Interactions between M protein and other structural proteins of severe, acute respiratory syndrome-associated coronavirus

Hsieh, Yi-Ching; Li, Huichun; Chen, Shih‐Chi; Lo, Shih-Yen

doi:10.1007/s11373-008-9278-3

Cited by 34 publications

(55 citation statements)

References 38 publications

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“…The construction of the full-length membrane protein into the pcDNA3.1/V5-His A vector (Invitrogen, USA) or pcDNA3-myc vector (constructed in our laboratory) was described previously [12,13,20]. The construction of the plasmid expressing the full-length envelope protein fused with a myc tag (containing 10 amino acids of the myc epitope and six amino acids of junction sequences) at the N-terminus was also described previously [13].…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…The construction of the plasmid expressing the full-length envelope protein fused with a myc tag (containing 10 amino acids of the myc epitope and six amino acids of junction sequences) at the N-terminus was also described previously [13].…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Experiments were repeated three times. To quantitate the average percentage of co-localization, similar to our previous reports [12,13], Image J (NIH web) program was used. P values were calculated by t test.…”

Section: Western Blotting Analysismentioning

confidence: 99%

“…Multiple regions in the M protein are able to interact with E protein [13]. In order to determine which region/s of E protein is/are important for interaction with M protein, different regions of E protein were deleted individually (E11-76, ED11-33, ED37-59, E1-59).…”

Section: Both Of the Transmembrane Domains Of Sars-cov E Protein Are mentioning

confidence: 99%

“…Expression and membrane integration of SARS-CoV M protein was characterized previously [12]. The interacting domains of SARS-CoV M with other structural proteins (S, E, and NC proteins) were also reported previously [13]. The envelope protein of coronaviruses is a small membrane protein with multiple functions.…”

mentioning

confidence: 95%

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Expression and membrane integration of SARS-CoV E protein and its interaction with M protein

Chen

et al. 2009

Virus Genes

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The severe acute respiratory syndrome (SARS)-CoV E gene fragment was cloned and expressed as a recombinant protein fused with a myc tag at the N-terminus in vitro and in Vero E6 cells. Similar to other N-glycosylated proteins, the glycosylation of SARS-CoV E protein occurred co-translationally in the presence of microsomes. The SARS-CoV E protein is predicted to be a double-spanning membrane protein lacking a conventional signal peptide. Both of the transmembrane regions (a.a. 11-33 and 37-59) are predicted to be alpha-helices, which penetrate into membranes by themselves. As expected, these two transmembrane regions inserted a cytoplasmic protein into the endoplasmic reticulum membrane. Either of these two transmembrane domains co-localized with M protein. Both the transmembrane domains of E protein are required to interact with M protein, while either of the hydrophilic regions (a.a. 1-10 or 60-76) is dispensable as shown by co-immunoprecipitation assay. These results are important for the study of SARS-CoV assembly.

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Section: Plasmid Constructionmentioning

confidence: 99%

Section: Plasmid Constructionmentioning

confidence: 99%

Section: Western Blotting Analysismentioning

confidence: 99%

Section: Both Of the Transmembrane Domains Of Sars-cov E Protein Are mentioning

confidence: 99%

mentioning

confidence: 95%

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Expression and membrane integration of SARS-CoV E protein and its interaction with M protein

Chen

et al. 2009

Virus Genes

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Studying Coronavirus–Host Protein Interactions

Yang

Hung

et al. 2015

Coronaviruses

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To understand the molecular mechanisms of viral replication and pathogenesis, it is necessary to establish the virus-host protein interaction networks. The yeast two-hybrid system is a powerful proteomic approach to study protein-protein interactions. After the identification of specific cellular factors interacting with the target viral protein using the yeast two-hybrid screening system, co-immunoprecipitation and confocal microscopy analyses are often used to verify the virus-host protein interactions in cells. Identification of the cellular factors required for viral survival or eliminating virus infected cells could help scientists develop more effective antiviral drugs. Here we summarize a standard protocol used in our lab to study the coronavirus-host protein interactions, including yeast two-hybrid screening, co-immunoprecipitation, and immunofluorescence microscopy analyses.

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SARS-CoV nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity

Wei

Chen

et al. 2012

Arch Virol

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The pathogenesis of SARS-CoV remains largely unknown. To study the function of the SARS-CoV nucleocapsid protein, we have conducted a yeast two-hybrid screening experiment to identify cellular proteins that may interact with the SARS-CoV nucleocapsid protein. Pyruvate kinase (liver) was found to interact with SARS-CoV nucleocapsid protein in this experiment. The binding domains of these two proteins were also determined using the yeast two-hybrid system. The physical interaction between the SARS-CoV nucleocapsid and cellular pyruvate kinase (liver) proteins was further confirmed by GST pull-down assay, co-immunoprecipitation assay and confocal microscopy. Cellular pyruvate kinase activity in hepatoma cells was repressed by SARS-CoV nucleocapsid protein in either transiently transfected or stably transfected cells. PK deficiency in red blood cells is known to result in human hereditary non-spherocytic hemolytic anemia. It is reasonable to assume that an inhibition of PKL activity due to interaction with SARS-CoV N protein is likely to cause the death of the hepatocytes, which results in the elevation of serum alanine aminotransferase and liver dysfunction noted in most SARS patients. Thus, our results suggest that SARS-CoV could reduce pyruvate kinase activity via its nucleocapsid protein, and this may in turn cause disease.

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Interactions between M protein and other structural proteins of severe, acute respiratory syndrome-associated coronavirus

Cited by 34 publications

References 38 publications

Expression and membrane integration of SARS-CoV E protein and its interaction with M protein

Expression and membrane integration of SARS-CoV E protein and its interaction with M protein

Studying Coronavirus–Host Protein Interactions

SARS-CoV nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity

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