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Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV replication.

Pan J, Peng X, Gao Y, Li Z, Lu X, Chen Y, Ishaq M, Liu D, Dediego ML, Enjuanes L, Guo D - PLoS ONE (2008)

Bottom Line: The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses.Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription.Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology and Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.

ABSTRACT
Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

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The effect of additional N protein on viral genome replication and transcription.(A) Kinetics of luciferase activity of the reporter replicon. 2×105 BHK21 cells were transfected with pRL-TK plasmid (0.1 µg) and pBAC-Rep-SCV-luc/neo (0.4 µg). After transfection, the cells were collected for luciferase assays at different time points (6 h to 72 h). (B) Reporter gene activity in presence or absence of additional N protein provided in trans. 2×105 BHK21 cells were transfected with pRL-TK plasmid (0.05 µg), pBAC-Rep-SCV-luc/neo (0.25 µg) and pcDNA3.0-N (0.2 µg) or pcDNA3.0 (0.2 µg). After transfection, the cells were harvested for luciferase assays at different time points (6 h to 38 h). (C) The ratios of luciferase activities of Rep-SCV-luc/neo in presence of N protein related to that in absence of N protein at different time points (6 h to 38 h). Error bars represent standard deviations of the mean of three experiments.
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pone-0003299-g005: The effect of additional N protein on viral genome replication and transcription.(A) Kinetics of luciferase activity of the reporter replicon. 2×105 BHK21 cells were transfected with pRL-TK plasmid (0.1 µg) and pBAC-Rep-SCV-luc/neo (0.4 µg). After transfection, the cells were collected for luciferase assays at different time points (6 h to 72 h). (B) Reporter gene activity in presence or absence of additional N protein provided in trans. 2×105 BHK21 cells were transfected with pRL-TK plasmid (0.05 µg), pBAC-Rep-SCV-luc/neo (0.25 µg) and pcDNA3.0-N (0.2 µg) or pcDNA3.0 (0.2 µg). After transfection, the cells were harvested for luciferase assays at different time points (6 h to 38 h). (C) The ratios of luciferase activities of Rep-SCV-luc/neo in presence of N protein related to that in absence of N protein at different time points (6 h to 38 h). Error bars represent standard deviations of the mean of three experiments.

Mentions: To make further analysis on the enhancement effect of N protein on the viral replication and transcription, we examined the dynamic changes of the luciferase activity expressed from the reporter replicon Rep-SCV-luc/neo. When the replicon plasmid pBAC-Rep-SCV-luc/neo was transfected alone, the luciferase activity could be detected 13 h post transfection (more than 10 times compared with background levels) while the highest value of activity was reached around 32 h post transfection (Fig. 5A). The luciferase activity became stable 72 h post transfection and the activity level was as low as that of 14 h post transfection (Fig. 5A).


Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV replication.

Pan J, Peng X, Gao Y, Li Z, Lu X, Chen Y, Ishaq M, Liu D, Dediego ML, Enjuanes L, Guo D - PLoS ONE (2008)

The effect of additional N protein on viral genome replication and transcription.(A) Kinetics of luciferase activity of the reporter replicon. 2×105 BHK21 cells were transfected with pRL-TK plasmid (0.1 µg) and pBAC-Rep-SCV-luc/neo (0.4 µg). After transfection, the cells were collected for luciferase assays at different time points (6 h to 72 h). (B) Reporter gene activity in presence or absence of additional N protein provided in trans. 2×105 BHK21 cells were transfected with pRL-TK plasmid (0.05 µg), pBAC-Rep-SCV-luc/neo (0.25 µg) and pcDNA3.0-N (0.2 µg) or pcDNA3.0 (0.2 µg). After transfection, the cells were harvested for luciferase assays at different time points (6 h to 38 h). (C) The ratios of luciferase activities of Rep-SCV-luc/neo in presence of N protein related to that in absence of N protein at different time points (6 h to 38 h). Error bars represent standard deviations of the mean of three experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553179&req=5

pone-0003299-g005: The effect of additional N protein on viral genome replication and transcription.(A) Kinetics of luciferase activity of the reporter replicon. 2×105 BHK21 cells were transfected with pRL-TK plasmid (0.1 µg) and pBAC-Rep-SCV-luc/neo (0.4 µg). After transfection, the cells were collected for luciferase assays at different time points (6 h to 72 h). (B) Reporter gene activity in presence or absence of additional N protein provided in trans. 2×105 BHK21 cells were transfected with pRL-TK plasmid (0.05 µg), pBAC-Rep-SCV-luc/neo (0.25 µg) and pcDNA3.0-N (0.2 µg) or pcDNA3.0 (0.2 µg). After transfection, the cells were harvested for luciferase assays at different time points (6 h to 38 h). (C) The ratios of luciferase activities of Rep-SCV-luc/neo in presence of N protein related to that in absence of N protein at different time points (6 h to 38 h). Error bars represent standard deviations of the mean of three experiments.
Mentions: To make further analysis on the enhancement effect of N protein on the viral replication and transcription, we examined the dynamic changes of the luciferase activity expressed from the reporter replicon Rep-SCV-luc/neo. When the replicon plasmid pBAC-Rep-SCV-luc/neo was transfected alone, the luciferase activity could be detected 13 h post transfection (more than 10 times compared with background levels) while the highest value of activity was reached around 32 h post transfection (Fig. 5A). The luciferase activity became stable 72 h post transfection and the activity level was as low as that of 14 h post transfection (Fig. 5A).

Bottom Line: The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses.Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription.Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology and Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.

ABSTRACT
Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

Show MeSH
Related in: MedlinePlus