Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro.
Tomato ringspot nepovirus RNA-1-encoded polyprotein (P1) contains the domains for the putative NTP-binding protein, VPg, 3C-like protease and a putative RNA-dependent RNA polymerase in its C-terminal region. The N-terminal region of P1, with a coding capacity for a protein (or a precursor) of 67 kDa, has not been characterized. Using partial cDNA clones, it is shown that the 3C-like protease can process the N-terminal region of P1 at a novel cleavage site in vitro, allowing the release of two proteins, X1 (located at the N terminus of P1) and X2 (located immediately upstream of the NTB domain). P1 precursors in which the protease was inactive or absent were not cleaved by exogenously added protease, suggesting that P1 processing was predominantly in cis. Results from site-directed mutagenesis of putative cleavage sites suggest that dipeptides Q(423)/G and Q(620)/G are the X1-X2 and X2-NTB cleavage sites, respectively. The putative X1 protein contains a previously identified alanine-rich sequence which is present in nepoviruses but not in the related comoviruses. The putative X2 protein contains a region with similarity to the comovirus 32 kDa protease co-factor (the only mature protein released from the N terminus of comovirus P1 polyproteins) and to the corresponding region of other nepovirus P1 polyproteins. These results raise the possibility that the presence of two distinct protein domains in the N-terminal part of the P1 polyprotein may be a common feature of nepoviruses.
Wang
Aiming
A
Department of Botany, The University of British Columbia, Vancouver, BC, , CanadaV6T 1Z41.
Sanfaçon
Hélène
H
Pacific Agri-Food Research Centre, Summerland, BC, , CanadaV0H 1Z02.
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D018551:Solanum lycopersicum / Q000821:virology*; D008969:Molecular Sequence Data; D017860:Nepovirus / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D012367:RNA, Viral; D014764:Viral Proteins; C121154:polyprotein, comovirus; D012321:DNA-Directed RNA Polymerases; D010450:Endopeptidases