Protein composition of 6K2-induced membrane structures formed during Potato virus A infection

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Lohmus , A , Varjosalo , M & Mäkinen , K 2016 , ' Protein composition of 6K2-induced membrane structures formed during Potato virus A infection ' , Molecular Plant Pathology , vol. 17 , no. 6 , pp. 943-958 . https://doi.org/10.1111/mpp.12341

Title: Protein composition of 6K2-induced membrane structures formed during Potato virus A infection
Author: Lohmus, Andres; Varjosalo, Markku; Mäkinen, Kristiina
Contributor: University of Helsinki, Department of Food and Nutrition
University of Helsinki, Institute of Biotechnology
University of Helsinki, Department of Food and Nutrition
Date: 2016-08
Language: eng
Number of pages: 16
Belongs to series: Molecular Plant Pathology
ISSN: 1464-6722
URI: http://hdl.handle.net/10138/297758
Abstract: The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein-induced membranous structures from Potato virus A (PVA)-infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N-terminal Twin-Strep-tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non-tagged Cerulean-6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep-tag-based affinity chromatography was developed. Both (+)- and (-)-strand PVA RNA and viral protein VPg were co-purified specifically with the affinity tagged PVA-SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA-SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2-induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co-purified with PVA-derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication.
Subject: 6K2 protein
Potato virus A
potyvirus
proteome
viral replication complex
DEPENDENT RNA-POLYMERASE
TURNIP-MOSAIC-VIRUS
HOST METABOLIC ENZYME
LARGE GENE LISTS
ENDOPLASMIC-RETICULUM
VIRAL PROTEIN
GENOME AMPLIFICATION
MUTATIONAL ANALYSIS
6K2 PROTEIN
REPLICATION
1182 Biochemistry, cell and molecular biology
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