Proteolytic processing of the plum pox potyvirus polyprotein by the NIa protease at a novel cleavage site.
The expression of potyvirus genomic RNA takes place through translation of its unique long and functional open reading frame into a large polyprotein that undergoes extensive proteolytic processing. Most of the cleavages are performed by the virus-encoded NIa protease, which cuts the polyprotein at defined sites that are characterized by conserved heptapeptide sequences. We have demonstrated in vitro cleavage activity by the plum pox potyvirus (PPV) NIa protease at a novel site, previously identified by sequence analysis, thus allowing a further refinement of the potyviral genetic map. This novel site is located 52 amino acids upstream from the site corresponding to the N-terminus of the CI protein (the NIa cleavage site previously considered the closest to the beginning of the polyprotein). The specificity of the processing was demonstrated by its abolishment when the Gln at position -1 of the cleavage site was changed to His. This novel NIa cleavage site was only partially processed, a characteristic that was not altered when its heptapeptide sequence was modified to become that of the efficiently cleaved NIb-CP junction. On the contrary, substitutions at the nonconserved position +3 had notable effects, positive or negative, on the efficiency of processing. These results show the relevance of sequence and/or conformational context outside the conserved heptapeptide for modulating the cleavage reaction catalyzed by the NIa protease.
García
J A
JA
Centro de Biología Molecular (CSIC-UAM), Madrid, Spain.
Martín
M T
MT
Cervera
M T
MT
Riechmann
J L
JL
D000595:Amino Acid Sequence; D010450:Endopeptidases; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D011506:Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses