Purification of viral genome-linked protein VPg from potato virus A-infected plants reveals several post-translationally modified forms of the protein.
In order to be able to analyse post-translational modifications and protein interactions of viral genome-linked protein VPg taking place during potato virus A (PVA) infection, an affinity tag-based purification system was developed by inserting a sequence encoding a six-histidine and haemagglutinin (HisHA) tag to the 3' end of the VPg coding sequence within the infectious cDNA clone of PVA. The engineered virus was fully functional and the HisHA tag-encoding sequence remained stable in the PVA genome throughout the infection process. Purification under denaturing conditions resulted in a protein sample that contained multiple VPg and NIa forms carrying post-translational modifications that altered their isoelectric points. Non-modified tagged VPg (pI 8) was a minor product in the protein sample derived from total leaf proteins, but when the replication-associated membranes were used as starting material, its relative amount increased. Further characterization demonstrated that some of the PVA VPg isoforms were modified by multiple phosphorylation events. Purity of the proteins derived from the native purifications with either of the tags was evaluated. A clearly purer VPg sample was obtained by performing tandem affinity purification utilizing both tags sequentially. NIb, CI and HC-Pro co-purified in an affinity-tagged VPg-dependent manner, indicating that the system was able to isolate protein complexes operating during PVA infection.
Hafrén
Anders
A
Department of Applied Chemistry and Microbiology, Latokartanonkaari 11, PO Box 27, University of Helsinki, FIN-00014 Helsinki, Finland.
Mäkinen
Kristiina
K
Department of Applied Chemistry and Microbiology, Latokartanonkaari 11, PO Box 27, University of Helsinki, FIN-00014 Helsinki, Finland.
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D054884:Host-Pathogen Interactions; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010935:Plant Diseases / Q000821:virology*; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017800:Potyvirus / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D012261:Ribonucleoproteins / Q000235:genetics* / Q000302:isolation & purification*; D016622:Silver Staining; D014026:Nicotiana / Q000378:metabolism / Q000821:virology*; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000302:isolation & purification*
D006388:Hemagglutinins; D010940:Plant Proteins; D012261:Ribonucleoproteins; D017361:Viral Nonstructural Proteins; C102206:genome-linked viral protein, potato virus Y; D006639:Histidine