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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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Related in: MedlinePlus

Protein interactions of SARS-CoV detected by mammalian two-hybrid assays.(A) Interaction matrix of SARS-CoV proteins. The grey squares indicate the novel interactions detected in this work. The black squares represent the interactions which have also been reported previously, including nsp5–nsp5[19], [68], [69], nsp5–nsp7 and nsp8[9], nsp7–nsp7[21], [70], nsp7–nsp8[21], nsp7–nsp9[9], nsp8–9b[9], nsp10–nsp14 and nsp10–nsp16[10], nsp15–nsp15 [54], [56], [71], [72], nsp7-E and 7a-M[73], N-N[55], [57] and N-N[55], [57]. (B) A typical result for a positive interaction with the example of nsp10–nsp14. The combination of pM-53 and pVP16-T represents a positive control. (C) A typical interaction inhibition assay performed to confirm that the interaction was not resulted from self-activation. Error bars represent standard deviations from three independent experiments.
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pone-0003299-g001: Protein interactions of SARS-CoV detected by mammalian two-hybrid assays.(A) Interaction matrix of SARS-CoV proteins. The grey squares indicate the novel interactions detected in this work. The black squares represent the interactions which have also been reported previously, including nsp5–nsp5[19], [68], [69], nsp5–nsp7 and nsp8[9], nsp7–nsp7[21], [70], nsp7–nsp8[21], nsp7–nsp9[9], nsp8–9b[9], nsp10–nsp14 and nsp10–nsp16[10], nsp15–nsp15 [54], [56], [71], [72], nsp7-E and 7a-M[73], N-N[55], [57] and N-N[55], [57]. (B) A typical result for a positive interaction with the example of nsp10–nsp14. The combination of pM-53 and pVP16-T represents a positive control. (C) A typical interaction inhibition assay performed to confirm that the interaction was not resulted from self-activation. Error bars represent standard deviations from three independent experiments.

Mentions: In total, 1024 interaction combinations between all SARS-CoV proteins were examined in a pairwise matrix. As a result, 40 different interactions were detected using the mammalian two-hybrid assays (Fig. 1A). All the interactions shown (Fig. 1) were most likely specific for the viral protein domain of the fusion proteins as the activities of the reporter genes were reduced to background levels when one interacting partner in any of the combinations was replaced with non-relevant fusion protein (negative control) (data not shown). To further show the specificity of the interactions (Fig. 1), a quantitative assay for a typical positive interaction exemplified by nsp10–nsp14 and various controls were shown (Fig. 1B). To test the specificity of individual interactions, competitive assays were performed by co-expression of viral proteins using a different vector within the cells transformed with the bait and prey constructs. The co-expressed partner proteins interfered with the nsp10–nsp14 interaction, leading to reduced reporter activity (Fig. 1C), indicating that the same proteins with different fusion domains competed with each other and impaired the specific interactions needed for activation of the reporter genes.


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)

Protein interactions of SARS-CoV detected by mammalian two-hybrid assays.(A) Interaction matrix of SARS-CoV proteins. The grey squares indicate the novel interactions detected in this work. The black squares represent the interactions which have also been reported previously, including nsp5–nsp5[19], [68], [69], nsp5–nsp7 and nsp8[9], nsp7–nsp7[21], [70], nsp7–nsp8[21], nsp7–nsp9[9], nsp8–9b[9], nsp10–nsp14 and nsp10–nsp16[10], nsp15–nsp15 [54], [56], [71], [72], nsp7-E and 7a-M[73], N-N[55], [57] and N-N[55], [57]. (B) A typical result for a positive interaction with the example of nsp10–nsp14. The combination of pM-53 and pVP16-T represents a positive control. (C) A typical interaction inhibition assay performed to confirm that the interaction was not resulted from self-activation. Error bars represent standard deviations from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003299-g001: Protein interactions of SARS-CoV detected by mammalian two-hybrid assays.(A) Interaction matrix of SARS-CoV proteins. The grey squares indicate the novel interactions detected in this work. The black squares represent the interactions which have also been reported previously, including nsp5–nsp5[19], [68], [69], nsp5–nsp7 and nsp8[9], nsp7–nsp7[21], [70], nsp7–nsp8[21], nsp7–nsp9[9], nsp8–9b[9], nsp10–nsp14 and nsp10–nsp16[10], nsp15–nsp15 [54], [56], [71], [72], nsp7-E and 7a-M[73], N-N[55], [57] and N-N[55], [57]. (B) A typical result for a positive interaction with the example of nsp10–nsp14. The combination of pM-53 and pVP16-T represents a positive control. (C) A typical interaction inhibition assay performed to confirm that the interaction was not resulted from self-activation. Error bars represent standard deviations from three independent experiments.
Mentions: In total, 1024 interaction combinations between all SARS-CoV proteins were examined in a pairwise matrix. As a result, 40 different interactions were detected using the mammalian two-hybrid assays (Fig. 1A). All the interactions shown (Fig. 1) were most likely specific for the viral protein domain of the fusion proteins as the activities of the reporter genes were reduced to background levels when one interacting partner in any of the combinations was replaced with non-relevant fusion protein (negative control) (data not shown). To further show the specificity of the interactions (Fig. 1), a quantitative assay for a typical positive interaction exemplified by nsp10–nsp14 and various controls were shown (Fig. 1B). To test the specificity of individual interactions, competitive assays were performed by co-expression of viral proteins using a different vector within the cells transformed with the bait and prey constructs. The co-expressed partner proteins interfered with the nsp10–nsp14 interaction, leading to reduced reporter activity (Fig. 1C), indicating that the same proteins with different fusion domains competed with each other and impaired the specific interactions needed for activation of the reporter genes.

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