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Journal of Virology, November 2004, p. 11766-11777, Vol. 78, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.21.11766-11777.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Membrane Binding Properties and Terminal Residues of the Mature Hepatitis C Virus Capsid Protein in Insect Cells

Tomoaki Ogino,1,{dagger} Hiroyuki Fukuda,2 Shinobu Imajoh-Ohmi,2 Michinori Kohara,3 and Akio Nomoto1*

Department of Microbiology, Graduate School of Medicine, The University of Tokyo,1 Department of Microbiology, The Tokyo Metropolitan Institute of Medical Science,3 Division of Cell Biology and Biochemistry, Department of Basic Medical Science, Institute of Medical Science, The University of Tokyo, Tokyo, Japan2

Received 27 February 2004/ Accepted 24 June 2004

The immature core protein (p23, residues 1 to 191) of hepatitis C virus undergoes posttranslational modifications including intramembranous proteolysis within its C-terminal signal sequence by signal peptide peptidase to generate the mature form (p21). In this study, we analyzed the cleavage site and other amino acid modifications that occur on the core protein. To produce the posttranslationally modified core protein, we used a baculovirus-insect cell expression model system. As previously reported, p23 is processed to form p21 in insect as well as in mammalian cells. p21 was found to be associated with the cytoplasmic membrane, and its significant portion behaved as an integral membrane protein. The protein was purified from the membrane by a simple and unique procedure on the basis of its membrane-binding properties and solubility in detergents. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of purified p21 showed that the average molecular mass (m/z 19,307) of its single-charged ion differs by m/z 1,457 from that calculated for p23. To determine the posttranslational modifications, tryptic p21 peptides were analyzed by MALDI-TOF MS. We found three peptides that did not match the theoretically derived peptides of p23. Analysis of these peptides by MALDI-TOF tandem MS revealed that they correspond to N-terminal peptides (residues 2 to 9 and 2 to 10) starting with {alpha}-N-acetylserine and C-terminal peptide (residues 150 to 177) ending with phenylalanine. These results suggest that the mature core protein (molecular mass of 19,306 Da) includes residues 2 to 177 and that its N terminus is blocked with an acetyl group.

* Corresponding author. Mailing address: Department of Microbiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: 81 3 5841 3413. Fax: 81 3 5841 3374. E-mail: anomoto{at}m.u-tokyo.ac.jp.

{dagger} Present address: Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195.

Journal of Virology, November 2004, p. 11766-11777, Vol. 78, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.21.11766-11777.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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