Two viral proteins involved in the proteolytic processing of the cowpea mosaic virus polyproteins.
A series of specific deletion mutants derived from a full-length cDNA clone of cowpea mosaic virus (CPMV) B RNA was constructed with the aim to study the role of viral proteins in the proteolytic processing of the primary translation products. For the same purpose cDNA clones were constructed having sequences derived from both M and B RNA of CPMV. In vitro transcripts prepared from these clones with T7 RNA polymerase, were efficiently translated in rabbit reticulocyte lysates. The translation products obtained were processed in the lysate by specific proteolytic cleavages into smaller products, which made it possible to study subsequently the effect of the various mutations on this process. The results obtained indicate that the B RNA-encoded 24K polypeptide represents a protease responsible for all cleavages in the polyproteins produced by both CPMV B and M RNA. For efficient cleavage of the glutamine-methionine site in the M RNA encoded polyprotein the presence of a second B RNA encoded protein, the 32K polypeptide, is essential, although the 32K polypeptide itself does not have proteolytic activity. A number of cleavage-site mutants were constructed in which the coding sequence for the glutamine-glycine cleavage site between the two capsid proteins was changed. Subsequent in vitro transcription and translation of these cleavage site mutants show that a correct dipeptide sequence is a prerequisite for efficient cleavage but that the folding of the polypeptide chain also plays an important role in the formation of a cleavage site.
Vos
P
P
Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands.
Verver
J
J
Jaegle
M
M
Wellink
J
J
van Kammen
A
A
Goldbach
R
R
D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D005814:Genes, Viral; D008970:Molecular Weight; D009029:Mosaic Viruses / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D011506:Proteins; D012367:RNA, Viral; D014764:Viral Proteins; D010447:Peptide Hydrolases