Abstract

The co-evolution of virus and host may partly be understood in terms of “cross-recognition”; that is, as a virus evolves to recognize and infiltrate a suitable cellular environment, the host reciprocally has evolved to recognize and thwart such an invasion. While this interplay is complex, audacious structural biologists may reduce it to discrete physical interactions between the virus and specific host factors, such as cognate receptors (i.e., viral recognition of host) or antibodies (i.e., host recognition of virus). These interactions underlie basic features of viral infection, including cellular tropism and antiviral immunity, as well as critical phenomena of viral evolution, such as cross-species transmission and immune evasion. Detailed study of these interactions may thus provide fundamental insights in virology and guide the optimization of clinical tools. To these ends, I explore in this dissertation the structural and functional properties of receptors and antibodies specific to two genera of RNA viruses, Betacoronavirus and Alphavirus. SARS-CoV-2, a Betacoronavirus which causes the respiratory syndrome COVID-19, emerged in 2019 and has caused over 770 million infections and 7 million deaths worldwide. While efficacious vaccines and monoclonal antibodies (mAbs) were rapidly developed, viral variants have emerged with resistance to these interventions. Here, we developed a panel of 64 murine mAbs targeting SARS-CoV-2 spike (S), a class I fusion protein that mediates viral attachment and entry. We identified 43 mAbs that engage the receptor-binding domain (RBD) of S and generally block virus attachment to the angiotensin-converting enzyme 2 (ACE2) receptor. One mAb, SARS2-38, neutralized most SARS-CoV-2 variants tested. We found via cryo-EM that this mAb engages a conserved epitope on the apex of the RBD; however, point mutations at this site abrogate SARS2-38 binding to some Omicron subvariants. Among the 21 mAbs targeting non-RBD epitopes, we found that many bind the N-terminal domain (NTD) of S and can inhibit viral entry after ACE2 attachment. Using cryo-EM, we showed that one mAb, SARS2-57, binds a known antigenic supersite on the NTD; while this mAb recognizes most viral variants, some harbor deletions that remodel this site and abrogate binding. Together, this work highlights mechanistic and structural features of antibodies targeting different domains of SARS-CoV-2 S, as well as corresponding strategies of immune evasion. Notably, combining RBD and NTD mAbs limits viral evasion in vitro, suggesting a potential clinical approach. We likewise have explored host-alphavirus interactions. Alphaviruses are mosquito-borne members of the Togaviridae family, characterized structurally by an icosahedral arrangement of glycoproteins E1 (a class II fusion protein) and E2 on the viral surface. Alphaviruses are subcategorized based on geographic origin and clinical presentation. “Old World” alphaviruses (e.g., Chikungunya, CHIKV) primarily cause rheumatic disease, whereas “New World” alphaviruses (e.g., Eastern equine encephalitis virus, EEEV) cause neurologic disease. While a live-attenuated CHIKV vaccination was recently approved, it carries a minor risk of severe CHIKV-like reactions, and there are no approved tools to combat encephalitic alphaviruses or to treat any ongoing alphavirus infection. Here, we analyzed the human B cell response to a noninfectious virus-like particle (VLP) CHIKV vaccine candidate that recently concluded phase 3 trials. We found that the vaccine induces high titers of neutralizing antibodies, and we generated mAbs from three subjects for targeted evaluation. Some of these mAbs recognized multiple arthritogenic alphaviruses, and we examined two such mAbs, 506.A08 and 506.C01, by cryo-EM. In contrast to previously described broadly reactive mAbs, which bind laterally to the B domain of E2, 506.A08 and 506.C01 define a new, conserved epitope on the apex of the B domain. Our structural and mutagenesis analyses elucidate the basis for the cross-reactivity profile of each mAb, identifying critical polymorphisms mediating the escape of specific arthritogenic alphaviruses. Lastly, we have determined how EEEV engages the very low-density lipoprotein receptor (VLDLR), which features 8 LDLR class A (LA) domains. By resolving multiple cryo-EM structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we found unexpectedly that EEEV uses two sites (E1/E2 cleft, E2 A domain) to engage different LA domains interchangeably and simultaneously, and some strains feature an additional LA-binding site on the E2 B domain enabled by a single amino acid substitution. At all three sites, EEEV engages LA domains using an electrostatic approach similar to that observed also for unrelated viruses that use LA domain-containing lipoprotein receptors, suggesting convergence upon a shared binding strategy. Ultimately, we defined the principal mode of VLDLR engagement across EEEV strains, informing the design of a minimal VLDLR decoy that protects mice from lethal challenge. In summary, our antibody studies have expanded our understanding of immune recognition and evasion of important human pathogens, and our receptor studies demonstrate how the notion of a sole “receptor-binding domain” may be oversimplified. Furthermore, we highlight a mode of receptor engagement shared by unrelated viruses. Together, these studies have deepened our understanding of viral pathogenesis and may inform therapeutic development for coronaviruses, alphaviruses, and potentially other viruses that use lipoprotein receptors for entry.

Committee Chair

Daved Fremont

Committee Members

Michael Diamond

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Immunology)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

3-13-2026

Language

English (en)

Author's ORCID

https://orcid.org/0000-0002-1724-8120

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