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postgraduate thesis: Role of double-stranded RNA-binding protein PACT in MDA5-mediated antiviral innate immune response

TitleRole of double-stranded RNA-binding protein PACT in MDA5-mediated antiviral innate immune response
Authors
Issue Date2014
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Lui, P. [呂柏賢]. (2014). Role of double-stranded RNA-binding protein PACT in MDA5-mediated antiviral innate immune response. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5387955
AbstractImmunity is an evolutionary conserved ability to protect organisms from infection by pathogens that co-exist in the same ecosystem. In mammals, the immune system is well developed and divided into innate and adaptive immune responses which are initiated and programmed to be biphasic. The innate immunity depends on the use of germ-line encoded machinery which can give a swift response to infection and thus is regarded as the first-line defense. During viral infection, type-I interferon (IFN) system is activated to establish local antiviral state and restrict virus spread within the host. This depends greatly on the initiation step which involves the detection of viral pathogen-associated molecular patterns (PAMPs) by the host pattern-recognition receptors (PRRs). Among numerous host PRRs which sense different viral PAMPs ranging from their surface proteins to their genome, RIG-I-like receptors (RLRs), comprising of three members RIGHI, MDA5 and LGP2, are a family of cytosolic helicases which are specialized in the detection of double-stranded RNA (dsRNA) found in the life cycle of some viruses inside the host cell. Although dsRNA is the prototypic agonist for RLRs, other cellular proteins have been demonstrated to be essential activators for them. Recently, our group has identified a dsRNA-binding protein PACT as an activator of RIG-I. However, in view of many other protein activators that do not cross-react with alternative RLRs, whether PACT also activates MDA5 is not known. In this study, I have shown that PACT is a cellular activator of MDA5. Briefly, ectopic expression of PACT activated IFNβ promoter activity, specifically through IRF3-arm but not NF-κB-arm of the signaling pathway, and enhanced MDA5-mediated response against dsRNA analogue polyinosinic:polycytidylic acid (polyI:C). This was supported by loss-of-function experiments in which PACT-deficient cells showed impaired IRF3-dependent IFN gene expression but not NF-κB-dependent cytokine gene expression in response to polyI:C induction, and the defects were reversed with the reconstitution of PACT into deficient cells. In biochemical assays, it was observed that polyI:C recruited PACT and MDA5, and PACT intrinsically associated with MDA5 and stimulated its oligomerization likely independent of dsRNA ligand. Dissecting the physiological role of PACT in antiviral immune response revealed that deficiency in PACT abolished the ability to induce IFN and cytokine gene expression in response to MDA5-dependent encephalomyocarditis virus (EMCV) infection. Virus replication was also favored in PACT-deficient cells during the course of infection. Taken together, I have shown an antiviral role of PACT in activating MDA5-mediated antiviral response which results in a more favorable outcome against virus infection. The knowledge derived from this study leads to a better understanding of the virus-host arms race and logical design of immunostimulatory pharmaceutical agents in the future.
DegreeDoctor of Philosophy
SubjectImmune response
RNA-protein interactions
Dept/ProgramBiochemistry
Persistent Identifierhttp://hdl.handle.net/10722/222199

 

DC FieldValueLanguage
dc.contributor.authorLui, Pak-yin-
dc.contributor.author呂柏賢-
dc.date.accessioned2016-01-02T23:34:50Z-
dc.date.available2016-01-02T23:34:50Z-
dc.date.issued2014-
dc.identifier.citationLui, P. [呂柏賢]. (2014). Role of double-stranded RNA-binding protein PACT in MDA5-mediated antiviral innate immune response. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5387955-
dc.identifier.urihttp://hdl.handle.net/10722/222199-
dc.description.abstractImmunity is an evolutionary conserved ability to protect organisms from infection by pathogens that co-exist in the same ecosystem. In mammals, the immune system is well developed and divided into innate and adaptive immune responses which are initiated and programmed to be biphasic. The innate immunity depends on the use of germ-line encoded machinery which can give a swift response to infection and thus is regarded as the first-line defense. During viral infection, type-I interferon (IFN) system is activated to establish local antiviral state and restrict virus spread within the host. This depends greatly on the initiation step which involves the detection of viral pathogen-associated molecular patterns (PAMPs) by the host pattern-recognition receptors (PRRs). Among numerous host PRRs which sense different viral PAMPs ranging from their surface proteins to their genome, RIG-I-like receptors (RLRs), comprising of three members RIGHI, MDA5 and LGP2, are a family of cytosolic helicases which are specialized in the detection of double-stranded RNA (dsRNA) found in the life cycle of some viruses inside the host cell. Although dsRNA is the prototypic agonist for RLRs, other cellular proteins have been demonstrated to be essential activators for them. Recently, our group has identified a dsRNA-binding protein PACT as an activator of RIG-I. However, in view of many other protein activators that do not cross-react with alternative RLRs, whether PACT also activates MDA5 is not known. In this study, I have shown that PACT is a cellular activator of MDA5. Briefly, ectopic expression of PACT activated IFNβ promoter activity, specifically through IRF3-arm but not NF-κB-arm of the signaling pathway, and enhanced MDA5-mediated response against dsRNA analogue polyinosinic:polycytidylic acid (polyI:C). This was supported by loss-of-function experiments in which PACT-deficient cells showed impaired IRF3-dependent IFN gene expression but not NF-κB-dependent cytokine gene expression in response to polyI:C induction, and the defects were reversed with the reconstitution of PACT into deficient cells. In biochemical assays, it was observed that polyI:C recruited PACT and MDA5, and PACT intrinsically associated with MDA5 and stimulated its oligomerization likely independent of dsRNA ligand. Dissecting the physiological role of PACT in antiviral immune response revealed that deficiency in PACT abolished the ability to induce IFN and cytokine gene expression in response to MDA5-dependent encephalomyocarditis virus (EMCV) infection. Virus replication was also favored in PACT-deficient cells during the course of infection. Taken together, I have shown an antiviral role of PACT in activating MDA5-mediated antiviral response which results in a more favorable outcome against virus infection. The knowledge derived from this study leads to a better understanding of the virus-host arms race and logical design of immunostimulatory pharmaceutical agents in the future.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subject.lcshImmune response-
dc.subject.lcshRNA-protein interactions-
dc.titleRole of double-stranded RNA-binding protein PACT in MDA5-mediated antiviral innate immune response-
dc.typePG_Thesis-
dc.identifier.hkulb5387955-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiochemistry-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b5387955-

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