Carol M. Bator

Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor.

Two human rhinovirus serotypes complexed with two‐ and five‐domain soluble fragments of the cellular receptor, intercellular adhesion molecule‐1, have been investigated by X‐ray crystallographic analyses of the individual components and by cryo‐electron microscopy of the complexes. The three‐dimensional image reconstructions provide a molecular envelope within which the crystal structures of the viruses and the receptor fragments can be positioned with accuracy. The N‐terminal domain of the receptor binds to the rhinovirus ‘canyon’ surrounding the icosahedral 5‐fold axes. Fitting of molecular models into the image reconstruction density identified the residues on the virus that interact with those on the receptor surface, demonstrating complementarity of the electrostatic patterns for the tip of the N‐terminal receptor domain and the floor of the canyon. The complexes seen in the image reconstructions probably represent the first stage of a multistep binding process. A mechanism is proposed for the subsequent viral uncoating process.

Interaction of coxsackievirus B3 with the full length coxsackievirus-adenovirus receptor

Group B coxsackieviruses (CVB) utilize the coxsackievirus-adenovirus receptor (CAR) to recognize host cells. CAR is a membrane protein with two Ig-like extracellular domains (D1 and D2), a transmembrane domain and a cytoplasmic domain. The three-dimensional structure of coxsackievirus B3 (CVB3) in complex with full length human CAR and also with the D1D2 fragment of CAR were determined to ∼22 Å resolution using cryo-electron microscopy (cryo-EM). Pairs of transmembrane domains of CAR associate with each other in a detergent cloud that mimics a cellular plasma membrane. This is the first view of a virus–receptor interaction at this resolution that includes the transmembrane and cytoplasmic portion of the receptor. CAR binds with the distal end of domain D1 in the canyon of CVB3, similar to how other receptor molecules bind to entero- and rhinoviruses. The previously described interface of CAR with the adenovirus knob protein utilizes a side surface of D1.

Complexes of Poliovirus Serotypes with Their Common Cellular Receptor, CD155

ABSTRACT Structures of all three poliovirus (PV) serotypes (PV1, PV2, and PV3) complexed with their cellular receptor, PV receptor (PVR or CD155), were determined by cryoelectron microscopy. Both glycosylated and fully deglycosylated CD155 exhibited similar binding sites and orientations in the viral canyon for all three PV serotypes, showing that all three serotypes use a common mechanism for cell entry. Difference maps between the glycosylated and deglycosylated CD155 complexes determined the sites of the carbohydrate moieties that, in turn, helped to verify the position of the receptor relative to the viral surface. The proximity of the CD155 carbohydrate site at Asn105 to the viral surface in the receptor-virus complex suggests that it might interfere with receptor docking, an observation consistent with the properties of mutant CD155. The footprints of CD155 on PV surfaces indicate that the south rim of the canyon dominates the virus-receptor interactions and may correspond to the initial CD155 binding state of the receptor-mediated viral uncoating. In contrast, the interaction of CD155 with the north rim of the canyon, especially the region immediately outside the viral hydrophobic pocket that normally binds a cellular “pocket factor,” may be critical for the release of the pocket factor, decreasing the virus stability and hence initiating uncoating. The large area of the CD155 footprint on the PV surface, in comparison with other picornavirus-receptor interactions, could be a potential limitation on the viability of PV escape mutants from antibody neutralization. Many of these are likely to have lost their ability to bind CD155, resulting in there being only three PV serotypes.

Interaction of Coxsackievirus A21 with Its Cellular Receptor, ICAM-1

ABSTRACT Coxsackievirus A21 (CAV21), like human rhinoviruses (HRVs), is a causative agent of the common cold. It uses the same cellular receptor, intercellular adhesion molecule 1 (ICAM-1), as does the major group of HRVs; unlike HRVs, however, it is stable at acid pH. The cryoelectron microscopy (cryoEM) image reconstruction of CAV21 is consistent with the highly homologous crystal structure of poliovirus 1; like other enteroviruses and HRVs, CAV21 has a canyon-like depression around each of the 12 fivefold vertices. A cryoEM reconstruction of CAV21 complexed with ICAM-1 shows all five domains of the extracellular component of ICAM-1. The known atomic structure of the ICAM-1 amino-terminal domains D1 and D2 has been fitted into the cryoEM density of the complex. The site of ICAM-1 binding within the canyon of CAV21 overlaps the site of receptor recognition utilized by rhinoviruses and polioviruses. Interactions within this common region may be essential for triggering viral destabilization after attachment to susceptible cells.

Crystal structure of CD155 and electron microscopic studies of its complexes with polioviruses.

When poliovirus (PV) recognizes its receptor, CD155, the virus changes from a 160S to a 135S particle before releasing its genome into the cytoplasm. CD155 is a transmembrane protein with 3 Ig-like extracellular domains, D1–D3, where D1 is recognized by the virus. The crystal structure of D1D2 has been determined to 3.5-Å resolution and fitted into ≈8.5-Å resolution cryoelectron microscopy reconstructions of the virus–receptor complexes for the 3 PV serotypes. These structures show that, compared with human rhinoviruses, the virus–receptor interactions for PVs have a greater dependence on hydrophobic interactions, as might be required for a virus that can inhabit environments of different pH. The pocket factor was shown to remain in the virus during the first recognition stage. The present structures, when combined with earlier mutational investigations, show that in the subsequent entry stage the receptor moves further into the canyon when at a physiological temperature, thereby expelling the pocket factor and separating the viral subunits to form 135S particles. These results provide a detailed analysis of how a nonenveloped virus can enter its host cell.

Structure of decay-accelerating factor bound to echovirus 7: A virus-receptor complex

Echoviruses are enteroviruses that belong to Picornaviridae. Many echoviruses use decay-accelerating factor (DAF) as their cellular receptor. DAF is a glycosylphosphatidyl inositol-anchored complement regulatory protein found on most cell surfaces. It functions to protect cells from complement attack. The cryo-electron microscopy reconstructions of echovirus 7 complexed with DAF show that the DAF-binding regions are located close to the icosahedral twofold axes, in contrast to other enterovirus complexes where the viral canyon is the receptor binding site. This novel receptor binding position suggests that DAF is important for the attachment of viral particles to host cells, but probably not for initiating viral uncoating, as is the case with canyon-binding receptors. Thus, a different cell entry mechanism must be used for enteroviruses that bind DAF.

Structural and virological studies of the stages of virus replication that are affected by antirhinovirus compounds

ABSTRACT Pleconaril is a broad-spectrum antirhinovirus and antienterovirus compound that binds into a hydrophobic pocket within viral protein 1, stabilizing the capsid and resulting in the inhibition of cell attachment and RNA uncoating. When crystals of human rhinovirus 16 (HRV16) and HRV14 are incubated with pleconaril, drug occupancy in the binding pocket is lower than when pleconaril is introduced during assembly prior to crystallization. This effect is far more marked in HRV16 than in HRV14 and is more marked with pleconaril than with other compounds. These observations are consistent with virus yield inhibition studies and radiolabeled drug binding studies showing that the antiviral effect of pleconaril against HRV16 is greater on the infectivity of progeny virions than the parent input viruses. These data suggest that drug integration into the binding pocket during assembly, or at some other late stage in virus replication, may contribute to the antiviral activity of capsid binding compounds.

Interaction of Decay-Accelerating Factor with Echovirus 7

ABSTRACT Echovirus 7 (EV7) belongs to the Enterovirus genus within the family Picornaviridae. Many picornaviruses use IgG-like receptors that bind in the viral canyon and are required to initiate viral uncoating during infection. However, in addition, some of the enteroviruses use an alternative or additional receptor that binds outside the canyon. Decay-accelerating factor (DAF) has been identified as a cellular receptor for EV7. The crystal structure of EV7 has been determined to 3.1-Å resolution and used to interpret the 7.2-Å-resolution cryo-electron microscopy reconstruction of EV7 complexed with DAF. Each DAF binding site on EV7 is near a 2-fold icosahedral symmetry axis, which differs from the binding site of DAF on the surface of coxsackievirus B3, indicating that there are independent evolutionary processes by which DAF was selected as a picornavirus accessory receptor. This suggests that there is an advantage for these viruses to recognize DAF during the initial process of infection.

Sample preparation induced artifacts in cryo-electron tomographs.

We investigated the effects of sample preparation and of the exposure to an electron beam on particles in cryo-electron tomographs. Various virus particles with icosahedral symmetry were examined, allowing a comparison of symmetrically related components that should be identical in structure but might be affected differently by these imaging artifacts. Comparison of tomographic reconstructions with previously determined structures established by an independent method showed that neither freezing nor electron beam exposure produced a significant amount of shrinkage along the z axis (thickness). However, we observed damage to regions of the particles located close to the surface of the vitreous ice.