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Enveloped viruses are the root of many issues in human society, with infections driving disease, stigma, financial burden, persistent sequelae, and death. Understanding the mechanisms by which viruses initiate human disease is critical to designing effective tools with which we can counter negative outcomes or prevent infection in the first place. In this work we describe three sets of novel observations regarding two major human pathogens: SARS-CoV-2 and Ebola virus (EBOV)., Chapter II details a previously undescribed mechanism of SARS-CoV-2 entry that relies on phosphatidylserine (PS) receptors and PS in the viral envelope. We show that 1) this class of host receptors serves to bind to viral PS and facilitate virion attachment and internalization, 2) AXL is the primary PS receptor enhancing SARS-CoV-2 entry in the absence of TMPRSS2, and 3) preventing AXL internalization therapeutically reduces infection, inhibits replication, and significantly lowers the amount of infectious virus produced in human lung cells. The implications of these data are manifold, from identifying an undescribed cofactor for SARS-CoV-2 infection with the potential to expand the range of infectable cells and the conditions under which such entry is possible, to the realization of a potential therapy for COVID-19 that has already shown some promise in clinical settings. The observations in this chapter serve to expand the number of pathogens that utilize PS receptors to enhance entry to include three betacoronaviruses and imply that future pandemic betacoronaviruses may be similarly susceptible to PS receptor-based inhibition of entry., Chapter III centers around the novel observation that EBOV glycoprotein (GP) is sufficient to allow infection of mammalian skin. Previously supported only by post-hoc epidemiological studies, our data conclusively demonstrate that 1) EBOV GP can enable infection of keratinocytes in-vitro and in the context of the full epidermis, 2) EBOV GP is sufficient to penetrate the dermoepidermal junction and subsequently infect cells in the dermis, and 3) decornification and/or abrasion of the epidermis lower the barrier to EBOV GP-mediated infection. These data have major impacts on our understand of the readiness with which EBOV can transmit from person to person and to the potential avenues by which infection may be established. We thus provide insights for a route of infection that was previously uncharacterized with clear repercussions for public health and epidemiology for this highly lethal pathogen., Chapter IV delineates the tropism of the EBOV GP with specific examination of dendritic cells (DCs). Long believed to be an indispensable cell type for EBOV replication and dissemination, we describe the first subset-level evidence of splenic DC infection in an EBOV GP-dependent manner. We show that 1) EBOV GP is sufficient to infect cDCs but does not confer an enhanced tropism for splenic conventional DCs (cDCs) relative to the broad tropism of Vesicular stomatitis virus glycoprotein (VSV G), 2) the presence of EBOV GP specifically elicits an accumulation of monocyte-origin DCs (moDC) in the murine spleen, and 3) this population of moDC is highly permissive to recombinant VSV-EBOV GP (rVSV/EBOV) infection, supporting high levels of viral replication virion production, and the depletion of these cells during infection significantly improves all disease and mortality readouts. These results show that EBOV infection of cDCs does not exceed the typical rates for broadly tropic viruses, and thus the importance of cDCs as early targets of infection may have been overestimated. We also demonstrate a novel role for moDC during EBOV GP-mediate infection, suggesting that this population of elicited antigen presenting cells (APCs) may play a pathogenic role during bona-fide EBOV infection and represent an interesting target for therapeutic abatement., Altogether we present three complete sets of observations that lay groundwork for deployment of therapeutic and/or epidemiological modalities. By leveraging these observations we may be able to: develop PS receptor-targeting therapies to be better equipped should the next pandemic pathogen utilize these receptors during entry as EBOV and SARS-CoV-2 have; update public health messaging during EBOV outbreaks regarding the potential for skin-skin transmission and reduce the likelihood with which the virus can be transmitted; critically examine the role of cDC and moDC during EBOV infection, viewing them not as early footholds for the virus, but rather as elicited virus factories that should be targeted on a subset-specific level to reduce pathology.

Currently circulating SARS-CoV-2 variants acquired convergent mutations at receptor-binding domain (RBD) hot spots. Their impact on viral infection, transmission, and efficacy of vaccines and therapeutics remains poorly understood. Here, we demonstrate that recently emerged BQ.1.1. and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1 and XBB.1 RBDs bound to human ACE2 and S309 Fab (sotrovimab parent) explain the altered ACE2 recognition and preserved antibody binding through conformational selection. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1, the variant displaying the greatest loss of neutralization. Moreover, in several donors vaccine-elicited plasma antibodies cross-react with and trigger effector functions against Omicron variants despite reduced neutralizing activity. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring persistent immune imprinting. Our findings suggest that this previously overlooked class of cross-reactive antibodies, exemplified by S309, may contribute to protection against disease caused by emerging variants through elicitation of effector functions.

Phosphatidylserine (PS) receptors are PS binding proteins that mediate uptake of apoptotic bodies. Many enveloped viruses utilize this PS/PS receptor mechanism to adhere to and internalize into the endosomal compartment of cells and this is termed apoptotic mimicry. For viruses that have a mechanism(s) of endosomal escape, apoptotic mimicry is a productive route of virus entry. We evaluated if PS receptors serve as cell surface receptors for SARS-CoV-2 and found that the PS receptors, AXL, TIM-1 and TIM-4, facilitated virus infection when low concentrations of the SARS-CoV-2 cognate receptor, ACE2, was present. Consistent with the established mechanism of PS receptor utilization by other viruses, PS liposomes competed with SARS-CoV-2 for binding and entry. We demonstrated that this PS receptor enhances SARS-CoV-2 binding to and infection of an array of human lung cell lines and is an under-appreciated but potentially important host factor facilitating SARS-CoV-2 entry.