Hyunwook Lee

Secretion of genome-free hepatitis B virus--single strand blocking model for virion morphogenesis of para-retrovirus.

As a para-retrovirus, hepatitis B virus (HBV) is an enveloped virus with a double-stranded (DS) DNA genome that is replicated by reverse transcription of an RNA intermediate, the pregenomic RNA or pgRNA. HBV assembly begins with the formation of an “immature” nucleocapsid (NC) incorporating pgRNA, which is converted via reverse transcription within the maturing NC to the DS DNA genome. Only the mature, DS DNA-containing NCs are enveloped and secreted as virions whereas immature NCs containing RNA or single-stranded (SS) DNA are not enveloped. The current model for selective virion morphogenesis postulates that accumulation of DS DNA within the NC induces a “maturation signal” that, in turn, triggers its envelopment and secretion. However, we have found, by careful quantification of viral DNA and NCs in HBV virions secreted in vitro and in vivo, that the vast majority of HBV virions (over 90%) contained no DNA at all, indicating that NCs with no genome were enveloped and secreted as empty virions (i.e., enveloped NCs with no DNA). Furthermore, viral mutants bearing mutations precluding any DNA synthesis secreted exclusively empty virions. Thus, viral DNA synthesis is not required for HBV virion morphogenesis. On the other hand, NCs containing RNA or SS DNA were excluded from virion formation. The secretion of DS DNA-containing as well as empty virions on one hand, and the lack of secretion of virions containing single-stranded (SS) DNA or RNA on the other, prompted us to propose an alternative, “Single Strand Blocking” model to explain selective HBV morphogenesis whereby SS nucleic acid within the NC negatively regulates NC envelopment, which is relieved upon second strand DNA synthesis.

Enterovirus 71 Binding to PSGL-1 on Leukocytes: VP1- 145 Acts as a Molecular Switch to Control Receptor Interaction

Some strains of enterovirus 71 (EV71), but not others, infect leukocytes by binding to a specific receptor molecule: the P-selectin glycoprotein ligand-1 (PSGL-1). We find that a single amino acid residue within the capsid protein VP1 determines whether EV71 binds to PSGL-1. Examination of capsid sequences of representative EV71 strains revealed that the PSGL-1-binding viruses had either a G or a Q at residue 145 within the capsid protein VP1 (VP1-145G or Q), whereas PSGL-1-nonbinding viruses had VP1-145E. Using site-directed mutagenesis we found that PSGL-1-binding strains lost their capacity to bind when VP1-145G/Q was replaced by E; conversely, nonbinding strains gained the capacity to bind PSGL-1 when VP1-145E was replaced with either G or Q. Viruses with G/Q at VP1-145 productively infected a leukocyte cell line, Jurkat T-cells, whereas viruses with E at this position did not. We previously reported that EV71 binds to the N-terminal region of PSGL-1, and that binding depends on sulfated tyrosine residues within this region. We speculated that binding depends on interaction between negatively charged sulfate groups and positively charged basic residues in the virus capsid. VP1-145 on the virus surface is in close proximity to conserved lysine residues at VP1-242 and VP1-244. Comparison of recently published crystal structures of EV71 isolates with either Q or E at VP1-145 revealed that VP1-145 controls the orientation of the lysine side-chain of VP1-244: with VP1-145Q the lysine side chain faces outward, but with VP1-145E, the lysine side chain is turned toward the virus surface. Mutation of VP1-244 abolished virus binding to PSGL-1, and mutation of VP1-242 greatly reduced binding. We propose that conserved lysine residues on the virus surface are responsible for interaction with sulfated tyrosine residues at the PSGL-1 N-terminus, and that VP1-145 acts as a switch, controlling PSGL-1 binding by modulating the exposure of VP1-244K.

A Strain-Specific Epitope of Enterovirus 71 Identified by Cryo-Electron Microscopy of the Complex with Fab from Neutralizing Antibody

ABSTRACT Enterovirus 71 (EV71) is a picornavirus that causes outbreaks of hand, foot, and mouth disease (HFMD), primarily in the Asia-Pacific area. Unlike coxsackievirus A16, which also causes HFMD, EV71 induces severe neuropathology leading to high fatalities, especially among children under the age of 6 years. Currently, no established vaccines or treatments are available against EV71 infection. The monoclonal antibody MA28-7 neutralizes only specific strains of EV71 that have a conserved glycine at amino acid VP1-145, a surface-exposed residue that maps to the 5-fold vertex and that has been implicated in receptor binding. The cryo-electron microscopy structure of a complex between EV71 and the Fab fragment of MA28-7 shows that only one Fab fragment occupies each 5-fold vertex. A positively charged patch, which has also been implicated in receptor binding, lies within the Fab footprint. We identify the strain-specific epitope of EV71 and discuss the possible neutralization mechanisms of the antibody.

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.V5 conformational 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.

Structures of the Procapsid and Mature Virion of Enterovirus 71 Strain 1095

ABSTRACT Enterovirus 71 (EV71) is an important emerging human pathogen with a global distribution and presents a disease pattern resembling poliomyelitis with seasonal epidemics that include cases of severe neurological complications, such as acute flaccid paralysis. EV71 is a member of the Picornaviridae family, which consists of icosahedral, nonenveloped, single-stranded RNA viruses. Here we report structures derived from X-ray crystallography and cryoelectron microscopy (cryo-EM) for the 1095 strain of EV71, including a putative precursor in virus assembly, the procapsid, and the mature virus capsid. The cryo-EM map of the procapsid provides new structural information on portions of the capsid proteins VP0 and VP1 that are disordered in the higher-resolution crystal structures. Our structures solved from virus particles in solution are largely in agreement with those from prior X-ray crystallographic studies; however, we observe small but significant structural differences for the 1095 procapsid compared to a structure solved in a previous study (X. Wang, W. Peng, J. Ren, Z. Hu, J. Xu, Z. Lou, X. Li, W. Yin, X. Shen, C. Porta, T. S. Walter, G. Evans, D. Axford, R. Owen, D. J. Rowlands, J. Wang, D. I. Stuart, E. E. Fry, and Z. Rao, Nat. Struct. Mol. Biol. 19:424–429, 2012) for a different strain of EV71. For both EV71 strains, the procapsid is significantly larger in diameter than the mature capsid, unlike in any other picornavirus. Nonetheless, our results demonstrate that picornavirus capsid expansion is possible without RNA encapsidation and that picornavirus assembly may involve an inward radial collapse of the procapsid to yield the native virion.

Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization.

Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope. Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays.

The novel asymmetric entry intermediate of a picornavirus captured with nanodiscs.

Nanodiscs used as a mock membrane captured a picornavirus undergoing global and local changes to enter host cells. Many nonenveloped viruses engage host receptors that initiate capsid conformational changes necessary for genome release. Structural studies on the mechanisms of picornavirus entry have relied on in vitro approaches of virus incubated at high temperatures or with excess receptor molecules to trigger the entry intermediate or A-particle. We have induced the coxsackievirus B3 entry intermediate by triggering the virus with full-length receptors embedded in lipid bilayer nanodiscs. These asymmetrically formed A-particles were reconstructed using cryo-electron microscopy and a direct electron detector. These first high-resolution structures of a picornavirus entry intermediate captured at a membrane with and without imposing icosahedral symmetry (3.9 and 7.8 Å, respectively) revealed a novel A-particle that is markedly different from the classical A-particles. The asymmetric receptor binding triggers minimal global capsid expansion but marked local conformational changes at the site of receptor interaction. In addition, viral proteins extrude from the capsid only at the site of extensive protein remodeling adjacent to the nanodisc. Thus, the binding of the receptor triggers formation of a unique site in preparation for genome release.

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

ABSTRACT The human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformation-dependent neutralizing epitope of HPV thought to include the L1 protein C terminus. The structure of human papillomavirus 16 (HPV16) complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab bound only to epitopes located around the icosahedral five-fold vertex of the capsid. Thus, the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers. IMPORTANCE Human papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This monoclonal antibody (MAb) is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus infection. Our analyses precisely define important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines.

Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus.

ABSTRACT Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. IMPORTANCE Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time.

MOPS and coxsackievirus B3 stability

Study of coxsackievirus B3 strain 28 (CVB3/28) stability using MOPS to improve buffering in the experimental medium revealed that MOPS (3-morpholinopropane-1-sulfonic acid) increased CVB3 stability and the effect was concentration dependent. Over the pH range 7.0-7.5, virus stability was affected by both pH and MOPS concentration. Computer-simulated molecular docking showed that MOPS can occupy the hydrophobic pocket in capsid protein VP1 where the sulfonic acid head group can form ionic and hydrogen bonds with Arg95 and Asn211 near the pocket opening. The effects of MOPS and hydrogen ion concentrations on the rate of virus decay were modeled by including corresponding parameters in a recent kinetic model. These results indicate that MOPS can directly associate with CVB3 and stabilize the virus, possibly by altering capsid conformational dynamics.