Cell-free translation and proteolytic processing of the hepatitis A virus polyprotein.
Virus-encoded proteinase activity of hepatitis A virus (HAV) was studied in vitro. Genomic regions coding for segments of the viral polyprotein were expressed by in vitro transcription and translation in rabbit reticulocyte lysates. Polyproteins translated from synthetic transcripts encoding P1-P2 or delta VP1-P2 were not processed indicating that no proteolytic activity is encoded within P2 of HAV, in contrast to other picornaviruses. Proteinase activity was, however, detected in the genomic region encoding 3C. Mutant transcripts (mu) which encode an alanine in place of the cysteine residue at amino acid position 172 of 3C did not yield proteolytic activity, consistent with the hypothesis that proteinase 3C is a cysteine-containing trypsin-like proteinase. Processing products 3ABC and P3 were identified by immunoprecipitation, providing evidence that proteolytic cleavage occurs at the 2C/3A and less frequently at the 3C/3D junction. For cleavages at either site, the complete 3D moiety was not required. In general, analysis of cleavage products was made difficult by the presence of polypeptides which were translated from internal start sites, predominantly within the P3 region. Since only small amounts of the full-length products P1-P2-P3 or P2-P3 were translated, possible cleavage of P1 and P2 by 3C could not be resolved in this system. Furthermore, no intermolecular cleavage could be detected when in vitro translated polypeptides of the P3 region were incubated with P1, P1-P2 or P2-P3 mu as substrates.
Institut für Medizinische Mikrobiologie, Medizinischen Universität Lübeck, Germany.
D
D
Jürgensen
Y Y
YY
Kusov
M
M
Fäcke
H G
HG
Kräusslich
V
V
Gauss-Müller
D000595:Amino Acid Sequence; D002474:Cell-Free System; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D005814:Genes, Viral; D006507:Hepatovirus / Q000235:genetics*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D015678:Viral Structural Proteins / Q000235:genetics
D011506:Proteins; D015678:Viral Structural Proteins; D010450:Endopeptidases