Alphaviruses are arthropod-borne, single-stranded positive-sense RNA viruses of the Togaviridae family that infect various vertebrates worldwide in tropical and temperate areas, causing emerging and reemerging diseases in humans. Mature virions are 70 nm in diameter and contain a ~11-kilobase genome encapsidated within a nucleocapsid core, a host-derived lipid bilayer, and an envelope comprised of heterodimers of the glycoproteins E1 and E2 arranged into trimeric spikes with T=4 icosahedral symmetry. Alphaviruses are categorized into two groups based on their clinical symptoms: the arthritogenic alphaviruses, such as chikungunya (CHIKV), Mayaro (MAYV), Ross River (RRV), Semliki Forest (SFV), and O’nyong-nyong (ONNV) viruses, which induce arthritis, polyarthralgia, and musculoskeletal-associated diseases, and the encephalitic alphaviruses, including Venezuelan (VEEV), Eastern (EEEV), and Western (WEEV) equine encephalitic viruses, which lead to meningitis, encephalitis, and long-term neurological sequelae in survivors. The global distribution of alphaviruses has increased in recent decades due to international travel, expansion of mosquito vectors, deforestation, and urbanization. Currently, there are no approved vaccines or treatments available to mitigate alphavirus infection and disease. Further understanding of host-alphavirus interactions may inform the development of such therapies for multiple members of this family. The studies encompassed in this dissertation describe how alphaviruses engage two different entry receptors and how a panel neutralizing anti-MAYV monoclonal antibodies protect against infection.Mxra8 is a receptor for multiple arthritogenic alphaviruses such as CHIKV, MAYV, RRV, and ONNV. We determined a 2.2 Å resolution X-ray crystal structure of Mxra8 and 4 to 5 Å resolution cryo-electron microscopy reconstructions of Mxra8 bound to CHIKV virus-like particles (VLPs) and infectious virus. Our structures revealed that the Mxra8 ectodomain contains two strand-swapped Ig-like domains oriented in a unique disulfide-linked head-to-head arrangement, and that Mxra8 binds CHIKV by wedging into a cleft created by two adjacent E2-E1 heterodimers in one trimeric spike while also engaging a neighboring spike. Furthermore, we observed two binding modes with the fully mature VLP, with one Mxra8 binding with unique additional contacts. This high- and low- binding-site model was supported by our surface plasmon resonance measurements. Lastly, we found that the low-affinity binding sites were sterically obscured by the retention of the E3 glycoprotein on infectious CHIKV, suggesting that viral maturation and E3 occupancy influences receptor binding-site usage. In later studies, we also determined near-atomic resolution cryo-electron microscopy reconstructions of VEEV VLPs alone and complexed with its entry receptor, LDLRAD3. We showed that domain 1 (D1) of LDLRAD3, a low-density lipoprotein receptor type-A (LA) module, binds VEEV by wedging into a cleft created by two adjacent E2-E1 heterodimers in one trimeric spike, specifically engaging domains A and B of E2 and the fusion loop in E1. Our atomic modeling of this interface was supported by mutagenesis and anti-VEEV antibody binding competition assays. These studies demonstrated that VEEV engages LDLRAD3 in a manner that is remarkably similar to CHIKV with the Mxra8 receptor, but with an exceptionally smaller interface. We speculate that the common positioning of these receptors near the fusion loop might serve to modulate viral fusion during endocytosis. Our studies are among the first to structurally characterize alphavirus-receptor complexes. Additionally, we generated a panel of neutralizing monoclonal antibodies (mAbs) against MAYV, over half of which had “elite” activity that inhibited infection with EC50 values of \u3c10 ng/ml. We demonstrated that antibodies with the greatest inhibitory capacity in vitro mapped to epitopes near the fusion peptide of E1 and in domain B of E2. Unexpectedly, many of the elite neutralizing mAbs failed to prevent MAYV infection and disease in vivo. Instead, protection required fragment crystallizable (Fc) effector functions, as isotype-switched or aglycosyl variants with less or no capacity to interact with the complement component C1q or activating Fc-γ receptors lost protective activity in vivo. These results demonstrated that an optimally protective antibody response to MAYV and possibly other alphaviruses may require tandem optimization of virus neutralization by the Fab moiety and effector functions of the Fc region. Altogether, these studies establish how alphaviruses interact with the entry receptors and humoral responses of their hosts, which may inform the basis of future therapies and improved vaccine designs
