Identification of a Human Papillomavirus Type 16-Specific Epitope on the C-Terminal Arm of the Major Capsid Protein L1

Abstract

To characterize epitopes on human papillomavirus (HPV) virus-like particles (VLPs), a panel of mutated HPV-16 VLPs was created. Each mutated VLP had residues substituted from HPV-31 or HPV-52 L1 sequences to the HPV-16 L1 backbone. Mutations were created on the HPV-31 and ؊52 L1 proteins to determine if HPV-16 type-specific recognition could be transferred. Correct folding of the mutated proteins was verified by resistance to trypsin digestion and by binding to one or more conformation-dependent monoclonal antibodies. Several of the antibodies tested were found to bind to regions already identified as being important for HPV VLP recognition (loops DE, EF, FG, and HI). Sequences at both ends of the long FG loop (amino acids 260 to 290) were required for both H16.V5 and H16.E70 reactivity. A new antibody-binding site was discovered on the C-terminal arm of L1 between positions 427 and 445. Recognition of these residues by the H16.U4 antibody suggests that this region is surface exposed and supports a recently proposed molecular model of HPV VLPs.The human papillomavirus (HPV) virion is composed of major (L1) and minor (L2) capsid proteins. The L1 protein self-assembles into virus-like particles (VLPs) that appear identical to infectious virus both by electron microscopy and immunologically (8, 9, 11). The L2 protein is important for assembly of infectious virus (20) but does not contain the conformation-dependent and type-specific epitopes most frequently recognized by anti-HPV sera (2, 9).The VLP is a T ϭ 7 icosahedron composed of 72 pentamers of L1, termed capsomers. Sixty of the capsomers subunits are at hexavalent positions, interacting with six neighboring capsomers with the remaining 12 capsomers at pentavalent positions (1). The only HPV L1 crystal structure solved to date is of T1 particles (3), in which all capsomers are at pentavalent positions. In the T1 particle, capsomers interact through a portion of the C-terminal tail. This flexible arm extends away from the capsomer of origin, interacts with similar regions from neighboring capsomers and returns to the capsomer from which it came. The remainder of this C-terminal region extends around the circumference of the capsomer, participating in the core ␤-sheet structure, forming a short helix (h5) and finally reinserting into the core of the pentamer from which it came. Missing from this model is an intercapsomer disulfide bond, shown by others to help stabilize the VLP structure (12, 21).A revised model for HPV VLPs was proposed by Modis et al. (18). In this model, the C-terminal extension adopts a conformation similar to its conformation in the T1 structure, but instead of returning to the capsomer of origin, the arm is displaced onto, and ultimately invades, a neighboring capsomer. The C-terminal arm is anchored by the previously described disulfide bond between the cysteine from this region (amino acids 428) and cysteine 175 of a neighboring capsomer. The new model was termed the "invading arm" model because of its similarity to the simian virus 40 a...

Humoral Immune Response Recognizes a Complex Set of Epitopes on Human Papillomavirus Type 6 L1 Capsomers

“…The crystal structure of T ϭ 1 L1 particles showed that the C-terminal arms were involved in intercapsomeric junctions but not surface exposed (7), while a more recent atomic model blending cryo-electron microscopic data and the crystallographic structure of HPV16 L1 resulted in a reassessment, suggesting the C-terminal arm is surface exposed in intercapsomeric junctions, as is seen in polyomavirus capsids (38). Recently, mapping of the epitope recognized by MAb H16.U4 identified a region within the C-terminal arm, supporting the model of a surface-exposed C-terminal arm (5).…”

“…An atomic model of the full-size HPV VLP, made using cryo-electron microscopic data of bovine papillomavirus VLPs and the coordinates from HPV16 L1, suggested that the C terminus of L1 is surface exposed in intercapsomeric connections (38). This potentially implicates the C terminus as an immunogenic target, as recently supported by MAb epitope mapping (5).…”

“…A trypsin digest previously used to assess viral capsid folding was followed with some modifications (5,26,33). Briefly, capsomers were diluted into digestion buffer (10 mM Tris-HCl, 2 mM EDTA, 15 mM 2-␤-mercaptoethanol, 100 mM NaCl, 100 mM NaHCO 3 , pH 7.8) to a final concentration of 30 g/ml.…”

“…To ascertain that capsomers with mutagenized loops were properly folded after purification from bacterial cultures, the capsomers were assayed by trypsin digestion (5,26,33). Properly folded capsomers have only peripheral trypsin cleavage sites exposed, yielding L1 fragments of 42 kDa.…”

Subtype HPV38b[FA125] demonstrates heterogeneity of human papillomavirus type 38

“…Similarly, the substitution at position 291 was immediately before the surface-exposed hypervariable residue 285 in the FG loop of HPV16, which has been implicated as an important residue for binding of a major neutralizing antibody. 34,35 The possible surface exposure of the adjacent substitution at residue 288 in HPV38 is difficult to predict since the FG loop of HPV38 contains an insertion of seven amino acids seven residues before this position, compared to that of HPV16.…”