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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

Confirmation of the novel interactions by pull-down assays.The two test proteins were fused with glutathione S-transferase (GST) and maltose-binding protein (MBP), respectively, and mixed for binding in PBS buffer as described in the Materials and Methods section. The protein mixture was pulled down with glutathione-Sepharose that binds GST and GST fusion proteins. Proteins bound by glutathione-Sepharose were resolved in SDS-PAGE, transferred to PVDF membrane and then was detected by anti-MBP rabbit serum. For every assay, GST protein was used as a negative control. For example, to examine the interaction between nsp10 and nsp14, the mixtures of GST-nsp10/MBP-nsp14 and GST/MBP-nsp14 were incubated with glutathione-Sepharose and the proteins pulled down by glutathione-Sepharose were identified by anti-MBP rabbit serum, respectively. The proteins indicated on the left side of the vertical line were MBP fusions and that on the right are GST fusions with “-” indicating non-fused GST as negative control. The star signs indicate the expected bands for MBP-fusion proteins. The smaller bands observed are probably premature proteins or degradation products of the same proteins.
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pone-0003299-g002: Confirmation of the novel interactions by pull-down assays.The two test proteins were fused with glutathione S-transferase (GST) and maltose-binding protein (MBP), respectively, and mixed for binding in PBS buffer as described in the Materials and Methods section. The protein mixture was pulled down with glutathione-Sepharose that binds GST and GST fusion proteins. Proteins bound by glutathione-Sepharose were resolved in SDS-PAGE, transferred to PVDF membrane and then was detected by anti-MBP rabbit serum. For every assay, GST protein was used as a negative control. For example, to examine the interaction between nsp10 and nsp14, the mixtures of GST-nsp10/MBP-nsp14 and GST/MBP-nsp14 were incubated with glutathione-Sepharose and the proteins pulled down by glutathione-Sepharose were identified by anti-MBP rabbit serum, respectively. The proteins indicated on the left side of the vertical line were MBP fusions and that on the right are GST fusions with “-” indicating non-fused GST as negative control. The star signs indicate the expected bands for MBP-fusion proteins. The smaller bands observed are probably premature proteins or degradation products of the same proteins.

Mentions: To confirm the interactions which were newly detected by mammalian two-hybrid assays, pull-down assays were performed. Proteins that were related to the interactions not reported previously were expressed in different bacterial systems including pET30a (His-tagged), pGEX-6P-1 (GST fusion) and pMAL-c2X (MBP fusion). Using pull-down assays that were described in Materials and Methods section, six protein-protein interactions were confirmed, including those between: nsp10 and nsp14, nsp10 and nsp16, nsp13 and 3b, nsp8 and 3b, nsp16 and N, and 8b and N (Fig. 2). Due to difficulties in bacteria expression, the interactions involving nsp12, nsp3.2 and nsp3.3 could not be confirmed.


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)

Confirmation of the novel interactions by pull-down assays.The two test proteins were fused with glutathione S-transferase (GST) and maltose-binding protein (MBP), respectively, and mixed for binding in PBS buffer as described in the Materials and Methods section. The protein mixture was pulled down with glutathione-Sepharose that binds GST and GST fusion proteins. Proteins bound by glutathione-Sepharose were resolved in SDS-PAGE, transferred to PVDF membrane and then was detected by anti-MBP rabbit serum. For every assay, GST protein was used as a negative control. For example, to examine the interaction between nsp10 and nsp14, the mixtures of GST-nsp10/MBP-nsp14 and GST/MBP-nsp14 were incubated with glutathione-Sepharose and the proteins pulled down by glutathione-Sepharose were identified by anti-MBP rabbit serum, respectively. The proteins indicated on the left side of the vertical line were MBP fusions and that on the right are GST fusions with “-” indicating non-fused GST as negative control. The star signs indicate the expected bands for MBP-fusion proteins. The smaller bands observed are probably premature proteins or degradation products of the same proteins.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003299-g002: Confirmation of the novel interactions by pull-down assays.The two test proteins were fused with glutathione S-transferase (GST) and maltose-binding protein (MBP), respectively, and mixed for binding in PBS buffer as described in the Materials and Methods section. The protein mixture was pulled down with glutathione-Sepharose that binds GST and GST fusion proteins. Proteins bound by glutathione-Sepharose were resolved in SDS-PAGE, transferred to PVDF membrane and then was detected by anti-MBP rabbit serum. For every assay, GST protein was used as a negative control. For example, to examine the interaction between nsp10 and nsp14, the mixtures of GST-nsp10/MBP-nsp14 and GST/MBP-nsp14 were incubated with glutathione-Sepharose and the proteins pulled down by glutathione-Sepharose were identified by anti-MBP rabbit serum, respectively. The proteins indicated on the left side of the vertical line were MBP fusions and that on the right are GST fusions with “-” indicating non-fused GST as negative control. The star signs indicate the expected bands for MBP-fusion proteins. The smaller bands observed are probably premature proteins or degradation products of the same proteins.
Mentions: To confirm the interactions which were newly detected by mammalian two-hybrid assays, pull-down assays were performed. Proteins that were related to the interactions not reported previously were expressed in different bacterial systems including pET30a (His-tagged), pGEX-6P-1 (GST fusion) and pMAL-c2X (MBP fusion). Using pull-down assays that were described in Materials and Methods section, six protein-protein interactions were confirmed, including those between: nsp10 and nsp14, nsp10 and nsp16, nsp13 and 3b, nsp8 and 3b, nsp16 and N, and 8b and N (Fig. 2). Due to difficulties in bacteria expression, the interactions involving nsp12, nsp3.2 and nsp3.3 could not be confirmed.

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