Dissertation
ADP-ribosylation during coronavirus infection: connections to viral protein modification, innate immunity, and the toxin response
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2021
DOI: 10.17077/etd.006058
Abstract
Coronaviruses (CoVs) are responsible for several diseases of economic and medical significance, and understanding the host response to CoV infection is key to developing antiviral strategies. One of these processes, ADP-ribosylation, is the post-translational addition of ADP-ribose (ADPr) to proteins to regulate their function. This addition is catalyzed by poly-ADPr polymerases (PARPs), which have been implicating in regulating antiviral host defenses. Because all CoVs encode a viral macrodomain that removes ADPr from proteins, we hypothesized that ADP-ribosylation was integral to CoV replication or to the host immune response.
During a screen for ADP-ribosylated proteins in cells infected with mouse hepatitis virus (MHV), a prototypical CoV, we identified a ~55 kD APD-ribosylated protein that was highly expressed. Co-immunoprecipitation verified the protein to be the MHV nucleocapsid (N) protein, which stabilizes the viral genome. This modification was conserved on N protein in the MHV virion and in cells infected with CoVs of other lineages. Interestingly, N protein produced by Venezuelan equine encephalitis virus replicon particles but not by transfected plasmid was modified, implying that this modification only occurs during infection conditions. In summary, the CoV N protein is ADP-ribosylated, suggesting a possible point of regulation between viral replication and host ADP-ribosylation.
Because the CoV macrodomain counters host PARPs, we next investigated how deficiency of the macrodomain affects infection. Recapitulating previous results in mice, we found that bone marrow-derived macrophages (BMDMs) infected with MHV encoding a catalytically inactive macrodomain showed reduced virus replication and increased interferon type 1 (IFN-I) production. Inhibition of global ADP-ribosylation attenuated these differences. Screening of cellular PARP expression identified several PARPs highly induced by infection. Individual knockdown of these PARPs showed that PARP12 and PARP14 were responsible for restricting MHV replication in the absence of macrodomain activity. Furthermore, we discovered that PARP14 facilitates IFN-I production in MHV-infected BMDMs and in cell lines stimulated with poly(I:C). Taken together, we identified two antiviral proteins that are countered by the activity of the MHV macrodomain.
In the PARP expression screen described above, we found that TiPARP was upregulated in MHV infection of BMDMs even in the absence of IFN-I signaling. TiPARP is also downstream of activation of the aryl hydrocarbon receptor (AhR), a cytosolic toxin receptor and transcription factor. Other proteins known to be downstream of AhR were upregulated during MHV infection, and these increases were attenuated with chemical inhibition of AhR, suggesting that AhR was activated during CoV infection. TiPARP expression was attenuated by AhR inhibition in IFN-I-deficient cells but not in wild-type cells, indicating that AhR and IFN-I can both upregulate TiPARP in a compensatory manner. Interestingly, while AhR ligand production is known to be catalyzed by indoleamine 2,3-dioxygenase (IDO1), loss of IDO1 did not affect transcription of proteins downstream of AhR. Finally, inhibition or activation of AhR affected transcription of cytokines including TNF, IL-1, and IL-10. These results demonstrate that activation of the AhR receptor modulates the immune response during MHV infection.
Taken as a whole, our results identify distinct proteins and interactions that link cellular ADP-ribosylation, the toxin response, and host immunity to CoV infection. Future projects will further characterize these pathways and will investigate their antiviral therapeutic potential.
Details
- Title: Subtitle
- ADP-ribosylation during coronavirus infection: connections to viral protein modification, innate immunity, and the toxin response
- Creators
- Matthew Ellis Grunewald
- Contributors
- Stanley Perlman (Advisor)Jon Houtman (Committee Member)Aloysius Klingelhutz (Committee Member)Wendy Maury (Committee Member)Richard Roller (Committee Member)Todd Washington (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Microbiology
- Date degree season
- Spring 2021
- DOI
- 10.17077/etd.006058
- Publisher
- University of Iowa
- Number of pages
- xiv, 201 pages
- Copyright
- Copyright 2019 Matthew Ellis Grunewald
- Comment
- This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (pages 157-182).
- Public Abstract (ETD)
Coronaviruses (CoVs) are a group of viruses that cause important diseases in humans and agriculture. Our goal is to understand how CoVs cause infection in order to eventually create new strategies to fight CoV infection. We found that ADP-ribosylation, a cellular process that adds a molecule called ADP-ribose (ADPr) to a protein to change how the protein functions, was important to CoV infection. First, we discovered that a CoV protein that helps to stabilize the virus was modified with ADPr in infected cells and in the virus itself. Though it is unclear how this modification affects the virus protein, it only occurs during infection and therefore may be vital to the virus itself. Second, we found that ADP-ribosylation in infected cells has a generally antiviral effect on CoVs. Though several proteins that add ADPr to other proteins (called PARPs) are induced by CoV infection, two specific PARPs are responsible for limiting the amount of virus produced. One of these PARP also helps to produce interferons, which are vital to help fight infection in humans. This could potentially be utilized to help infected cells fight CoV infection. Finally, we found that one PARP in infected cells can be regulated by a cellular receptor that is responsible for aiding in clearance of harmful toxins. During CoV infection, this receptor also regulates several proteins including ones that help to fight infection. This receptor could also be a potential target in future antiviral strategies.
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984097369502771
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