Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase.
The expression and processing of hepatitis C virus core protein was analyzed. Two protein bands, 21 kDa (P21), corresponding to the full-length core, and 19 kDa (P19), were detected as major products when core protein was expressed in the standard rabbit reticulocyte lysate system or in Sf9 insect cells. Core proteins with amino-terminal hexa-histidine tags were expressed which allowed the purification of the hexa-histidine P19 core with NI(2+)-NTA columns. With the help of mass spectrometry, the molecular weight of hexa-histidine-P19 was analyzed and its carboxy-terminus could be calculated. Fusion proteins of truncated core/core-E1 species fused to mouse dihydrofolate reductase (mDHFR) showed cleavage in the expected region. Cleavage sites could be determined by amino-terminal protein sequencing of the DHFR-fusion partner. Our data show that there are not one but two core products with an apparent molecular weight of about 19 kDa, ending either at amino acid leucine 179 or leucine 182, respectively. These cleavages in the hydrophobic, carboxy-terminal region of HCV core suggest processing by (a) recently proposed eucaryotic signal peptide peptidase(s) (F. Lyko et al. (1995) J. Biol. Chem. 270, 19873-19878). Furthermore, our results demonstrate that cleavage at these sites and the formation of the P19 species does not require previous processing at the signalase site (position 191/192) of the HCV-polyprotein.
Hüssy
P
P
F. Hoffmann-La Roche Ltd, Basel, Switzerland.
Langen
H
H
Mous
J
J
Jacobsen
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002874:Chromosome Mapping; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
C087035:E1 protein, Hepatitis C virus; D008565:Membrane Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; C069130:nucleocapsid protein, Hepatitis C virus; C091015:glycoprotein E2, Hepatitis C virus; D013762:Tetrahydrofolate Dehydrogenase; D012697:Serine Endopeptidases; C026515:type I signal peptidase