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postgraduate thesis: Role of deubiquitylases in influenza A virus infection and immunity
Title | Role of deubiquitylases in influenza A virus infection and immunity |
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Authors | |
Advisors | |
Issue Date | 2019 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Akhee, S. J.. (2019). Role of deubiquitylases in influenza A virus infection and immunity. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | Ubiquitylation is a cellular post-translational modification (PTM), routinely targeted by pathogens to deregulate and hijack host intracellular pathways. During Influenza A virus (IAV) infection, the host ubiquitylation machinery is significantly altered and exploited for RIG-I mediated viral sensing and immune response. To comprehensively characterize the ubiquitylation machinery hijacked by IAV in mammalian cells, we designed and employed a chemoenzymatic strategy to identify specific deubiquitylases (DUBs) that are induced upon IAV infections. Using a C-terminal vinyl methyl ester modified HA-tagged ubiquitin (HA-Ub-vme) combined with large-scale immunoprecipitation and mass spectrometry we identified DUBs that are upregulated in IAV infected cells.
Among others, OTUB1 was identified as a deubiquitylase that is upregulated upon IAV infection. Interferon β treatment alone could also induce OTUB1 expression in A549 cells, suggesting it is an IFN-I induced host factor. To functionally characterize OTUB1 in IAV infection, CRISPR/Cas9 genome editing was utilized to generate OTUB1-deleted A549 cells. OTUB1-deficient (OTUB1-/- ) cells produced significantly less viral progeny, which were rescued by introduction of OTUB1 in OTUB1-/- cells. Further investigation showed severely defective polymerase activity in OTUB1-/- cells compared to WT. Influenza A viral protein PB2 which is essential for viral polymerase activity interacted directly with OTUB1 as measured by co-immunoprecipitation and co-localization score. Lack of OTUB1 was shown to be associated with a higher K-48 linked polyubiquitylation of PB2, suggesting a vital role of OTUB1 in regulating polymerase activity through deubiquitylation of PB2.
Additionally, OTUB1-/- cells were defective in production of both IFN-α as well as pro-inflammatory cytokines compared to the WT when subjected to IAV infection. Further investigation of OTUB1-/- cells upon infection showed a significant redution in IRF and NFB activation, upstream of IFN and cytokines respectively. Through the use of proximity-based labeling, components of the RIG-I signaling pathway were found to be highly enriched in the OTUB1 interactome. Furthermore, OTUB1 was shown to directly interact with IAV viral protein NS1 known for its RIG-I antagonism and was shown to be degraded without proteasome inhibitor treatment upn IAV infection. Immunoprecipitated NS1 samples were shown to be enriched in K-48 and K-63 linked polyubiquitylation and formed complexes with TRAF3 and RIG-I, which have been previously reported as NS1 interactors. In OTUB1-/- cells, neither the ubiquitylated material, nor the RIG-I complex was detectable upon NS1 pull-down. The NS1-RIG-I interaction is known to be integral in the function of NS1 to limit IRF and NFB activation, eventually leading to dampened antiviral response. We find OTUB1 to be an essential determinant in the formation of NS1-RIG-I complex and mediation of the downstream antiviral responses.
Apart from innate immune responses associated with IAV, ubiquitylation also plays a significant role in the adaptive immune responses, such as T cell receptor (TCR) signaling. We identified Ubiquitin-specific peptidase (Usp) 12 as a positive regulator highly enriched T lymphocytes and a crucial component of TCR expression at the cell surface. These findings provided novel insights in fine-tuning host signaling cascades both in IAV infection and immunity, identifying new avenues for the development of potential therapeutic targets.
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Degree | Doctor of Philosophy |
Subject | Influenza A virus Proteinase Ubiquitin |
Dept/Program | Public Health |
Persistent Identifier | http://hdl.handle.net/10722/278458 |
DC Field | Value | Language |
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dc.contributor.advisor | Bruzzone, R | - |
dc.contributor.advisor | Sanyal, S | - |
dc.contributor.author | Akhee, Sabiha Jahan | - |
dc.date.accessioned | 2019-10-09T01:17:48Z | - |
dc.date.available | 2019-10-09T01:17:48Z | - |
dc.date.issued | 2019 | - |
dc.identifier.citation | Akhee, S. J.. (2019). Role of deubiquitylases in influenza A virus infection and immunity. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/278458 | - |
dc.description.abstract | Ubiquitylation is a cellular post-translational modification (PTM), routinely targeted by pathogens to deregulate and hijack host intracellular pathways. During Influenza A virus (IAV) infection, the host ubiquitylation machinery is significantly altered and exploited for RIG-I mediated viral sensing and immune response. To comprehensively characterize the ubiquitylation machinery hijacked by IAV in mammalian cells, we designed and employed a chemoenzymatic strategy to identify specific deubiquitylases (DUBs) that are induced upon IAV infections. Using a C-terminal vinyl methyl ester modified HA-tagged ubiquitin (HA-Ub-vme) combined with large-scale immunoprecipitation and mass spectrometry we identified DUBs that are upregulated in IAV infected cells. Among others, OTUB1 was identified as a deubiquitylase that is upregulated upon IAV infection. Interferon β treatment alone could also induce OTUB1 expression in A549 cells, suggesting it is an IFN-I induced host factor. To functionally characterize OTUB1 in IAV infection, CRISPR/Cas9 genome editing was utilized to generate OTUB1-deleted A549 cells. OTUB1-deficient (OTUB1-/- ) cells produced significantly less viral progeny, which were rescued by introduction of OTUB1 in OTUB1-/- cells. Further investigation showed severely defective polymerase activity in OTUB1-/- cells compared to WT. Influenza A viral protein PB2 which is essential for viral polymerase activity interacted directly with OTUB1 as measured by co-immunoprecipitation and co-localization score. Lack of OTUB1 was shown to be associated with a higher K-48 linked polyubiquitylation of PB2, suggesting a vital role of OTUB1 in regulating polymerase activity through deubiquitylation of PB2. Additionally, OTUB1-/- cells were defective in production of both IFN-α as well as pro-inflammatory cytokines compared to the WT when subjected to IAV infection. Further investigation of OTUB1-/- cells upon infection showed a significant redution in IRF and NFB activation, upstream of IFN and cytokines respectively. Through the use of proximity-based labeling, components of the RIG-I signaling pathway were found to be highly enriched in the OTUB1 interactome. Furthermore, OTUB1 was shown to directly interact with IAV viral protein NS1 known for its RIG-I antagonism and was shown to be degraded without proteasome inhibitor treatment upn IAV infection. Immunoprecipitated NS1 samples were shown to be enriched in K-48 and K-63 linked polyubiquitylation and formed complexes with TRAF3 and RIG-I, which have been previously reported as NS1 interactors. In OTUB1-/- cells, neither the ubiquitylated material, nor the RIG-I complex was detectable upon NS1 pull-down. The NS1-RIG-I interaction is known to be integral in the function of NS1 to limit IRF and NFB activation, eventually leading to dampened antiviral response. We find OTUB1 to be an essential determinant in the formation of NS1-RIG-I complex and mediation of the downstream antiviral responses. Apart from innate immune responses associated with IAV, ubiquitylation also plays a significant role in the adaptive immune responses, such as T cell receptor (TCR) signaling. We identified Ubiquitin-specific peptidase (Usp) 12 as a positive regulator highly enriched T lymphocytes and a crucial component of TCR expression at the cell surface. These findings provided novel insights in fine-tuning host signaling cascades both in IAV infection and immunity, identifying new avenues for the development of potential therapeutic targets. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | Influenza A virus | - |
dc.subject.lcsh | Proteinase | - |
dc.subject.lcsh | Ubiquitin | - |
dc.title | Role of deubiquitylases in influenza A virus infection and immunity | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Doctor of Philosophy | - |
dc.description.thesislevel | Doctoral | - |
dc.description.thesisdiscipline | Public Health | - |
dc.description.nature | published_or_final_version | - |
dc.identifier.doi | 10.5353/th_991044146573103414 | - |
dc.date.hkucongregation | 2019 | - |
dc.identifier.mmsid | 991044146573103414 | - |