses. These antibodies are non-neutralizing yet bind avidly to cells infected by arthritogenic and encephalitic alphaviruses. In vivo administration of these antibodies protected mice in a CHIKV-induced arthritis model, MAYV-induced arthritis model, and a lethal VEEV challenge model. Competition binding studies revealed that these antibodies recognize an epitope proximal to the E1 fusion loop, a highly conserved region amongst all alphaviruses. Altogether, these studies reveal important determinants of alphavirus-host interactions and humoral immune responses which can inform therapeutic and immunogen design strategies to combat this global epidemic.
Chair and Committee
Michael S. Diamond, Daved H. Fremont
Ali H. Ellebedy, Deborah J. Lenschow, Jonathan J. Miner
Kim, Arthur S., "Structural and Functional Insights into Alphavirus-Host Interactions" (2020). Arts & Sciences Electronic Theses and Dissertations. 2934.