Expression and membrane integration of SARS-CoV M protein

Chieh, Hsin; Fang, Chiu Ping; Hsieh, Yu‐Chia; Chen, Shih‐Chi; Li, Hui Chun; Lo, Shih-Yen

doi:10.1007/s11373-008-9235-1

Cited by 18 publications

(25 citation statements)

References 31 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%

“…Microsomes were added to study glycosylation. To stop the translation, CaCl 2 was added to the reaction mixture at a final concentration of 5 mM [12].…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…SARS-CoV M protein could not be detected with SDS-PAGE after regular boiling treatment [22]. Therefore, non-heated treatments were used in antigen preparations (sample buffer containing 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol; no boiling) to detect the expression of SARS-CoV M protein [12]. After electrophoresis, the SDS-PAGE gel was transferred to a PVDF (polyvinylidene difluoride) membrane (Pall Corporation, USA).…”

Section: Western Blotting Analysismentioning

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%

“…Both SARS-CoV M and E proteins are integral membrane proteins, though neither of them contains a conventional signal peptide [10,11]. 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].…”

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confidence: 91%

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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%

“…Microsomes were added to study glycosylation. To stop the translation, CaCl 2 was added to the reaction mixture at a final concentration of 5 mM [12].…”

Section: Plasmid Constructionmentioning

confidence: 99%

Section: Western Blotting Analysismentioning

confidence: 99%

Section: Western Blotting Analysismentioning

confidence: 99%

mentioning

confidence: 91%

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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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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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Influenza A virus upregulates PRPF8 gene expression to increase virus production

Yang

Shiu

et al. 2017

Arch Virol

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A ddRT-PCR analysis was performed to detect cellular genes that are differentially expressed after influenza A virus (H1N1) infection of A549 cells. After ddRT-PCR, eight DNA fragments were identified. PRPF8, one of the cellular genes that were upregulated after virus infection, was further analyzed since it has previously been identified as a cellular factor required for influenza virus replication. The upregulation of PRPF8 gene expression after viral infection was confirmed using real-time RT-PCR for mRNA detection and Western blot analysis for protein detection. Influenza A virus also upregulated the PRPF8 promoter in a reporter assay. In addition to H1N1, influenza A virus H3N2 and influenza B virus could also activate PRPF8 expression. Therefore, upregulation of PRPF8 expression might be important for the replication of different influenza viruses. Indeed, overexpression of PRPF8 gene enhanced virus production, while knockdown of expression of this gene reduced viral production significantly. To determine which viral protein could enhance PRPF8 gene expression, individual viral genes were cloned and expressed. Among the different viral proteins, expression of either the viral NS1 or PB1 gene could upregulate the PRPF8 expression. Our results from this study indicate that influenza A virus upregulates cellular PRPF8 gene expression through viral NS1 and PB1 proteins to increase virus production.

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Expression and membrane integration of SARS-CoV M protein

Cited by 18 publications

References 31 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

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

Influenza A virus upregulates PRPF8 gene expression to increase virus production

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