Date of Award


Degree Type


Degree Name

Master of Science (MS)


Microbiology and Immunology


Two coronaviruses (CoVs)—severe acute respiratory syndrome (SARS) virus and Middle East respiratory syndrome (MERS) virus—have emerged in the 21st century from animal reservoirs into the human population, each causing an epidemic associated with significant disease and mortality. CoV epidemics are currently only controllable by rigorous public health measures; no targeted therapeutics or vaccines exist to treat or prevent any human CoV infection. One method of generating attenuated CoV strains to be studied as vaccine candidates involves specifically disrupting CoV-encoded interferon (IFN) antagonists, thereby rendering the virus vulnerable to host innate antiviral immunity. Deubiquitinating (DUB) activity encoded within CoV nonstructural protein (nsp) 3 and endoribonuclease (EndoU) activity encoded within nsp15 are both reported to suppress IFN-mediated antiviral immunity during infection. Using murine hepatitis virus (MHV) as a model CoV, we generated viruses that encode enzymatically-deficient forms of these proteins and have shown that EndoU-mutant- and DUB-mutant-MHV elicit significantly increased type I IFN responses relative to the parental wild type (WT) strain. However, despite similar patterns of IFN induction by both mutant viruses, we previously found that only the EndoU-mut virus is attenuated and does not cause detectable disease in mice, whereas DUB-mut-MHV is not attenuated in vivo and causes disease similar to WT-MHV. The purpose of this project was to investigate the host transcriptional response to infection with EndoU-mut-, DUB-mut-, and WT-MHV in primary murine macrophages using RNA-sequencing (RNA-seq) technology to examine the cellular dynamics that underlie the remarkably distinct phenotypes of EndoU-mut- and DUB-mut-MHV infections in mice.

The results of our RNA-seq experiments and subsequent bioinformatic analyses demonstrate that WT-MHV infection led to profound transcriptional dysregulation of thousands of host genes, many of which encode proteins that are involved in inflammation, antiviral defense, signaling pathways, and transcription regulation. Similarly, DUB-mut-MHV elicited a statistically indistinguishable transcriptional response of all but a select few genes relative to WT-MHV. In stark contrast, EndoU-mut-MHV-infected cells exhibited markedly diminished transcription of the vast majority of genes that were induced by DUB-mut- and WT-MHV, leading to a profoundly distinct transcriptional response overall. Both EndoU-mut- and DUB-mut-MHV induced significantly higher expression of type I IFN isoforms relative to the WT virus, with the EndoU-mut strain prompting a dramatically higher IFN response than even the DUB-mut virus. Together, the results of this work suggest that the induction of IFN alone is not a sufficient marker for mutant CoV attenuation or of widespread dysregulation of host gene expression in the context of studying interferon antagonist-deficient coronavirus strains; rather, the magnitude and timing of IFN expression are critical. We conclude that there is a threshold of interferon expression that must be crossed before a host macrophage mounts a differential response to an IFN antagonist-deficient coronavirus that is capable of limiting the infection. Furthermore, we propose that MHV encodes a hierarchy of IFN antagonists that suppress and/or evade the host immune response in different ways and to different degrees. The results of this project advance what is known about how coronavirus-encoded interferon antagonists fine-tune the host response to viral infection and we hope that this work will guide future studies involving interferon antagonist-deficient coronaviruses being evaluated as vaccine candidates.

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