AUTOCATALYTIC PROCESSING OF THE 223-KDA PROTEIN OF BLUEBERRY SCORCH CARLAVIRUS BY A PAPAIN-LIKE PROTEINASE

摘要

The first open reading frame of the blueberry scorch carlavirus (BBScV) genome encodes a putative replication-associated protein of 223 kDa (p223). A pulse-chase analysis of viral RNA translated in vitro in rabbit reticulocyte lysate revealed that p223 was proteolytically processed. Using a full-length ORF 1 cDNA clone in a coupled in vitro transcription/translation reaction, we confirmed that the ORF 1 gene product of BBScV processes autocatalytically. From sequence alignments with phylogenetically related viruses, including tymoviruses, we predicted that p223 contained a papain-like proteinase domain with a putative catalytic cysteine(994) and histidine(1075). A second possible proteinase domain, which contained cysteine(895) and histidine(984) residues with similar spacing but was otherwise less similar to the viral papin-like proteinases, was identified immediately upstream of the predicted catalytic site. The cleavage site of the proteinase was predicted to be between the putative helicase and the polymerase domains, possibly between or close to glycine(1472) and alanine(1473). Supporting these predictions, deletion of the 2091 nucleotides encoding the C-terminal region of p223, which contained the putative RNA polymerase domain and the putative cleavage site of the polyprotein, abolished autoproteolysis. Deletion of the 2061 nucleotides encoding the N-terminal region, which contained the putative methyltransferase domain, did not affect autoproteolysis. Alteration of cysteine(994), histidine(1075), or glycine(1472) abolished autoproteolysis in vitro, supporting the involvement of these residues at the catalytic site and cleavage site. Alteration of the upstream cysteine(895) and histidine(984) residues did not affect processing in vitro. Capped BBScV full-length transcripts containing mutations in the codons for either cysteine(994) or histidine(1075) were not infectious in the systemic host plants Chenopodium quinoa and C. amaranticolor, whereas alteration of glycine(1472) significantly decreased but did not abolish infectivity. Transcripts containing mutations in the codons for either cysteine(895) or histidine(984) also were infectious, but resulted in delayed symptom expression in plants. (C) 1995 Academic Press, Inc.