Characterization of dominant-negative and temperature-sensitive mutants of tombusvirus replication proteins affecting replicase assembly

Kunj B. Pathak; Zuodong Jiang; Verena Ochanine; Monika Sharma; Judit Pogany; Peter D. Nagy

doi:10.1016/j.virol.2012.12.009

Characterization of dominant-negative and temperature-sensitive mutants of tombusvirus replication proteins affecting replicase assembly

Kunj B. Pathak, Zuodong Jiang, Verena Ochanine, Monika Sharma, Judit Pogany, Peter D. Nagy

Plant Pathology

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

9 Scopus citations

Abstract

The assembly of the viral replicase complex (VRC) on subcellular membranes is a key step in the replication process of plus-stranded RNA viruses. In this work, we have identified lethal and temperature sensitive (ts) point mutations within the essential p33:p33/p92 interaction domain of p33 and p92 replication proteins of Cucumber necrosis virus, a tombusvirus. Mutations within the p33:p33/p92 interaction domain also affected viral RNA recombination in yeast model host. An in vitro approach based on yeast cell free extract demonstrated that several p33 and p92 mutants behaved as dominant-negative during VRC assembly, and they showed reduced binding to the viral (+)RNA and affected activation of the p92 RdRp protein, while they did not directly influence (-) or (+)-strand synthesis. Overall, the presented data provide direct evidence that the p33:p33/p92 interaction domains in p33 and p92 are needed for the early stage of virus replication and also influence viral recombination.

Original language	English
Pages (from-to)	48-61
Number of pages	14
Journal	Virology
Volume	437
Issue number	1
DOIs	https://doi.org/10.1016/j.virol.2012.12.009
State	Published - Mar 1 2013

Bibliographical note

Funding Information:
The authors thank Dr. Natalia Shapka for initiating this project and Dr. Judit Pogany for the FHV reagents and for helpful discussions. This work was supported by NIH-NIAID ( AI05767001A1 and 5R21AI079457-02 ), and by the University of Kentucky to PDN. KBP was also supported by Philip Morris fellowship.

Keywords

Cell-free
Cucumber necrosis virus
Dominant negative mutant
In vitro
Recombination
Replicase assembly
Replication
Temperature-sensitive
Tombusvirus
Yeast

ASJC Scopus subject areas

Virology

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10.1016/j.virol.2012.12.009

Cite this

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title = "Characterization of dominant-negative and temperature-sensitive mutants of tombusvirus replication proteins affecting replicase assembly",

abstract = "The assembly of the viral replicase complex (VRC) on subcellular membranes is a key step in the replication process of plus-stranded RNA viruses. In this work, we have identified lethal and temperature sensitive (ts) point mutations within the essential p33:p33/p92 interaction domain of p33 and p92 replication proteins of Cucumber necrosis virus, a tombusvirus. Mutations within the p33:p33/p92 interaction domain also affected viral RNA recombination in yeast model host. An in vitro approach based on yeast cell free extract demonstrated that several p33 and p92 mutants behaved as dominant-negative during VRC assembly, and they showed reduced binding to the viral (+)RNA and affected activation of the p92 RdRp protein, while they did not directly influence (-) or (+)-strand synthesis. Overall, the presented data provide direct evidence that the p33:p33/p92 interaction domains in p33 and p92 are needed for the early stage of virus replication and also influence viral recombination.",

keywords = "Cell-free, Cucumber necrosis virus, Dominant negative mutant, In vitro, Recombination, Replicase assembly, Replication, Temperature-sensitive, Tombusvirus, Yeast",

author = "Pathak, {Kunj B.} and Zuodong Jiang and Verena Ochanine and Monika Sharma and Judit Pogany and Nagy, {Peter D.}",

note = "Funding Information: The authors thank Dr. Natalia Shapka for initiating this project and Dr. Judit Pogany for the FHV reagents and for helpful discussions. This work was supported by NIH-NIAID ( AI05767001A1 and 5R21AI079457-02 ), and by the University of Kentucky to PDN. KBP was also supported by Philip Morris fellowship.",

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language = "English",

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AU - Jiang, Zuodong

AU - Ochanine, Verena

AU - Sharma, Monika

AU - Pogany, Judit

AU - Nagy, Peter D.

N1 - Funding Information: The authors thank Dr. Natalia Shapka for initiating this project and Dr. Judit Pogany for the FHV reagents and for helpful discussions. This work was supported by NIH-NIAID ( AI05767001A1 and 5R21AI079457-02 ), and by the University of Kentucky to PDN. KBP was also supported by Philip Morris fellowship.

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Y1 - 2013/3/1

N2 - The assembly of the viral replicase complex (VRC) on subcellular membranes is a key step in the replication process of plus-stranded RNA viruses. In this work, we have identified lethal and temperature sensitive (ts) point mutations within the essential p33:p33/p92 interaction domain of p33 and p92 replication proteins of Cucumber necrosis virus, a tombusvirus. Mutations within the p33:p33/p92 interaction domain also affected viral RNA recombination in yeast model host. An in vitro approach based on yeast cell free extract demonstrated that several p33 and p92 mutants behaved as dominant-negative during VRC assembly, and they showed reduced binding to the viral (+)RNA and affected activation of the p92 RdRp protein, while they did not directly influence (-) or (+)-strand synthesis. Overall, the presented data provide direct evidence that the p33:p33/p92 interaction domains in p33 and p92 are needed for the early stage of virus replication and also influence viral recombination.

AB - The assembly of the viral replicase complex (VRC) on subcellular membranes is a key step in the replication process of plus-stranded RNA viruses. In this work, we have identified lethal and temperature sensitive (ts) point mutations within the essential p33:p33/p92 interaction domain of p33 and p92 replication proteins of Cucumber necrosis virus, a tombusvirus. Mutations within the p33:p33/p92 interaction domain also affected viral RNA recombination in yeast model host. An in vitro approach based on yeast cell free extract demonstrated that several p33 and p92 mutants behaved as dominant-negative during VRC assembly, and they showed reduced binding to the viral (+)RNA and affected activation of the p92 RdRp protein, while they did not directly influence (-) or (+)-strand synthesis. Overall, the presented data provide direct evidence that the p33:p33/p92 interaction domains in p33 and p92 are needed for the early stage of virus replication and also influence viral recombination.

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Characterization of dominant-negative and temperature-sensitive mutants of tombusvirus replication proteins affecting replicase assembly

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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