Cauliflower mosaic virus produces an aspartic proteinase to cleave its polyproteins.
Cauliflower mosaic virus (CaMV), a plant pararetrovirus, produces polyproteins from its adjacent genes for the coat protein (ORF IV) and for enzymatic functions (ORF V). The N-terminal domain of the latter gene includes a sequence showing homology to the active site of other retroviral and acid proteases. We have now shown that this domain does indeed produce a functional aspartic protease that can process both the polyproteins. Mutations in the putative active site abolished virus infectivity. In transient expression studies in protoplasts, the N-terminal domain of ORF V was able to free active CAT enzyme from a precursor containing an N-terminal fusion of a portion of ORF IV. The junction between the two domains of this artificial polyprotein comprised sequences from the ORF IV product that had previously been shown to include a proteolytic processing site. The protease mutants were not able to free active CAT enzyme from this precursor. Direct analysis of cleavage at the same site in the ORF IV product using proteins expressed in Escherichia coli revealed the expected products. In vitro translation of a synthetic transcript covering ORF V was used to study the autocatalytic cleavage of the ORF product. Pulse-chase experiments showed that the 80 kd initial translation product was processed to yield a N-terminal doublet of polypeptides of 22 and 20 kd apparent mol. wt, which cover the protease domain. The mutants in the active site were not processed.
Torruella
M
M
Friedrich Miescher-Institut, Basel, Switzerland.
Gordon
K
K
Hohn
T
T
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000201:enzymology / Q000235:genetics*; D009154:Mutation; D010957:Plasmids; D011480:Protease Inhibitors; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D011480:Protease Inhibitors; D014764:Viral Proteins; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases