Mechanism of enterovirus VP0 maturation cleavage based on the structure of a stabilised assembly intermediate.

Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0.

Mechanism of enterovirus VP0 maturation cleavage based on the structure of a stabilised assembly intermediate.

Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0.

Real-Time Imaging of Polioviral RNA Translocation across a Membrane.

Genome transfer from a virus into a cell is a critical early step in viral replication. Enveloped viruses achieve the delivery of their genomes into the cytoplasm by merging the viral membrane with the cellular membrane via a conceptually simple mechanism called membrane fusion. In contrast, genome translocation mechanisms in nonenveloped viruses, which lack viral membranes, remain poorly understood.

Resistance to a Nucleoside Analog Antiviral Drug from More Rapid Extension of Drug-Containing Primers.

Nucleoside analogs are mainstays of antiviral therapy. Although resistance to these drugs hinders their use, understanding resistance can illuminate mechanisms of the drugs and their targets. Certain nucleoside analogs, such as ganciclovir (GCV), a leading therapy for human cytomegalovirus (HCMV), contain the equivalent of a 3'-hydoxyl moiety, yet their triphosphates can terminate genome synthesis (nonobligate chain termination).

Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate.

The virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and by the opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane-interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle.

Cryo-EM reveals the structural basis of long-range electron transport in a cytochrome-based bacterial nanowire.

Electrically conductive pili from species, termed bacterial nanowires, are intensely studied for their biological significance and potential in the development of new materials. Using cryo-electron microscopy, we have characterized nanowires from conductive pili preparations that are composed solely of head-to-tail stacked monomers of the six-heme C-type cytochrome OmcS. The unique fold of OmcS - closely wrapped around a continuous stack of hemes that can serve as an uninterrupted path for electron transport - generates a scaffold that supports the unbranched chain of hemes along the central axis of the filament.

A Small Covalent Allosteric Inhibitor of Human Cytomegalovirus DNA Polymerase Subunit Interactions.

Human cytomegalovirus DNA polymerase comprises a catalytic subunit, UL54, and an accessory subunit, UL44, the interaction of which may serve as a target for the development of new antiviral drugs. Using a high-throughput screen, we identified a small molecule, (5-((dimethylamino)methylene-3-(methylthio)-6,7-dihydrobenzo[c]thiophen-4(5H)-one), that selectively inhibits the interaction of UL44 with a UL54-derived peptide in a time-dependent manner, full-length UL54, and UL44-dependent long-chain DNA synthesis. A crystal structure of the compound bound to UL44 revealed a covalent reaction with lysine residue 60 and additional noncovalent interactions that cause steric conflicts that would prevent the UL44 connector loop from interacting with UL54.

Getting to and through the inner nuclear membrane during herpesvirus nuclear egress.

Herpesviruses, like most DNA viruses, replicate and package their genomes into capsids in the host cell nucleus. Capsids then transit to the cytoplasm in a fascinating process called nuclear egress, which includes several unusual steps: Movement of capsids from the nuclear interior to the periphery, disruption of the nuclear lamina, capsid budding through the inner nuclear membrane, and fusion of enveloped particles with the outer nuclear membrane. Here, we review recent advances and emerging questions relating to herpesvirus nuclear egress, emphasizing controversies regarding mechanisms for capsid trafficking to the nuclear periphery, and implications of recent structures of the two-subunit, viral nuclear egress complex for the process, particularly at the step of budding through the inner nuclear membrane.

Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State.

Unlabelled: By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs bind to a site on the top surface of the capsid protein VP3, which is hidden in the native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the Nanobodies has sampled a range of low-energy structures available to the expanded virion.

Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

Unlabelled: Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site.

Unexpected features and mechanism of heterodimer formation of a herpesvirus nuclear egress complex.

Herpesvirus nucleocapsids escape from the nucleus in a process orchestrated by a highly conserved, viral nuclear egress complex. In human cytomegalovirus, the complex consists of two proteins, UL50 and UL53. We solved structures of versions of UL53 and the complex by X-ray crystallography.

Structure of a herpesvirus nuclear egress complex subunit reveals an interaction groove that is essential for viral replication.

Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50.

Characterization of Poliovirus Neutralization Escape Mutants of Single-Domain Antibody Fragments (VHHs).

To complete the eradication of poliovirus and to protect unvaccinated people subsequently, the development of one or more antiviral drugs will be necessary. A set of five single-domain antibody fragments (variable parts of the heavy chain of a heavy-chain antibody [VHHs]) with an in vitro neutralizing activity against poliovirus type 1 was developed previously (B. Thys, L.

Nectin-like interactions between poliovirus and its receptor trigger conformational changes associated with cell entry.

Unlabelled: Poliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane.

Mechanism of action and capsid-stabilizing properties of VHHs with an in vitro antipolioviral activity.

Unlabelled: Previously, we reported on the in vitro antiviral activity of single-domain antibody fragments (VHHs) directed against poliovirus type 1. Five VHHs were found to neutralize poliovirus type 1 in an in vitro setting and showed 50% effective concentrations (EC50s) in the nanomolar range. In the present study, we further investigated the mechanism of action of these VHHs.

Cryo-electron microscopy reconstruction shows poliovirus 135S particles poised for membrane interaction and RNA release.

During infection, binding of mature poliovirus to cell surface receptors induces an irreversible expansion of the capsid, to form an infectious cell-entry intermediate particle that sediments at 135S. In these expanded virions, the major capsid proteins (VP1 to VP3) adopt an altered icosahedral arrangement to open holes in the capsid at 2-fold and quasi-3-fold axes, and internal polypeptides VP4 and the N terminus of VP1, which can bind membranes, become externalized. Cryo-electron microscopy images for 117,330 particles were collected using Leginon and reconstructed using FREALIGN.

RNA transfer from poliovirus 135S particles across membranes is mediated by long umbilical connectors.

During infection, the binding of poliovirus to its cell surface receptor at 37°C triggers an expansion of the virus in which internal polypeptides that bind to membranes are externalized. Subsequently, in a poorly understood process, the viral RNA genome is transferred directly across an endosomal membrane, and into the host cell cytoplasm, to initiate infection. Here, cryoelectron tomography demonstrates the results of 37°C warming of a poliovirus-receptor-liposome model complex that was produced using Ni-nitrilotriacetic acid lipids and His-tagged receptor ectodomains.

Structure of the Fab-labeled "breathing" state of native poliovirus.

At 37°C, the structure of poliovirus is dynamic, and internal polypeptides VP4 and N terminus of VP1 (residues 1 to 53) externalize reversibly. An Fab fragment of a monospecific antibody, which binds to residues 39 to 55 of VP1, was utilized to locate the N termini of VP1 in native (160S) particles in this "breathing" state. Fab and virus were mixed and imaged via cryogenic electron microscopy.

An externalized polypeptide partitions between two distinct sites on genome-released poliovirus particles.

During cell entry, native poliovirus (160S) converts to a cell-entry intermediate (135S) particle, resulting in the externalization of capsid proteins VP4 and the amino terminus of VP1 (residues 1 to 53). Externalization of these entities is followed by release of the RNA genome (uncoating), leaving an empty (80S) particle. The antigen-binding fragment (Fab) of a monospecific peptide 1 (P1) antibody, which was raised against a peptide corresponding to amino-terminal residues 24 to 40 of VP1, was utilized to track the location of the amino terminus of VP1 in the 135S and 80S states of poliovirus particles via cryogenic electron microscopy (cryo-EM) and three-dimensional image reconstruction.

Poliovirus RNA is released from the capsid near a twofold symmetry axis.

After recognizing and binding to its host cell, poliovirus (like other nonenveloped viruses) faces the challenge of translocating its genome across a cellular membrane and into the cytoplasm. To avoid entanglement with the capsid, the RNA must exit via a single site on the virion surface. However, the mechanism by which a single site is selected (from among 60 equivalents) is unknown; and until now, even its location on the virion surface has been controversial.

Catching a virus in the act of RNA release: a novel poliovirus uncoating intermediate characterized by cryo-electron microscopy.

Poliovirus infection requires that the particle undergo a series of conformational transitions that lead to cell entry and genome release. In an effort to understand the conformational changes associated with the release of the RNA genome, we have used cryo-electron microscopy to characterize the structure of the 80S "empty" particles of poliovirus that are thought to represent the final product of the cell entry pathway. Using two-dimensional classification methods, we show that preparations of 80S particles contain at least two structures, which might represent snapshots from a continuous series of conformers.

The crystal structure of PF-8, the DNA polymerase accessory subunit from Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus is an emerging pathogen whose mechanism of replication is poorly understood. PF-8, the presumed processivity factor of Kaposi's sarcoma-associated herpesvirus DNA polymerase, acts in combination with the catalytic subunit, Pol-8, to synthesize viral DNA. We have solved the crystal structure of residues 1 to 304 of PF-8 at a resolution of 2.

The human cytomegalovirus UL44 C clamp wraps around DNA.

Processivity factors tether the catalytic subunits of DNA polymerases to DNA so that continuous synthesis of long DNA strands is possible. The human cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer intermediate in structure between monomeric herpes simplex virus UL42, which binds DNA directly via a basic surface, and the trimeric sliding clamp PCNA, which encircles DNA. To investigate how UL44 interacts with DNA, calculations were performed in which a 12 bp DNA oligonucleotide was docked to UL44.

Post-imaging fiducial markers aid in the orientation determination of complexes with mixed or unknown symmetry.

During the entry process many icosahedral viruses must adopt a lower-order symmetry or incur a symmetry mismatch to release their genome through a single site. A membrane model system in which poliovirus was bound to receptor-decorated liposomes was used to pioneer techniques that studied the break in the symmetry of the initial attachment complex by cryo-electron microscopy. Novel methods involving a fiducial marker for the membrane contact point were developed to objectively determine the symmetry of this complex and provide a starting model to initiate a bootstrap orientation refinement.