Maturation of the human papillomavirus 16 capsid - PubMed (original) (raw)

Maturation of the human papillomavirus 16 capsid

Giovanni Cardone et al. mBio. 2014.

Abstract

Papillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, under in vitro conditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized "suspended bridges" that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism. Importance: Human papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines.

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Figures

FIG 1

(a to c) Cryo-electron micrograph images of HPV16 capsids matured for 18 h (a), 4 h (b), and 1 h (c). (d) L1 C175S mutant capsids matured for 1 h. Note the presence of larger capsids in the 4-h and 1-h preparations. Larger capsids are indicated by asterisks. Bar (top left) = 200 Å.

FIG 2

Computer analysis of the capsids allowed to mature for 18 h provided a 3D reconstruction of mature HPV16 capsids (a) as well as two slightly larger, presumably less mature capsids (b and c). All images have had their internal DNA computationally removed in order to visualize the protein capsid. The mature capsid (a) contained 1,026 images and has a nominal diameter of 600 Å. The next capsid class (b) contained 1,106 images and has a 610-Å diameter. The least mature capsid (c) contained 1,296 images and has a 626-Å diameter. Bar = 200 Å.

FIG 3

Combining 18-h, 4-h, and 1-h capsids provided a 3D reconstruction of mature 600 Å capsids (a) as well as four intermediates ranging in size from (nominally) a 605-Å diameter to a 633-Å diameter (b to e). All images have had their internal DNA computationally removed in order to visualize the protein capsid shell. The relative size and number of particles for each class are as follows: (a) 600 Å, 1,675 particles; (b) 607 Å, 591 particles; (c) 612 Å, 588 particles; (d) 619 Å, 982 particles; (e) 633 Å, 1,252 particles. The first column shows the 2-fold view. The second column shows the same view with the front half removed. The third column shows radial density at a radius of 260 Å (a) and scaled by diameter (b to e). Bar = 200 Å. The same results are presented as an animated file in Fig. S3 in the supplemental material.

FIG 4

C175S mutant HPV16 capsids, which cannot form cystine 175 cross-links, provided the following final result starting from 3 wt models, a, d, and e (Fig. 3). Refinement converged into two models: smaller capsids (a) as well as larger, less mature capsids (b). There were 582 images in the smaller (a) and 771 images in the larger (b). Cutaway views of panels a and b are shown in panels c and d, respectively. Their respective resolutions are 34 Å and 29 Å (FSC = 0.3). Bar = 200 Å.

FIG 5

To facilitate the formation of intermolecular disulfide bonds between neighboring L1 molecules, lysates of 293TT cells expressing L1 were buffered with ammonium sulfate. Capsids derived from the buffered lysate exhibited a greater extent of maturation. This is reflected in a greater fraction of the slower-migrating ring trimer and faster-migrating reciprocal dimer.

FIG 6

Reconstruction of a fully mature HPV16 capsid at 9 Å resolution. (A) Surface rendering of the capsid, colored according to radius. Radius values (color bar) are in angstroms. (B) Radial sections of the reconstruction taken at 7-Å increments. Shown are radii from 299 Å (near the tip of the capsomers) to 250 Å (close to the floor of the canyons between capsomers). An image taken from one electron micrograph of fully mature HPV16 capsids is shown in Fig. S4.

FIG 7

Pseudoatomic modeling of the mature HPV16 capsid. (A and B) Two views of the surface rendering of the capsid, shown in semitransparency, overlapped with the models obtained for the six subunits in one asymmetric unit, with each chain colored differently. The models were obtained by molecular dynamics-based flexible fitting, using the atomic coordinates available for the core of the HPV16 major capsid protein L1 (PDB ID, 1DZL) as a starting reference. (C) Superposition of the six models obtained for the six nonequivalent locations of the protein L1 in one asymmetric unit. The differences between the models are only in the arrangement of the intercapsomeric connections formed by the N and C termini.

FIG 8

Comparison between models of HPV16 and BPV1. The model of BPV1 is from a cryo-EM reconstruction obtained by Wolf et al. (18) (PDB ID, 3IYJ). (A) Top view. (B) Side view. All the subunits of the BPV model are in black, while those of HPV16 are colored using the same scheme as in Fig. 7.

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Maturation of the human papillomavirus 16 capsid - PubMed (original) (raw)