Maturation of giardiavirus capsid protein involves posttranslational proteolytic processing by a cysteine protease.
The double-stranded RNA genome of giardiavirus (GLV) has only two large open reading frame (ORFs). The 100-kDa capsid polypeptide (p100) is encoded by ORF1, whereas the only other viral polypeptide, the 190-kDa GLV RNA-dependent RNA polymerase (p190), is synthesized as an ORF1-ORF2 fusion protein by a (-1) ribosomal frameshifting. Edman degradation revealed that p100 was N-terminally blocked except for 2 to 5% of it that showed free N terminus starting from amino acid residue 33 of ORF1. Studies using antiserum targeted against amino acid residues 6 to 27 indicated that this region (NT) is absent from viral p100 and p190, while pulse-labelling experiments showed that NT is present in nascent p100 synthesized in GLV-infected Giardia lamblia but removed subsequently. In contrast, this region was retained in the two viral proteins synthesized in vitro, and it was not removed upon prolonged incubation or inclusion of microsomal fraction in the in vitro translation reaction mixtures. These results suggest that endoplasmic reticulum is not involved in the protein processing and that the precursors of p100 and p190 are incapable of cleaving themselves or each other. This specific cleavage was reproduced when lysates from GLV-infected G. lamblia were added, but not those from uninfected cells. The cleavage activity was relatively insensitive to phenylmethylsulfonyl fluoride, but it was inhibitable by leupeptin or E-64, two known specific inhibitors of cysteine protease. The possible origin of this processing activity is discussed.
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Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D016679:Genome, Viral; D016829:Giardia lamblia / Q000821:virology*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012328:RNA Viruses / Q000235:genetics / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D011498:Protein Precursors; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D003546:Cysteine Endopeptidases