A Cryo-Electron Microscopy Study Identifies the Complete H16.V5 Epitope and Reveals Global Conformational Changes Initiated by Binding of the Neutralizing Antibody Fragment

Abstract

Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid-Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called "invading-arm" structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites. IMPORTANCE Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and "invading-arm" structures. This study advances the understanding of the neutralization mechanism used by H16.V5. Human papillomavirus (HPV) is a nonenveloped doublestranded DNA virus that can induce several epithelial cancers, especially cervical cancer (1-3). HPV16 is the most prevalent high-risk type of HPV (4, 5) and has been a primary target for the development of prophylactic vaccines (6, 7). HPV is epitheliotropic, and its replication is tightly associated with terminal differentiation of keratinocytes. This restricted tropism makes the production of high-titer preparations of authentic virion challenging. Alternative production methods have been developed to produce high-titer stocks of virus-like particles (VLP) (8), pseudovirions (PsV) (9), and quasivirions (QV) (10) while preserving the main attributes of the native capsid structure. These particles have been used successfully for vaccine development and for studies of antigenicity, receptor usage, entry mechanisms, and capsid structure.The infectious HPV has a Tϭ7 icosahedral capsid (55 to 60 nm in diameter), composed of 72 L1 capsid protein pentamers and up to 72 copies of L2 capsid protein located bene...

The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy

“…Subsequent structural studies have refined the C-terminal arm arrangement in HPV capsids (Fig. 8A) (9,17,18). Of the two types of available binding spaces (defined as type I and II in Results) for H16.U4 (Fig.…”

“…The centrifuged pellet was resuspended in 1 M NaCl-0.2 M Tris (pH 7.4). After CsCl gradient purification, the lower band was collected, concentrated, and dialyzed against phosphate-buffered saline (PBS), as described previously (9). The concentrated QV16 particles were applied to Formvarcoated copper grids, stained with 2% phosphotungstic acid, and analyzed for integrity and concentration on a JEOL JEM 1400 electron microscope.…”

“…Twelve capsomers lie on an icosahedral five-fold vertex and are referred to as pentavalent capsomers, whereas the remaining 60 capsomers are each surrounded by six other capsomers and referred to as hexavalent capsomers. The C terminus of each L1 protein, called the C-terminal arm, extends along the capsid floor to interact with the neighboring capsomer before returning to the original donor cap-somer (9,17,18). Intercapsomer disulfide bonds are formed between cysteine C428 and C175, which stabilize the icosahedral structure and play an important role in virus maturation (18,19).…”

“…Since the HPV life cycle depends on the differentiation of keratinocytes, it is difficult to purify high-titer virus stocks for structural studies. Virus-like particles (VLPs) that are devoid of viral genome (11) have been used successfully for structural studies (8,12,13), whereas both pseudovirus (PsV) and quasivirus (QV), which contain expression plasmid DNA (14,15), have been used for structural studies and infectivity assays (9,10). For the work presented here, quasivirus has been used throughout.…”

Modeling the Role of Epitope Arrangement on Antibody Binding Stoichiometry in Flaviviruses

“…In a study of cryo-EM structures of Ab-virus complexes, Thouvenin and Hewat found that the fitting of Ab structures to the cryo-EM density was highly unreliable at partial occupancies of <50% and that the variation in density at regions with full occupancy was 515% (39), which suggests minimal and maximal limits for detectable partial occupancy of 15% and 85%, respectively. Previous cryo-EM studies of Ab-virus complexes have reported partial occupancies of 40-50% (40,41), whereas structures with partial occupancy of 20% do not reliably show density in icosahedral averaged structures (42,43). In the latter cases, low partial occupancy was detected based on the presence of a high-occupancy non-Fab-like overlap density of multiple overlapping low-density Fabs (42) or through the use of asymmetric reconstruction methods (43).…”