Mutational analysis of human papillomavirus type 16 major capsid protein L1: the cysteines affecting the intermolecular bonding and structure of L1-capsids

• Published in: Virology (New York, N.Y.) Vol. 308; no. 1; pp. 128 - 136
• Main Authors: Ishii, Yoshiyuki, Tanaka, Keiko, Kanda, Tadahito
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 30-03-2003
• Subjects: Amino Acid Substitution; Animals; Capsid - metabolism; Capsid - ultrastructure; Capsid Proteins; Cell Line; Centrifugation, Density Gradient; Cysteine - analysis; Cysteine - metabolism; Electrophoresis, Polyacrylamide Gel; HPV-16; Insecta; L1-capsid; Major capsid protein L1; Mutation; Oncogene Proteins, Viral - chemistry; Oncogene Proteins, Viral - genetics; Oncogene Proteins, Viral - metabolism; Papillomaviridae - chemistry; Papillomaviridae - metabolism; Serine - metabolism; Trypsin; Virus Assembly; HPV-16; Major capsid protein L1; L1-capsid
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1016/S0042-6822(02)00099-5

Human papillomavirus 16 major capsid protein L1 (composed of 505 amino acids (aa) including 12 cysteines) assembles by itself into virion-like icosahedral particles (L1-capsids), each of which is dissociated into 72 pentameric capsomeres when intermolecular disulfide bonds are disrupted. To identify the cysteines affecting the bonding and the structural integrity of the L1-capsids, we constructed a series of L1 mutants with substitution of serine for cysteine, which were expressed from recombinant baculoviruses in the insect Sf9 cells. From infected cells, the self-assembled L1-capsid fractions were purified by CsCl-equilibrium centrifugation and examined for velocity sedimentation profiles, for the presence of intermolecular bonding by SDS–PAGE with or without a reducing agent, for morphology under an electron microscope, and for susceptibility to trypsin digestion. Mutants C175S (C at aa 175 was replaced with S) and C185S were sedimented in sucrose-density gradients slightly slower than the wild type (WT) capsids, and mutant C428S stayed near the top as WT-capsomeres did. In the nonreducing SDS gel, where WT-capsids were separated into two bands of L1-trimers and L1-dimers, the C175S-trimer band was not detected, the C185S-dimer band was much less dense, and the C428S-trimer and C428S-dimer bands were not detected. Thus, it seems likely that C175, C185, and C428 are involved in L1 trimerization, in L1 dimerization, and in both, respectively. Morphologically, the C175S, C185S, and C428S fractions appeared to consist mostly of heterogeneous rod-shaped tubules, of smaller spherical particles, and of only capsomeres, respectively, whereas C102S, C229S, and C379S resembled WT. The C161S, C175S, C185S, C229S, C379S, and C428S capsids were more sensitive to degradation caused by trypsin than WT. The results indicate that C175, C185, and C428 are required for the normal assembly of L1-capsids through trimerization and dimerization of L1 bound by the intercapsomeric disulfide bonds between cysteines, and that C161, C229, and C379 are necessary for the integrity of L1-capsids probably through intramolecular bonding.