Potent neutralization of hepatitis A virus reveals a receptor mimic mechanism and the receptor recognition site

Abstract

Hepatitis A virus (HAV) infects ∼1.4 million people annually and, although there is a vaccine, there are no licensed therapeutic drugs. HAV is unusually stable (making disinfection problematic) and little is known of how it enters cells and releases its RNA. Here we report a potent HAV-specific monoclonal antibody, R10, which neutralizes HAV infection by blocking attachment to the host cell. High-resolution cryo-EM structures of HAV full and empty particles and of the complex of HAV with R10 Fab reveal the atomic details of antibody binding and point to a receptor recognition site at the pentamer interface. These results, together with our observation that the R10 Fab destabilizes the capsid, suggest the use of a receptor mimic mechanism to neutralize virus infection, providing new opportunities for therapeutic intervention.picornavirus | entry | neutralizing mechanism | receptor recognition | uncoating H epatitis A virus (HAV) is an ancient and ubiquitous pathogen found in primates and small mammals (1). It is a picornavirus with many distinctive features: in the blood it is found in an enveloped form but is shed in feces as a naked, unenveloped particle (2); it possesses a low G/C ratio in the genome sequence and a strong codon bias (3); it grows poorly in tissue culture; it possesses a 67-residue carboxyl-terminal extension of VP1 (VP1-2A or VPX), which is important for viral assembly (4); and it has a very short, nonmyristoylated VP4 (∼23 residues) (5). Because of its unusual properties, HAV remains enigmatic and occupies an evolutionary position on the periphery of picornaviruses (6, 7).Our previous crystal structure of unenveloped HAV (7) showed no trace of the canyon that encircles the fivefold axes of enteroviruses and is often the site of receptor binding (8, 9). Indeed, the structure provided no clues as to where T-cell Ig and mucin 1 (TIM-1), the proposed receptor (10, 11), might attach. In addition, the virus capsid contains no pocket factor and can withstand remarkably high temperature and low pH, indicating an uncoating mechanism (7) unlike that of enteroviruses; heating of HAV particles does not transform virions to an expanded state in vitro. Structural characterization of a HAV-TIM-1 complex might elucidate the cell-entry mechanism, but difficulty in obtaining homogenous complex preparations because of the low binding affinity of TIM-1, at least for unenveloped particles, has made this challenging. However, it is known that neutralizing antibodies (NAbs) protect against virus infection by mechanisms that include blocking attachment to the cellular receptor, overstabilizing the virus, preventing viral genome release, or physically destabilizing the virus (12, 13). Structural studies of virus complexes with such antibodies can therefore help illuminate these underlying biological functions. Here we identify a highly potent Nab, R10, which can neutralize HAV infection efficiently by blocking attachment to the host cell. The complex structure of the HAV full particle and R10 Fab suggest where the re...

TIM1 (HAVCR1) Is Not Essential for Cellular Entry of Either Quasi-enveloped or Naked Hepatitis A Virions

“…First, it is possible that the quasi-enveloped capsid, which contains an 8-kDa extension on each of its 60 VP1 molecules ( 1 ), may differ structurally from the naked viral particle. Consistent with this, tandem YPX 3 L “late domains” present in VP2 that appear to interact with the ESCRT-associated protein ALIX during the process of quasi-envelopment are mostly buried below the surface of the X-ray structure of the naked HAV capsid ( 30 , 31 ). If their capsid structures do differ significantly, HAV and eHAV may interact with different receptors.…”

The binding of a monoclonal antibody to the apical region of SCARB2 blocks EV71 infection

“…Crystals of SCARB2-JL2 complex diffracted to 3.5 Å, belonging to the space group C2, and the crystals contained two complex molecules in the asymmetric unit with a solvent content of 58% (corresponding to a Matthews coefficient VM = 2.7 Å 3 ·Da −1 ) (Matthews, 1968 ). The complex structure was determined using the molecular replacement method based on the combination of the models of SCARB2 at neutral pH (PDB code: 4TW2) (Dang et al, 2014 ) and the mouse Fab (PDB code: 5WTG) (Wang et al, 2017 ). The final refined complex model had reasonable R-factors and very good stereochemistry.…”

“…The data sets were processed and scaled using the HKL2000 package (Otwinowski and Minor, 1997 ). The initial structure solutions of the SCARB2-Fab complex were obtained by molecular replacement using the program Phaser v2.1 (McCoy et al, 2007 ) with the crystal structure of SCARB2 (PDB code: 4TW2) (Dang et al, 2014 ) and the mouse Fab structure (Wang et al, 2017 ) (PDB code: 5WTG) as a search template. Manual model building and refinement were performed using COOT (Emsley and Cowtan, 2004 ) and PHENIX (Adams et al, 2010 ) following rigid body refinement, energy minimization, B-factor refinement, and group NCS constraints.…”

Epitope-associated and specificity-focused features of EV71-neutralizing antibody repertoires from plasmablasts of infected children

“…In contrast to canyon epitopes, 3- and 2-fold plateau epitopes are not within or adjacent to known receptor binding sites, although antibodies targeting these epitopes neutralize viral infectivity prior to virus attachment. It is possible that these epitopes are in the neighborhood of as-yet unidentified receptor-binding sites; 62 however, this neutralization may involve an alternative mechanism 63 , 64 .…”