Identification of the Cleavage Site Recognized by the Turnip Yellow Mosaic Virus Protease

Abstract

The noncapsid protein expressed from ORF-206 of turnip yellow mosaic virus (TYMV) is autocatalytically processed by a papain-like protease, producing N-terminal 150-kDa and C-terminal 70-kDa proteins. By introducing two methionine residues near the N-terminus of the 70-kDa protein, we have obtained N-terminal amino acid sequence of that protein produced from [35S]methionine-labeled in vitro translations. The introduction of methionine residues was demonstrated to not interfere with viral replication or proteolysis, as assayed by inoculating mutant RNA transcripts onto whole plants and protoplasts, as well as by translating the RNAs in a rabbit reticulocyte lysate. This has allowed us to determine that the TYMV protease cleaves between alanine1259 and threonine1260 of the precursor protein p206, yielding proteins of calculated Mr 140,618 and 66,037, which will be referred to henceforth as p141 and p66, respectively. The sequence context around the cleavage site is LNGA/TP.

Targeting of the Turnip Yellow Mosaic Virus 66K Replication Protein to the Chloroplast Envelope Is Mediated by the 140K Protein

“…1). First, a stop codon was introduced at amino acid 1259 of the 206K ORF, i.e., at the C terminus of the 140K protein (5,30). This stop codon was followed by a 494-nt deletion (nt 3872 to 4366 of the TYMV genome), the region that formerly encoded the first 165 amino acids of the 66K protein.…”

“…This large nonstructural protein also contains a papain-like cysteine proteinase domain located between the methyltransferase and the NTPase/helicase domains (54) that is responsible for the cotranslational proteolytic cleavage of the 206K protein in vitro (6,43). Mapping of the cleavage site (between A1259 and T1260) revealed that the resulting N-terminal protein product of 140 kDa (140K) contains the methyltransferase, proteinase, and NTPase/helicase motifs, whereas the C-terminal 66K protein encompasses the RdRp domain (5,30). This cleavage was also demonstrated to be functional in vivo (50), and both the 140K and 66K viral proteins appear to be essential for the replication of TYMV RNA genome (65).…”

Genomic and biological characterization of chiltepín yellow mosaic virus, a new tymovirus infecting Capsicum annuum var. aviculare in Mexico

“…These regions of the RP gene contain the conserved methyltransferase and polymerase functional domains with signatures typical of the tymoviruses, which may explain their lower variability across species due to functional constrains [39]. In TYMV it has been demonstrated that the RP polyprotein is autocatalytically cleaved by the papainelike protease, yielding two replication-associated proteins of 140 kDa, containing the methyltransferase, the protease and the helicase domains, and 66 kDa, comprising the RNA-dependent RNA polymerase domains [40]. One cysteine and one histidine within the RP papaine-like domain are the putative active site residues [40,41].…”

“…In TYMV it has been demonstrated that the RP polyprotein is autocatalytically cleaved by the papainelike protease, yielding two replication-associated proteins of 140 kDa, containing the methyltransferase, the protease and the helicase domains, and 66 kDa, comprising the RNA-dependent RNA polymerase domains [40]. One cysteine and one histidine within the RP papaine-like domain are the putative active site residues [40,41]. These two residues were located also in the RP gene of ChiYMV, which strongly suggest that a similar mechanism of polyprotein processing is operating in this virus.…”

Assembly of Turnip Yellow Mosaic Virus Replication Complexes: Interaction between the Proteinase and Polymerase Domains of the Replication Proteins

“…The methyltransferase and proteinase enzymatic modules are separated by an ϳ200-amino-acid-long proline-rich sequence with a predicted nonregular secondary structure (36) that may constitute a hinge between these two domains. The papain-like cysteine proteinase is responsible for the proteolytic cleavage of the 206K protein in vitro (6,41), leading to the synthesis of a 140-kDa N-terminal protein product (140K) containing the methyltransferase, proteinase, and NTPase/helicase domains and a 66-kDa C-terminal protein product (66K) encompassing the RdRp domain (5,26) (Fig. 1A).…”