Please use this identifier to cite or link to this item: https://doi.org/10.1128/JVI.78.12.6498-6508.2004
Title: Membrane association of greasy grouper nervous necrosis virus protein A and characterization of its mitochondrial localization targeting signal
Authors: Guo, Y.X.
Chan, S.-W. 
Kwang, J. 
Issue Date: Jun-2004
Source: Guo, Y.X., Chan, S.-W., Kwang, J. (2004-06). Membrane association of greasy grouper nervous necrosis virus protein A and characterization of its mitochondrial localization targeting signal. Journal of Virology 78 (12) : 6498-6508. ScholarBank@NUS Repository. https://doi.org/10.1128/JVI.78.12.6498-6508.2004
Abstract: Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. The betanodavirus greasy grouper (Epinephelus tauvina) nervous necrosis virus (GGNNV) is a positive-RNA virus with one of the smallest genomes among RNA viruses replicating in fish cells. To understand the localization of GGNNV replication complexes, we generated polyclonal antisera against protein A, the GGNNV RNA-dependent RNA polymerase. Protein A was detected at 5 h postinfection in infected sea bass cells. Biochemical fractionation experiments revealed that GGNNV protein A sedimented with intracellular membranes upon treatment with an alkaline pH and a high salt concentration, indicating that GGNNV protein A is tightly associated with intracellular membranes in infected cells. Confocal immunofluorescence microscopy and bromo-UTP incorporation studies identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. In addition, protein A fused with green fluorescent protein (GFP) was detected in the mitochondria in transfected cells and was demonstrated to be tightly associated with intracelfular membranes by biochemical fractionation analysis and membrane flotation assays, indicating that protein A alone was sufficient for mitochondrial localization in the absence of RNA replication, nonstructural protein B, or capsid proteins. Three sequence analysis programs showed two regions of hydrophobic amino acid residues, amino acids 153 to 173 and 229 to 249, to be transmembrane domains (TMD) that might contain a membrane association domain. Membrane fraction analysis showed that the major domain is N-terminal amino acids 215 to 255, containing the predicted TMD from amino acids 229 to 249. Using GFP as the reporter by systematically introducing deletions of these two regions in the constructs, we further confirmed that the N-terminal amino acids 215 to 255 of protein A function as a mitochondrial targeting signal.
Source Title: Journal of Virology
URI: http://scholarbank.nus.edu.sg/handle/10635/131226
ISSN: 0022538X
DOI: 10.1128/JVI.78.12.6498-6508.2004
Appears in Collections:Staff Publications

Show full item record
Files in This Item:
There are no files associated with this item.

SCOPUSTM   
Citations

28
checked on Mar 27, 2018

WEB OF SCIENCETM
Citations

26
checked on Mar 27, 2018

Page view(s)

6
checked on Mar 11, 2018

Google ScholarTM

Check

Altmetric


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.