Limits...
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.

Show MeSH

Related in: MedlinePlus

Structure and activity assay of the reporter replicon construct pBAC-Rep-SCV-luc/neo.(A) Schematic structure of the replicon. The coding sequence of luciferase-neomycin fusion under the control of M gene TRS was inserted into the basic replicon construct pBAC-SARS-CoV-REP between AscI and BamHI sites (For details, see the Materials and Methods). (B) Luciferase assays of the reporter replicons. 2×105 BHK21 cells were transfected with the three kinds of replicon plasmids (0.4 µg each), respectively, and pRL-TK plasmid (0.1 µg) as an internal control. The luciferase activity assays were performed 24 h post transfection. Error bars represent standard deviations of the mean of three experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2553179&req=5

pone-0003299-g003: Structure and activity assay of the reporter replicon construct pBAC-Rep-SCV-luc/neo.(A) Schematic structure of the replicon. The coding sequence of luciferase-neomycin fusion under the control of M gene TRS was inserted into the basic replicon construct pBAC-SARS-CoV-REP between AscI and BamHI sites (For details, see the Materials and Methods). (B) Luciferase assays of the reporter replicons. 2×105 BHK21 cells were transfected with the three kinds of replicon plasmids (0.4 µg each), respectively, and pRL-TK plasmid (0.1 µg) as an internal control. The luciferase activity assays were performed 24 h post transfection. Error bars represent standard deviations of the mean of three experiments.

Mentions: Although a large number of protein-protein interactions were detected for SARS-CoV in virtue of the large-scale screening analysis in mammalian two-hybrid system, the roles of these interactions in the viral replication and transcription were still not clarified. To obtain more clues to the general roles of individual proteins, we constructed a SARS-CoV replicon (Rep-SCV-luc/neo) that expresses the firefly luciferase gene, a sensitive reporter, under the control of M gene transcription regulatory sequence (TRS) as described in the Materials and Methods section (Fig. 3A). The effects of individual proteins or protein domains provided in trans on the replication/transcription of the SARS-CoV replicon were evaluated. Any effect of the protein provided in trans on the luciferase expression levels could in principle be due either to changes in the extent of the replication, the transcription, or a combination of both.


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)

Structure and activity assay of the reporter replicon construct pBAC-Rep-SCV-luc/neo.(A) Schematic structure of the replicon. The coding sequence of luciferase-neomycin fusion under the control of M gene TRS was inserted into the basic replicon construct pBAC-SARS-CoV-REP between AscI and BamHI sites (For details, see the Materials and Methods). (B) Luciferase assays of the reporter replicons. 2×105 BHK21 cells were transfected with the three kinds of replicon plasmids (0.4 µg each), respectively, and pRL-TK plasmid (0.1 µg) as an internal control. The luciferase activity assays were performed 24 h post transfection. 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-g003: Structure and activity assay of the reporter replicon construct pBAC-Rep-SCV-luc/neo.(A) Schematic structure of the replicon. The coding sequence of luciferase-neomycin fusion under the control of M gene TRS was inserted into the basic replicon construct pBAC-SARS-CoV-REP between AscI and BamHI sites (For details, see the Materials and Methods). (B) Luciferase assays of the reporter replicons. 2×105 BHK21 cells were transfected with the three kinds of replicon plasmids (0.4 µg each), respectively, and pRL-TK plasmid (0.1 µg) as an internal control. The luciferase activity assays were performed 24 h post transfection. Error bars represent standard deviations of the mean of three experiments.
Mentions: Although a large number of protein-protein interactions were detected for SARS-CoV in virtue of the large-scale screening analysis in mammalian two-hybrid system, the roles of these interactions in the viral replication and transcription were still not clarified. To obtain more clues to the general roles of individual proteins, we constructed a SARS-CoV replicon (Rep-SCV-luc/neo) that expresses the firefly luciferase gene, a sensitive reporter, under the control of M gene transcription regulatory sequence (TRS) as described in the Materials and Methods section (Fig. 3A). The effects of individual proteins or protein domains provided in trans on the replication/transcription of the SARS-CoV replicon were evaluated. Any effect of the protein provided in trans on the luciferase expression levels could in principle be due either to changes in the extent of the replication, the transcription, or a combination of both.

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