Interaction between the replicase proteins of Tomato Bushy Stunt virus in vitro and in vivo

K. S. Rajendran; Peter D. Nagy

doi:10.1016/j.virol.2004.06.018

Interaction between the replicase proteins of Tomato Bushy Stunt virus in vitro and in vivo

K. S. Rajendran, Peter D. Nagy

Plant Pathology

Research output: Contribution to journal › Article › peer-review

45 Scopus citations

Abstract

Tomato bushy stunt virus (TBSV), a tombusvirus with a non-segmented, plus-stranded RNA genome, codes for p33 and p92 replicase proteins. The sequence of p33 overlaps with the N-terminal domain of p92, which also contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its non-overlapping C-terminal portion. In this research, we demonstrate in vitro interactions between p33:p33 and p33:p92 using surface plasmon resonance analysis with purified recombinant p33 and p92. The sequence in p33 involved in the above protein-protein interactions was mapped to the C-terminal region, which also contains an RNA-binding site. Using the yeast two-hybrid assay, we confirmed that two short regions within p33 could promote p33:p33 and p33:p92 interactions in vivo. Mutations in either p33 or p92 within the short regions involved in p33:p33 and p33:p92 interactions decreased the replication of a TBSV defective interfering RNA in yeast, a model host, supporting the significance of these protein interactions in tombusvirus replication.

Original language	English
Pages (from-to)	250-261
Number of pages	12
Journal	Virology
Volume	326
Issue number	2
DOIs	https://doi.org/10.1016/j.virol.2004.06.018
State	Published - Sep 1 2004

Bibliographical note

Funding Information:
We thank Drs. Judit Pogany, Saulius Serva and Jozsef Gal for critical reading of the manuscript and for very helpful suggestions. The authors also thank Drs. A. White and H. Scholthof for providing constructs T100 and pHS-175 and Dr. Serva for his help with using the Anti-His monoclonal antibodies. This work was supported by NSF (MCB0078152) and by the Kentucky Tobacco Research and Development Center at the University of Kentucky, awarded to PDN.

Keywords

Protein-protein interactions
RdRp
Replication
Surface plasmon resonance
Two-hybrid assay
Yeast host

ASJC Scopus subject areas

Virology

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10.1016/j.virol.2004.06.018

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title = "Interaction between the replicase proteins of Tomato Bushy Stunt virus in vitro and in vivo",

abstract = "Tomato bushy stunt virus (TBSV), a tombusvirus with a non-segmented, plus-stranded RNA genome, codes for p33 and p92 replicase proteins. The sequence of p33 overlaps with the N-terminal domain of p92, which also contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its non-overlapping C-terminal portion. In this research, we demonstrate in vitro interactions between p33:p33 and p33:p92 using surface plasmon resonance analysis with purified recombinant p33 and p92. The sequence in p33 involved in the above protein-protein interactions was mapped to the C-terminal region, which also contains an RNA-binding site. Using the yeast two-hybrid assay, we confirmed that two short regions within p33 could promote p33:p33 and p33:p92 interactions in vivo. Mutations in either p33 or p92 within the short regions involved in p33:p33 and p33:p92 interactions decreased the replication of a TBSV defective interfering RNA in yeast, a model host, supporting the significance of these protein interactions in tombusvirus replication.",

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note = "Funding Information: We thank Drs. Judit Pogany, Saulius Serva and Jozsef Gal for critical reading of the manuscript and for very helpful suggestions. The authors also thank Drs. A. White and H. Scholthof for providing constructs T100 and pHS-175 and Dr. Serva for his help with using the Anti-His monoclonal antibodies. This work was supported by NSF (MCB0078152) and by the Kentucky Tobacco Research and Development Center at the University of Kentucky, awarded to PDN.",

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N2 - Tomato bushy stunt virus (TBSV), a tombusvirus with a non-segmented, plus-stranded RNA genome, codes for p33 and p92 replicase proteins. The sequence of p33 overlaps with the N-terminal domain of p92, which also contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its non-overlapping C-terminal portion. In this research, we demonstrate in vitro interactions between p33:p33 and p33:p92 using surface plasmon resonance analysis with purified recombinant p33 and p92. The sequence in p33 involved in the above protein-protein interactions was mapped to the C-terminal region, which also contains an RNA-binding site. Using the yeast two-hybrid assay, we confirmed that two short regions within p33 could promote p33:p33 and p33:p92 interactions in vivo. Mutations in either p33 or p92 within the short regions involved in p33:p33 and p33:p92 interactions decreased the replication of a TBSV defective interfering RNA in yeast, a model host, supporting the significance of these protein interactions in tombusvirus replication.

AB - Tomato bushy stunt virus (TBSV), a tombusvirus with a non-segmented, plus-stranded RNA genome, codes for p33 and p92 replicase proteins. The sequence of p33 overlaps with the N-terminal domain of p92, which also contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its non-overlapping C-terminal portion. In this research, we demonstrate in vitro interactions between p33:p33 and p33:p92 using surface plasmon resonance analysis with purified recombinant p33 and p92. The sequence in p33 involved in the above protein-protein interactions was mapped to the C-terminal region, which also contains an RNA-binding site. Using the yeast two-hybrid assay, we confirmed that two short regions within p33 could promote p33:p33 and p33:p92 interactions in vivo. Mutations in either p33 or p92 within the short regions involved in p33:p33 and p33:p92 interactions decreased the replication of a TBSV defective interfering RNA in yeast, a model host, supporting the significance of these protein interactions in tombusvirus replication.

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Interaction between the replicase proteins of Tomato Bushy Stunt virus in vitro and in vivo

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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