"Host Immunity and Receptor Expression Determine RNA Virus Pathogenesis" by Madison Scott Strine

Date of Award

Spring 2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Immunobiology

First Advisor

Wilen, Craig

Abstract

Identifying host factors essential for viral infections has the potential to reveal novel mechanisms of viral pathogenesis and immune escape. Here, we use two mucosal positive-sense RNA viruses – murine norovirus (MNV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) – as probes to investigate mechanisms of viral pathogenesis at mucosal barrier surfaces. Among noroviruses, murine norovirus (MNV) is a model for human norovirus and a tractable system to elucidate fundamental mechanisms of viral tropism and immune evasion. The strain MNVCR6 infects rare chemosensory epithelial cells called tuft cells, which are dispensable for the canonical non-persistent strain MNVCW3. Herein, we investigate the determinants and consequences of tuft cell tropism on viral persistence and immune evasion. We show that tuft cells are required for enteric infection by diverse MNV strains and that serial infection of short-lived tuft cells rather than persistent infection of long-lived tuft cells mediates immune evasion. Upon extra-intestinal inoculation, MNVCR6 is controlled by adaptive immunity, suggesting that tuft cells serve as an immune privileged niche enabling chronic infection. We also show that non-persistent MNVCW3, thought to exclusively infect hematopoietic cells, can indeed infect tuft cells and establish chronic infection when STAT1 is deleted from tuft cells by restricting combinatorial signaling of type I and III interferon. We developed a novel in vitro enteroid model that supports MNV infection of tuft cells, and we unexpectedly found that MNVCR6 and MNVCW3 similarly infect tuft cells with equal susceptibility to type I, II, and III IFNs. These findings suggest tuft cell restriction of MNVCW3 is a consequence of immunomodulation by non-epithelial cells that signal via type I and III IFN on tuft cells in trans. Moreover, these data indicate that tropism for tuft cells is critical for MNV persistence in immunocompetent mice. Although MNVCR6 induces functional MNV-specific CD8+ T cells, these lymphocytes fail to clear viral infection, which we hypothesized could be attributed to tropism for tuft cells. To interrogate tuft cell interactions with CD8+ T cells, we adoptively transferred JEDI (Just EGFP Death Inducing) CD8+ T cells that detect EGFP200-208 presented on H2-Kd into tuft cell reporter mice (Gfi1b-GFP). Surprisingly, tuft cells at steady state can resist JEDI CD8+ T cell-mediated killing despite normal levels of surface MHC-I. In contrast, Lgr5+ intestinal stem cells and extraintestinal tuft cells are highly susceptible to clearance by JEDI CD8+ T cells. We further find that CD8+ T cells cannot clear nor prevent MNVCR6 infection in the colon, the site of viral persistence. These data support that colonic tuft cells offer a replicative niche for immune escape and chronic infection. Ultimately, we demonstrate that intestinal tuft cells resist CD8+ T cells independent of viral manipulation or exhaustion, suggesting a novel mechanism of CD8+ T cell dysfunction in chronic viral infection. For SARS-CoV-2 and other highly pathogenic human coronaviruses, we previously performed a genome-wide CRISPR/Cas9 screen to identify pro-viral host factors. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously unknown, DYRK1A encodes the Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the pro-viral activity of DYRK1A is conserved across species using cells of non-human primate or human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses. Collectively, we identify and mechanistically describe host factors that govern viral entry, cellular tropism, and immune evasion. As a whole, these findings highlight how tropism and receptor expression can dramatically alter viral pathogenesis at entry or post- entry stages. Together, these findings deepen our understanding of host-virus interactions and highlight novel mechanisms underlying viral persistence and viral susceptibility in the mucosa.

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