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postgraduate thesis: Identification of Middle East respiratory syndrome coronavirus : host protein-protein interactions by immunoprecipitation and liquid chromatography : tandem mass spectrometry

TitleIdentification of Middle East respiratory syndrome coronavirus : host protein-protein interactions by immunoprecipitation and liquid chromatography : tandem mass spectrometry
Authors
Issue Date2017
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Mak, L. [麥朗峰]. (2017). Identification of Middle East respiratory syndrome coronavirus : host protein-protein interactions by immunoprecipitation and liquid chromatography : tandem mass spectrometry. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractHuman infection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has recently emerged at Middle-East and Korea, causing severe respiratory disease in human with high morbidity and mortality rate. MERS-CoV belongs to Betacoronavirus lineage D under the order of Nidovirales and Coronaviridae family. MERS-CoV is an enveloped virus comprised of positive sense RNA genome approximately 30kb long. Upon infection, the viral genome encodes five accessory proteins (Orf3, Orf4a, Orf4b, Orf5, Orf8b), four structural proteins (Spike, Envelope, Membrane, Nucleoprotein), and two polyproteins (pp1a, pp1ab) of which undergo proteolytic cleavage and generate 16 non-structural proteins (NSP1-16). Some of the viral proteins are known to be hardly expressed in cultured cell expression system. In this study, we optimized the protein expression of total 25 MERS-CoV proteins and established the immunoprecipitation protocol for subsequent protein identification using liquid chromatography (LC) - tandem mass spectrometry (MS/MS). It is well known that some of the SARS-CoV proteins are difficult to express in non-infected cells. We had encountered similar problems in the expression of MERS-CoV NSP6, NSP12, NSP15 and Spike proteins. We found that some of these hard-to-express viral proteins are heat unstable and forming aggregates. We modified the protocol of protein expression and now all 25 viral proteins of MERS-CoV are successfully expressed. In addition, we found that immunoprecipitation of the viral membrane protein required detergent to maintain the protein solubility. Finally, we established a streamlined protocol of immunoprecipitation and LC-MS/MS for the identification of virus-host interactome. As exemplified by the interactome of viral Orf4b, we found that Orf4b interacted with the host Casein Kinases and importin complexes. Casein Kinase has been shown to negatively regulate Type-I interferon through TBK1 and IKKε. Further study on the interaction of Orf4b to Casein Kinases will reveal a novel molecular mechanism of the inhibitory role of Orf4b on type-I interferon signaling. Taken together, this virus - host interactome study may identify important host dependency factors that may be valuable for future drug development against MERS-CoV infection.
DegreeMaster of Philosophy
SubjectMERS (Disease)
Coronaviruses
Protein-protein interactions
Dept/ProgramMicrobiology
Persistent Identifierhttp://hdl.handle.net/10722/255476

 

DC FieldValueLanguage
dc.contributor.authorMak, Long-fung-
dc.contributor.author麥朗峰-
dc.date.accessioned2018-07-05T07:43:41Z-
dc.date.available2018-07-05T07:43:41Z-
dc.date.issued2017-
dc.identifier.citationMak, L. [麥朗峰]. (2017). Identification of Middle East respiratory syndrome coronavirus : host protein-protein interactions by immunoprecipitation and liquid chromatography : tandem mass spectrometry. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/255476-
dc.description.abstractHuman infection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has recently emerged at Middle-East and Korea, causing severe respiratory disease in human with high morbidity and mortality rate. MERS-CoV belongs to Betacoronavirus lineage D under the order of Nidovirales and Coronaviridae family. MERS-CoV is an enveloped virus comprised of positive sense RNA genome approximately 30kb long. Upon infection, the viral genome encodes five accessory proteins (Orf3, Orf4a, Orf4b, Orf5, Orf8b), four structural proteins (Spike, Envelope, Membrane, Nucleoprotein), and two polyproteins (pp1a, pp1ab) of which undergo proteolytic cleavage and generate 16 non-structural proteins (NSP1-16). Some of the viral proteins are known to be hardly expressed in cultured cell expression system. In this study, we optimized the protein expression of total 25 MERS-CoV proteins and established the immunoprecipitation protocol for subsequent protein identification using liquid chromatography (LC) - tandem mass spectrometry (MS/MS). It is well known that some of the SARS-CoV proteins are difficult to express in non-infected cells. We had encountered similar problems in the expression of MERS-CoV NSP6, NSP12, NSP15 and Spike proteins. We found that some of these hard-to-express viral proteins are heat unstable and forming aggregates. We modified the protocol of protein expression and now all 25 viral proteins of MERS-CoV are successfully expressed. In addition, we found that immunoprecipitation of the viral membrane protein required detergent to maintain the protein solubility. Finally, we established a streamlined protocol of immunoprecipitation and LC-MS/MS for the identification of virus-host interactome. As exemplified by the interactome of viral Orf4b, we found that Orf4b interacted with the host Casein Kinases and importin complexes. Casein Kinase has been shown to negatively regulate Type-I interferon through TBK1 and IKKε. Further study on the interaction of Orf4b to Casein Kinases will reveal a novel molecular mechanism of the inhibitory role of Orf4b on type-I interferon signaling. Taken together, this virus - host interactome study may identify important host dependency factors that may be valuable for future drug development against MERS-CoV infection. -
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.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.subject.lcshMERS (Disease)-
dc.subject.lcshCoronaviruses-
dc.subject.lcshProtein-protein interactions-
dc.titleIdentification of Middle East respiratory syndrome coronavirus : host protein-protein interactions by immunoprecipitation and liquid chromatography : tandem mass spectrometry-
dc.typePG_Thesis-
dc.description.thesisnameMaster of Philosophy-
dc.description.thesislevelMaster-
dc.description.thesisdisciplineMicrobiology-
dc.description.naturepublished_or_final_version-
dc.date.hkucongregation2018-
dc.identifier.mmsid991044019381303414-

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