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Title: Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: Implications for replication and genome packaging

Abstract

In Brome mosaic virus, it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein–protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. - Highlights: • YFP fusion proteins of BMV p1a and p2a are biologically active. • Self-interaction was observed for p1a, p2a and CP. • CP interacts with p2a but not p1a. • Majority of reconstituted YFP resulting from bona fide fusion protein partners localized on ER.

Authors:
;
Publication Date:
OSTI Identifier:
22435054
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 464-465; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; DIAGNOSTIC TECHNIQUES; ENDOPLASMIC RETICULUM; FLUORESCENCE; HYPOTHESIS; INTERACTIONS; LABELLING; PROTEINS; SPECIFICITY; VIRUSES

Citation Formats

Chaturvedi, Sonali, and Rao, A.L.N., E-mail: arao@ucr.edu. Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: Implications for replication and genome packaging. United States: N. p., 2014. Web. doi:10.1016/J.VIROL.2014.06.030.
Chaturvedi, Sonali, & Rao, A.L.N., E-mail: arao@ucr.edu. Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: Implications for replication and genome packaging. United States. doi:10.1016/J.VIROL.2014.06.030.
Chaturvedi, Sonali, and Rao, A.L.N., E-mail: arao@ucr.edu. Mon . "Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: Implications for replication and genome packaging". United States. doi:10.1016/J.VIROL.2014.06.030.
@article{osti_22435054,
title = {Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: Implications for replication and genome packaging},
author = {Chaturvedi, Sonali and Rao, A.L.N., E-mail: arao@ucr.edu},
abstractNote = {In Brome mosaic virus, it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein–protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. - Highlights: • YFP fusion proteins of BMV p1a and p2a are biologically active. • Self-interaction was observed for p1a, p2a and CP. • CP interacts with p2a but not p1a. • Majority of reconstituted YFP resulting from bona fide fusion protein partners localized on ER.},
doi = {10.1016/J.VIROL.2014.06.030},
journal = {Virology},
number = ,
volume = 464-465,
place = {United States},
year = {Mon Sep 15 00:00:00 EDT 2014},
month = {Mon Sep 15 00:00:00 EDT 2014}
}