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

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 rab bit reticulocyte lysate revealed that p223 was proteolytically process ed. Using a full-length ORF 1 cDNA clone in a coupled in vitro transcr iption/translation reaction, we confirmed that the ORF 1 gene product of BBScV processes autocatalytically. From sequence alignments with ph ylogenetically related viruses, including tymoviruses, we predicted th at p223 contained a papain-like proteinase domain with a putative cata lytic 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-lik e proteinases, was identified immediately upstream of the predicted ca talytic site. The cleavage site of the proteinase was predicted to be between the putative helicase and the polymerase domains, possibly bet ween or close to glycine(1472) and alanine(1473). Supporting these pre dictions, deletion of the 2091 nucleotides encoding the C-terminal reg ion of p223, which contained the putative RNA polymerase domain and th e putative cleavage site of the polyprotein, abolished autoproteolysis . Deletion of the 2061 nucleotides encoding the N-terminal region, whi ch contained the putative methyltransferase domain, did not affect aut oproteolysis. 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 affe ct processing in vitro. Capped BBScV full-length transcripts containin g 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 d ecreased but did not abolish infectivity. Transcripts containing mutat ions in the codons for either cysteine(895) or histidine(984) also wer e infectious, but resulted in delayed symptom expression in plants. (C ) 1995 Academic Press, Inc.