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Interferon-Mediated Regulation of RNA Virus Infection


Fernbach, Sonja. Interferon-Mediated Regulation of RNA Virus Infection. 2021, University of Zurich, Faculty of Medicine.

Abstract

Influenza A viruses (IAVs) and coronaviruses (CoVs) are respiratory pathogens that cause seasonal infections in humans and occasional pandemics, as last seen by the 2009 H1N1 influenza virus (Swine flu) pandemic and the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Both viruses heavily rely on the host cell machinery for replication, highlighting the tight interplay between host and pathogens. While viruses exploit host functions for their own benefit, the host cell in turn triggers effective antiviral signaling pathways, such as the interferon (IFN) response. The IFN pathway is an important barrier to infections and part of the innate immune defense system. The immediate activation upon infection triggers a signaling cascade that leads to the induction of hundreds of IFN-stimulated genes (ISGs), some of which possess profound antiviral activities. Understanding the mechanisms and pathways involved in the interplay between virus and host is a prerequisite in the development of antiviral therapies and vaccines.
Previous studies reported that the expression of the cellular receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2) is elevated by the action of IFN, implicating a possible increased susceptibility for infections either as the body triggers IFN to defend itself or in the context of using IFNs as antiviral therapies, which has recently been started in clinical trials. Thus, elucidation of the IFN-inducibility of ACE2 was of great importance. Using a cell line model, it was observed that treatment with IFNβ increased ACE2 mRNA and surface protein expression, but to a much lesser extent than was found for canonical ISGs. Furthermore, IFNλ1 induced antiviral ISGs, but had limited impact on ACE2 mRNA levels and showed no effect on ACE2 cell surface levels. IFNγ marginally increased canonical ISGs, but clearly induced ACE2 surface expression, similar to IFNβ. Strikingly, all types of IFN limited SARS-CoV-2 infection in a dose-dependent manner, highlighting that the antiviral function of IFNs outweighs any potential promotion of SARS-CoV-2 replication by increased ACE2 expression. The data from this first part of the thesis underscore the potential of IFN, specifically IFNλ1, as possible candidates for antiviral therapies against SARS-CoV-2 without increasing susceptibility to infection.
While many proteins have been classified as ISGs over the years, most of them remain uncharacterized. As part of an RNA interference (RNAi) screen targeting 100 putative ISGs, several potential ISGs with previously undescribed contributions to IFN-mediated restriction of IAVs were identified. In the second part of this thesis, I focused on studying the Tre2/Bub2/Cdc16 (TBC) domain-containing protein RAB GTPase-activating protein 1 like (RABGAP1L) as a potent host factor restricting IAV. Surprisingly, its expression itself was not detectably induced by IFN, indicating that RABGAP1L is not a classical highly inducible ISG, while its antiviral activity was greatly potentiated by the action of IFN. RABGAP1L further restricted several RNA viruses known to enter host cells via endocytosis. Truncation mapping and site-directed mutagenesis revealed that a catalytically active TBC domain is required for the antiviral activity. Using proximity labeling proteomics to study the RABGAP1L interactome, we discovered its association with proteins involved in endosomal sorting, maturation and trafficking. In line with this, immunofluorescence-based assays uncovered that RABGAP1L expression disrupts endosomal functions and restricts early IAV entry. The work described in this second part of the thesis reports the discovery of a previously undescribed IAV restriction factor that limits early infection by disruption of the endosomal pathway.

Abstract

Influenza A viruses (IAVs) and coronaviruses (CoVs) are respiratory pathogens that cause seasonal infections in humans and occasional pandemics, as last seen by the 2009 H1N1 influenza virus (Swine flu) pandemic and the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Both viruses heavily rely on the host cell machinery for replication, highlighting the tight interplay between host and pathogens. While viruses exploit host functions for their own benefit, the host cell in turn triggers effective antiviral signaling pathways, such as the interferon (IFN) response. The IFN pathway is an important barrier to infections and part of the innate immune defense system. The immediate activation upon infection triggers a signaling cascade that leads to the induction of hundreds of IFN-stimulated genes (ISGs), some of which possess profound antiviral activities. Understanding the mechanisms and pathways involved in the interplay between virus and host is a prerequisite in the development of antiviral therapies and vaccines.
Previous studies reported that the expression of the cellular receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2) is elevated by the action of IFN, implicating a possible increased susceptibility for infections either as the body triggers IFN to defend itself or in the context of using IFNs as antiviral therapies, which has recently been started in clinical trials. Thus, elucidation of the IFN-inducibility of ACE2 was of great importance. Using a cell line model, it was observed that treatment with IFNβ increased ACE2 mRNA and surface protein expression, but to a much lesser extent than was found for canonical ISGs. Furthermore, IFNλ1 induced antiviral ISGs, but had limited impact on ACE2 mRNA levels and showed no effect on ACE2 cell surface levels. IFNγ marginally increased canonical ISGs, but clearly induced ACE2 surface expression, similar to IFNβ. Strikingly, all types of IFN limited SARS-CoV-2 infection in a dose-dependent manner, highlighting that the antiviral function of IFNs outweighs any potential promotion of SARS-CoV-2 replication by increased ACE2 expression. The data from this first part of the thesis underscore the potential of IFN, specifically IFNλ1, as possible candidates for antiviral therapies against SARS-CoV-2 without increasing susceptibility to infection.
While many proteins have been classified as ISGs over the years, most of them remain uncharacterized. As part of an RNA interference (RNAi) screen targeting 100 putative ISGs, several potential ISGs with previously undescribed contributions to IFN-mediated restriction of IAVs were identified. In the second part of this thesis, I focused on studying the Tre2/Bub2/Cdc16 (TBC) domain-containing protein RAB GTPase-activating protein 1 like (RABGAP1L) as a potent host factor restricting IAV. Surprisingly, its expression itself was not detectably induced by IFN, indicating that RABGAP1L is not a classical highly inducible ISG, while its antiviral activity was greatly potentiated by the action of IFN. RABGAP1L further restricted several RNA viruses known to enter host cells via endocytosis. Truncation mapping and site-directed mutagenesis revealed that a catalytically active TBC domain is required for the antiviral activity. Using proximity labeling proteomics to study the RABGAP1L interactome, we discovered its association with proteins involved in endosomal sorting, maturation and trafficking. In line with this, immunofluorescence-based assays uncovered that RABGAP1L expression disrupts endosomal functions and restricts early IAV entry. The work described in this second part of the thesis reports the discovery of a previously undescribed IAV restriction factor that limits early infection by disruption of the endosomal pathway.

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Additional indexing

Item Type:Dissertation (monographical)
Referees:Münz Christian, Thiel Volker, Hale Benjamin G
Communities & Collections:04 Faculty of Medicine > Institute of Medical Virology
UZH Dissertations
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2021
Deposited On:25 Jan 2022 08:49
Last Modified:25 Jan 2022 15:47
OA Status:Closed
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