Atomic model of the papillomavirus capsid

Abstract

Papillomaviruses propagate in differentiating skin cells, and certain types are responsible for the onset of cervical cancer. We have combined image reconstructions from electron cryomicroscopy (cryoEM) of bovine papillomavirus at 9 A Ê resolution with coordinates from the crystal structure of small virus-like particles of the human papillomavirus type 16 L1 protein to generate an atomic model of the virion. The overall ®t of the L1 model into the cryoEM map is excellent, but residues 402±446 in the`C-terminal arm' must be rebuilt. We propose a detailed model for the structure of this arm, based on two constraints: the presence of an intermolecular disul®de bond linking residues 175 and 428, and the clear identi®cation of a feature in the image reconstruction corresponding to an a-helix near the C-terminus of L1. We have con®rmed the presence of the disul®de bond by mass spectrometry. Our`invading arm' model shows that papilloma-and polyomaviruses have a conserved capsid architecture. Most of the rebuilt C-terminal arm is exposed on the viral surface; it is likely to have a role in infection and in immunogenicity. Keywords: disul®de bond/electron microscopy/ papillomavirus/vaccine design/virus assembly IntroductionPapillomaviruses are double-stranded DNA viruses that propagate in differentiating skin cells. Over 100 human types been identi®ed, of which a fewÐnotably types 16, 18, 31 and 45Ðare known to be responsible for the onset of cervical cancer (Bosch et al., 1995;Parkin et al., 2000), the second most common cancer among women worldwide (Parkin et al., 2000). Papilloma virions are nonenveloped particles. Their outer shell,~600 A Ê in diameter, contains 72 pentamers of the protein L1, situated at the vertices of a T = 7dextro icosahedral lattice ( Figure 1B; Baker et al., 1991). Cellular histones condense the 8 kb genome into a double-stranded, covalently closed circular DNA minichromosome (Howley, 1996).Electron microscopy (EM) of virions in vitreous ice reveals that cottontail rabbit papillomavirus, bovine papillomavirus type 1 (BPV1) and human papillomavirus type 1 (HPV1) have essentially identical structures (Baker et al., 1991;Hagensee et al., 1994;Belnap et al., 1996;Trus et al., 1997); indeed, their L1 subunits have nearly 50% sequence identity. The atomic structure of recombinant HPV16 L1 has been determined from crystals of a 12-pentamer assembly, or small virus-like particle (small VLP), stable at low pH ( Figure 1C±F; Chen et al., 2000). Despite a lack of signi®cant sequence similarity, the architecture of the papillomavirus L1 pentamer closely resembles that of the VP1 pentamers of simian virus 40 (SV40) and murine polyomavirus: a ring of ®ve b-jellyroll domains, tightly linked by interacting loops between framework b-strands (Figure 1E and F;Liddington et al., 1991;Stehle et al., 1994). A C-terminal subdomain of the subunit mediates pentamer±pentamer contacts in the small VLPs of HPV16. The polypeptide chain exits the b-jellyroll, forms the protruding subdomain, and reinserts into the central ch...

Replication and Assembly of Human Papillomaviruses

“…Analysis of these data suggests a prominent role for Cys428 in the formation of interpentameric disulfide bonds, and reinforces previous assumptions made from the crystal structure of the HPV16, T=1 12-pentamer VLP (Chen et al, 2000;Kondo et al, 2007). These results contrast, however, with previous work using recombinant HPV33 VLPs, which depicted equivalent roles for Cys427 and Cys176 (i.e., the equivalent of Cys428 and Cys175 in HPV16) in the formation of interpentameric disulfide bonds, which supports previous claims that pentamers from HPV18 VLPs form interpentameric disulfide bonds with each other (Sapp et al, 1998;Modis et al, 2002). These results call into question whether disulfide-bonding patterns differ between HPV16 VLPs, and HPV18 and HPV33 VLPs, or whether the various techniques utilized to produce synthetic papillomavirus particles lead to disulfide interactions that do not normally occur.…”

“…HPV virions are non-enveloped, icosahedral particles approximately 50 to 60 nm in diameter (Hagensee et al, 1994;Chen et al, 2000;Modis et al, 2002). Each particle is composed of 72 pentamers of the L1 capsid protein.…”

“…However, analyses of recombinant VLPs suggest that a critical interpentameric disulfide bridge occurs between Cys428 and Cys175, and potentially Cys185 in HPV16, rather than between neighboring Cys428 residues (Sapp et al, 1998;Chen et al, 2000;Kondo et al, 2007). In addition, biochemical analyses assessing the disulfide bonding patterns of HPV virions utilized mass spectroscopy of VLP capsid proteins, coupled with cryoelectron microscopy image reconstructions of native virions, to determine which cysteine residue(s) is/are critical in the formation of interpentameric disulfide bonds (Modis et al, 2002). Briefly, HPV18 VLP L1 dimers were excised from a nonreducing polyacrylamide gel.…”

“…edu (Shepherd et al, 2006). trypsin-digested HPV18 VLP L1 dimers revealed a tryptic product that corresponded to an 11-residue tryptic fragment that flanked the Cys428 equivalent of HPV18, Cys429 (Modis et al, 2002). The tryptic fragment contained a 476-Da adduct on Cys429, which suggested that a tryptic peptide corresponding to Cys175 was linked to the Cys429 tryptic fragment via a disulfide bond (Modis et al, 2002).…”

“…trypsin-digested HPV18 VLP L1 dimers revealed a tryptic product that corresponded to an 11-residue tryptic fragment that flanked the Cys428 equivalent of HPV18, Cys429 (Modis et al, 2002). The tryptic fragment contained a 476-Da adduct on Cys429, which suggested that a tryptic peptide corresponding to Cys175 was linked to the Cys429 tryptic fragment via a disulfide bond (Modis et al, 2002). These results are in contrast to the assumptions made from the atomic structure of the HPV16, T=1, 12-pentamer VLP, in that it seems that interpentameric interactions take place via Cys175 and Cys428 rather than the predicted Cys428 and Cys428.…”