James Hogle | Harvard Medical School (original) (raw)

Papers by James Hogle

The Journal of biological chemistry, Jan 26, 2014

RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. T... more RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic expe...

PLoS pathogens, 2014

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantag... more Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA ...

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantag... more Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and footand-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

PLoS Pathogens, 2014

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantag... more Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and footand-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

Journal of Virology, 2014

Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egres... more Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC.

The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (dAg-S) is essential fo... more The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (dAg-S) is essential for genome replication, i.e., for the transcription, processing, and accumulation of HDV RNAs. To better understand this requirement, we used purified recombinant dAg-S and HDV RNA synthesized in vitro to assemble high- molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs into human cells, we detected

Nature Structural Biology, 1995

One approach to combinatorial ligand design begins by determining optimal locations (i.e., local ... more One approach to combinatorial ligand design begins by determining optimal locations (i.e., local potential energy minima) for functional groups in the binding site of a target macromolecule. MCSS and GRID are two methods, based on significantly different algorithms, which are used for this purpose. A comparison of the two methods for the same functional groups is reported. Calculations were performed for nonpolar and polar functional groups in the internal hydrophobic pocket of the poliovirus capsid protein, and on the binding surface of the src SH3 domain. The two approaches are shown to agree qualitatively; i.e., the global characteristics of the functional group maps generated by MCSS and GRID are similar. However, there are significant differences in the relative interaction energies of the two sets of minima, a consequence of the different functional form used to evaluate polar interactions (electrostatics and hydrogen bonding) in the two methods. The single sphere representation used by GRID affords only positional information, supplemented by the identification of hydrogen bonding interactions. By contrast, the multi-atom representation of most MCSS groups yields in both positional and orientational information. The two methods are most similar for small functional groups, while for larger functional groups MCSS yields results consistent with GRID but superior in detail. These results are in accord with the somewhat different purposes for which the two methods were developed. GRID has been used mainly to introduce functionalities at specific positions in lead compounds, in which case the orientation is predetermined by the structure of the latter. The orientational information provided by MCSS is important for its use in the de novo design of large, multi-functional ligands, as well as for improving lead compounds.

Computational methods were used to design structure-based combinatorial libraries of antipicornav... more Computational methods were used to design structure-based combinatorial libraries of antipicornaviral capsid-binding ligands. The multiple copy simultaneous search (MCSS) program was employed to calculate functionality maps for many diverse functional groups for both the poliovirus and rhinovirus capsid structures in the region of the known drug binding pocket. Based on the results of the MCSS calculations, small combinatorial libraries consisting of 10s or 100s of three-monomer compounds were designed and synthesized. Ligand binding was demonstrated by a noncell-based mass spectrometric assay, a functional immunoprecipitation assay, and crystallographic analysis of the complexes of the virus with two of the candidate ligands. The P1/Mahoney poliovirus strain was used in the experimental studies. A comparison showed that the MCSS calculations had correctly identified the observed binding site for all three monomer units in one ligand and for two out of three in the other ligand. The correct central monomer position in the second ligand was reproduced in calculations in which the several key residues lining the pocket were allowed to move. This study validates the computational methodology. It also illustrates that subtle changes in protein structure can lead to differences in docking results and points to the importance of including target flexibility, as well as ligand flexibility, in the design process. * Corresponding authors. (1) Joseph-McCarthy, D. Computational approaches to structure-based ligand design. Pharmacol. Ther. 1999, 84, 179-191. (2) Bohacek, R. S.; McMartin, C. Modern computational chemistry and drug discovery: structure generating programs. Curr. Opin. Chem. Biol. 1997, 1, 157-161. (3) Caflisch, A.; Karplus, M. Computational combinatorial chemistry for de novo ligand design: Review and assessment.

Current Topics in Microbiology and Immunology, 2010

The picornavirus family consists of a large number of small RNA viruses, many of which are signif... more The picornavirus family consists of a large number of small RNA viruses, many of which are significant pathogens of humans and livestock. They are amongst the simplest of vertebrate viruses comprising a single stranded positive sense RNA genome within a T = 1 (quasi T = 3) icosahedral protein capsid of approximately 30 nm diameter. The structures of a number of picornaviruses have been determined at close to atomic resolution by X-ray crystallography. The structures of cell entry intermediate particles and complexes of virus particles with receptor molecules or antibodies have also been obtained by X-ray crystallography or at a lower resolution by cryo-electron microscopy. Many of the receptors used by different picornaviruses have been identified, and it is becoming increasingly apparent that many use co-receptors and alternative receptors to bind to and infect cells. However, the mechanisms by which these viruses release their genomes and transport them across a cellular membrane to gain access to the cytoplasm are still poorly understood. Indeed, detailed studies of cell entry mechanisms have been made only on a few members of the family, and it is yet to be established how broadly the results of these are applicable across the full spectrum of picornaviruses. Working models of the cell entry process are being developed for the best studied picornaviruses, the enteroviruses. These viruses maintain particle integrity throughout the infection process and function as genome delivery modules. However, there is currently no model to explain how viruses such as cardio-and aphthoviruses that appear to simply dissociate into subunits during uncoating deliver their genomes into the cytoplasm.

Proteins: Structure, Function, and Genetics, 1997

A combinatorial ligand design approach based on the multiple copy simultaneous search (MCSS) meth... more A combinatorial ligand design approach based on the multiple copy simultaneous search (MCSS) method and a simple scheme for joining MCSS functional group sites was applied to the binding pocket of P3/Sabin poliovirus and rhinovirus 14. The MCSS method determines where specific functional (chemical) groups have local potential energy minima in the binding site. Before the virus application, test calculations were run to determine the optimal set of input parameters to be used in evaluating the MCSS results. The MCSS minima are analysed and selected minima are connected with (CH2)n linkers to form candidate ligands, whose structures are optimized in the binding site. Estimates of the binding strength were made for the ligands and compared with those for known drugs. The results indicate that the proposed ligands should bind to P3/Sabin poliovirus at least as well as the best of the existing drugs, and that they should also bind to P1/Mahoney poliovirus and rhinovirus 14. A detailed comparison of the poliovirus and rhinovirus binding pockets and an analysis of drug binding specificity is presented.

Proceedings of the National Academy of Sciences, 1999

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate in... more Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among alpha-like DNA polymerases. We measured Km and kcat values for dGTP and ACV triphosphate incorporation and Ki values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased Km values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced kcat values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered km and kcat values. In most cases, the fold increase in Ki of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in Ki. The effects of mutations on enzyme kinetics could be explained by using a model of an alpha-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.

PLoS Biology, 2007

Viruses initiate infection by transferring their genetic material across a cellular membrane and ... more Viruses initiate infection by transferring their genetic material across a cellular membrane and into the appropriate compartment of the cell. The mechanisms by which animal viruses, especially nonenveloped viruses, deliver their genomes are only poorly understood. This is due in part to technical difficulties involved in direct visualization of viral gene delivery and to uncertainties in distinguishing productive and nonproductive pathways caused by the high particle-to-plaque forming unit ratio of most animal viruses. Here, we combine an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based assay for RNA release to characterize the early events in the poliovirus (PV) infection. Effects on RNA genome delivery from inhibitors of cell trafficking pathways were probed systematically by both methods. Surprisingly, we observe that genome release by PV is highly efficient and rapid, and thus does not limit the overall infectivity or the infection rate. The results define a pathway in which PV binds to receptors on the cell surface and enters the cell by a clathrin-, caveolin-, flotillin-, and microtubuleindependent, but tyrosine kinase-and actin-dependent, endocytic mechanism. Immediately after the internalization of the virus particle, genome release takes place from vesicles or tightly sealed membrane invaginations located within 100-200 nm of the plasma membrane. These results settle a long-lasting debate of whether PV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell. We expect this imaging assay to be broadly applicable to the investigation of entry mechanisms for nonenveloped viruses.

Nature Structural & Molecular Biology, 2012

Two papers report the structure of the virion of emerging pathogen EV71, providing a three-dimens... more Two papers report the structure of the virion of emerging pathogen EV71, providing a three-dimensional context for understanding many of its biological functions.

Nano Letters, 2008

We describe a nanoplasmonic probing platform that exploits small-dimension (≤ 20 μm 2 ) ordered a... more We describe a nanoplasmonic probing platform that exploits small-dimension (≤ 20 μm 2 ) ordered arrays of subwavelength holes for multiplexed, high spatial resolution, and real-time analysis on biorecognition events. Nanohole arrays are perforated on a super smooth gold surface (roughness RMS < 2.7 Å) attached on a fluoropolymer (FEP) substrate fabricated by a replica technique. The smooth surface of gold provides a superb environment for fabricating nanometer features and uniform immobilization of biomolecules. The refractive index matching between FEP and biological solutions contributes to ∼ 20% improvement on the sensing performance. Spectral studies on a series of small-dimension nanohole arrays from 1 μm 2 to 20 μm 2 indicate that the plasmonic sensing sensitivity improves as the gold-solution contact area increases. Our results also demonstrate that nanohole arrays with dimension as small as 1 μm 2 can be used to effectively detect biomolecular binding events and analyze the binding kinetics. The future scientific opportunities opened by this nanohole platform include highly multiplexed analysis of ligand interactions with membrane proteins on high quality supported lipid bilayers.

Journal of Virology, 2006

The mechanism by which poliovirus infects the cell has been characterized by a combination of bio... more The mechanism by which poliovirus infects the cell has been characterized by a combination of biochemical and structural studies, leading to a working model for cell entry. Upon receptor binding at physiological temperature, native virus (160S) undergoes a conformational change to a 135S particle from which VP4 and the N terminus of VP1 are externalized. These components interact with the membrane and are proposed to form a membrane pore. An additional conformational change in the particle is accompanied by release of the infectious viral RNA genome from the particle and its delivery, presumably through the membrane pore into the cytoplasm, leaving behind an empty 80S particle. In this report, we describe the generation of a receptordecorated liposome system, comprising nickel-chelating nitrilotriacetic acid (NTA) liposomes and His-tagged poliovirus receptor, and its use in characterizing the early events in poliovirus infection. Receptor-decorated liposomes were able to capture virus and induce a temperature-dependent virus conversion to the 135S particle. Upon conversion, 135S particles became tethered to the liposome independently of receptor by a membrane interaction with the N terminus of VP1. Converted particles had lost VP4, which partitioned with the membrane. The development of a simple model membrane system provides a novel tool for studying poliovirus entry. The liposome system bridges the gap between previous studies using either soluble receptor or whole cells and offers a flexible template which can be extrapolated to electron microscopy experiments that analyze the structural biology of nonenveloped virus entry.

Journal of Virology, 2001

We examined the role of soluble poliovirus receptor on the transition of native poliovirus (160S ... more We examined the role of soluble poliovirus receptor on the transition of native poliovirus (160S or N particle) to an infectious intermediate (135S or A particle). The viral receptor behaves as a classic transition state theory catalyst, facilitating the N-to-A conversion by lowering the activation energy for the process by 50 kcal/mol. In contrast to earlier studies which demonstrated that capsid-binding drugs inhibit thermally mediated N-to-A conversion through entropic stabilization alone, capsid-binding drugs are shown to inhibit receptor-mediated N-to-A conversion through a combination of enthalpic and entropic effects.

Journal of Virology, 2000

Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicat... more Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at ϳ22-Å resolution by cryo-electron microscopy. The 135S and 80S particles are both ϳ4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 Å were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.

Journal of Virology, 2010

Poliovirus infection requires that the particle undergo a series of conformational transitions th... more 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. Using three-dimensional reconstruction methods, we have solved the structure of two distinct forms at subnanometric resolution, and we have built and refined pseudoatomic models into the reconstructions. The reconstructions and the derived models demonstrate that the two structural forms are both slightly expanded, resulting in partial disruption of interprotomer interfaces near their particle 2-fold axes, which may represent the site where RNA is released. The models demonstrate that each of the two 80S structures has undergone a unique set of movements of the capsid proteins, associated with rearrangement of flexible loops and amino-terminal extensions that participate in contacts between protomers, between pentamers, and with the viral RNA.

The Journal of biological chemistry, Jan 26, 2014

RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. T... more RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic expe...

PLoS pathogens, 2014

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantag... more Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA ...

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantag... more Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and footand-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

PLoS Pathogens, 2014

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantag... more Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and footand-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

Journal of Virology, 2014

Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egres... more Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC.

The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (dAg-S) is essential fo... more The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (dAg-S) is essential for genome replication, i.e., for the transcription, processing, and accumulation of HDV RNAs. To better understand this requirement, we used purified recombinant dAg-S and HDV RNA synthesized in vitro to assemble high- molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs into human cells, we detected

Nature Structural Biology, 1995

One approach to combinatorial ligand design begins by determining optimal locations (i.e., local ... more One approach to combinatorial ligand design begins by determining optimal locations (i.e., local potential energy minima) for functional groups in the binding site of a target macromolecule. MCSS and GRID are two methods, based on significantly different algorithms, which are used for this purpose. A comparison of the two methods for the same functional groups is reported. Calculations were performed for nonpolar and polar functional groups in the internal hydrophobic pocket of the poliovirus capsid protein, and on the binding surface of the src SH3 domain. The two approaches are shown to agree qualitatively; i.e., the global characteristics of the functional group maps generated by MCSS and GRID are similar. However, there are significant differences in the relative interaction energies of the two sets of minima, a consequence of the different functional form used to evaluate polar interactions (electrostatics and hydrogen bonding) in the two methods. The single sphere representation used by GRID affords only positional information, supplemented by the identification of hydrogen bonding interactions. By contrast, the multi-atom representation of most MCSS groups yields in both positional and orientational information. The two methods are most similar for small functional groups, while for larger functional groups MCSS yields results consistent with GRID but superior in detail. These results are in accord with the somewhat different purposes for which the two methods were developed. GRID has been used mainly to introduce functionalities at specific positions in lead compounds, in which case the orientation is predetermined by the structure of the latter. The orientational information provided by MCSS is important for its use in the de novo design of large, multi-functional ligands, as well as for improving lead compounds.

Computational methods were used to design structure-based combinatorial libraries of antipicornav... more Computational methods were used to design structure-based combinatorial libraries of antipicornaviral capsid-binding ligands. The multiple copy simultaneous search (MCSS) program was employed to calculate functionality maps for many diverse functional groups for both the poliovirus and rhinovirus capsid structures in the region of the known drug binding pocket. Based on the results of the MCSS calculations, small combinatorial libraries consisting of 10s or 100s of three-monomer compounds were designed and synthesized. Ligand binding was demonstrated by a noncell-based mass spectrometric assay, a functional immunoprecipitation assay, and crystallographic analysis of the complexes of the virus with two of the candidate ligands. The P1/Mahoney poliovirus strain was used in the experimental studies. A comparison showed that the MCSS calculations had correctly identified the observed binding site for all three monomer units in one ligand and for two out of three in the other ligand. The correct central monomer position in the second ligand was reproduced in calculations in which the several key residues lining the pocket were allowed to move. This study validates the computational methodology. It also illustrates that subtle changes in protein structure can lead to differences in docking results and points to the importance of including target flexibility, as well as ligand flexibility, in the design process. * Corresponding authors. (1) Joseph-McCarthy, D. Computational approaches to structure-based ligand design. Pharmacol. Ther. 1999, 84, 179-191. (2) Bohacek, R. S.; McMartin, C. Modern computational chemistry and drug discovery: structure generating programs. Curr. Opin. Chem. Biol. 1997, 1, 157-161. (3) Caflisch, A.; Karplus, M. Computational combinatorial chemistry for de novo ligand design: Review and assessment.

Current Topics in Microbiology and Immunology, 2010

The picornavirus family consists of a large number of small RNA viruses, many of which are signif... more The picornavirus family consists of a large number of small RNA viruses, many of which are significant pathogens of humans and livestock. They are amongst the simplest of vertebrate viruses comprising a single stranded positive sense RNA genome within a T = 1 (quasi T = 3) icosahedral protein capsid of approximately 30 nm diameter. The structures of a number of picornaviruses have been determined at close to atomic resolution by X-ray crystallography. The structures of cell entry intermediate particles and complexes of virus particles with receptor molecules or antibodies have also been obtained by X-ray crystallography or at a lower resolution by cryo-electron microscopy. Many of the receptors used by different picornaviruses have been identified, and it is becoming increasingly apparent that many use co-receptors and alternative receptors to bind to and infect cells. However, the mechanisms by which these viruses release their genomes and transport them across a cellular membrane to gain access to the cytoplasm are still poorly understood. Indeed, detailed studies of cell entry mechanisms have been made only on a few members of the family, and it is yet to be established how broadly the results of these are applicable across the full spectrum of picornaviruses. Working models of the cell entry process are being developed for the best studied picornaviruses, the enteroviruses. These viruses maintain particle integrity throughout the infection process and function as genome delivery modules. However, there is currently no model to explain how viruses such as cardio-and aphthoviruses that appear to simply dissociate into subunits during uncoating deliver their genomes into the cytoplasm.

Proteins: Structure, Function, and Genetics, 1997

A combinatorial ligand design approach based on the multiple copy simultaneous search (MCSS) meth... more A combinatorial ligand design approach based on the multiple copy simultaneous search (MCSS) method and a simple scheme for joining MCSS functional group sites was applied to the binding pocket of P3/Sabin poliovirus and rhinovirus 14. The MCSS method determines where specific functional (chemical) groups have local potential energy minima in the binding site. Before the virus application, test calculations were run to determine the optimal set of input parameters to be used in evaluating the MCSS results. The MCSS minima are analysed and selected minima are connected with (CH2)n linkers to form candidate ligands, whose structures are optimized in the binding site. Estimates of the binding strength were made for the ligands and compared with those for known drugs. The results indicate that the proposed ligands should bind to P3/Sabin poliovirus at least as well as the best of the existing drugs, and that they should also bind to P1/Mahoney poliovirus and rhinovirus 14. A detailed comparison of the poliovirus and rhinovirus binding pockets and an analysis of drug binding specificity is presented.

Proceedings of the National Academy of Sciences, 1999

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate in... more Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among alpha-like DNA polymerases. We measured Km and kcat values for dGTP and ACV triphosphate incorporation and Ki values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased Km values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced kcat values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered km and kcat values. In most cases, the fold increase in Ki of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in Ki. The effects of mutations on enzyme kinetics could be explained by using a model of an alpha-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.

PLoS Biology, 2007

Viruses initiate infection by transferring their genetic material across a cellular membrane and ... more Viruses initiate infection by transferring their genetic material across a cellular membrane and into the appropriate compartment of the cell. The mechanisms by which animal viruses, especially nonenveloped viruses, deliver their genomes are only poorly understood. This is due in part to technical difficulties involved in direct visualization of viral gene delivery and to uncertainties in distinguishing productive and nonproductive pathways caused by the high particle-to-plaque forming unit ratio of most animal viruses. Here, we combine an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based assay for RNA release to characterize the early events in the poliovirus (PV) infection. Effects on RNA genome delivery from inhibitors of cell trafficking pathways were probed systematically by both methods. Surprisingly, we observe that genome release by PV is highly efficient and rapid, and thus does not limit the overall infectivity or the infection rate. The results define a pathway in which PV binds to receptors on the cell surface and enters the cell by a clathrin-, caveolin-, flotillin-, and microtubuleindependent, but tyrosine kinase-and actin-dependent, endocytic mechanism. Immediately after the internalization of the virus particle, genome release takes place from vesicles or tightly sealed membrane invaginations located within 100-200 nm of the plasma membrane. These results settle a long-lasting debate of whether PV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell. We expect this imaging assay to be broadly applicable to the investigation of entry mechanisms for nonenveloped viruses.

Nature Structural & Molecular Biology, 2012

Two papers report the structure of the virion of emerging pathogen EV71, providing a three-dimens... more Two papers report the structure of the virion of emerging pathogen EV71, providing a three-dimensional context for understanding many of its biological functions.

Nano Letters, 2008

We describe a nanoplasmonic probing platform that exploits small-dimension (≤ 20 μm 2 ) ordered a... more We describe a nanoplasmonic probing platform that exploits small-dimension (≤ 20 μm 2 ) ordered arrays of subwavelength holes for multiplexed, high spatial resolution, and real-time analysis on biorecognition events. Nanohole arrays are perforated on a super smooth gold surface (roughness RMS < 2.7 Å) attached on a fluoropolymer (FEP) substrate fabricated by a replica technique. The smooth surface of gold provides a superb environment for fabricating nanometer features and uniform immobilization of biomolecules. The refractive index matching between FEP and biological solutions contributes to ∼ 20% improvement on the sensing performance. Spectral studies on a series of small-dimension nanohole arrays from 1 μm 2 to 20 μm 2 indicate that the plasmonic sensing sensitivity improves as the gold-solution contact area increases. Our results also demonstrate that nanohole arrays with dimension as small as 1 μm 2 can be used to effectively detect biomolecular binding events and analyze the binding kinetics. The future scientific opportunities opened by this nanohole platform include highly multiplexed analysis of ligand interactions with membrane proteins on high quality supported lipid bilayers.

Journal of Virology, 2006

The mechanism by which poliovirus infects the cell has been characterized by a combination of bio... more The mechanism by which poliovirus infects the cell has been characterized by a combination of biochemical and structural studies, leading to a working model for cell entry. Upon receptor binding at physiological temperature, native virus (160S) undergoes a conformational change to a 135S particle from which VP4 and the N terminus of VP1 are externalized. These components interact with the membrane and are proposed to form a membrane pore. An additional conformational change in the particle is accompanied by release of the infectious viral RNA genome from the particle and its delivery, presumably through the membrane pore into the cytoplasm, leaving behind an empty 80S particle. In this report, we describe the generation of a receptordecorated liposome system, comprising nickel-chelating nitrilotriacetic acid (NTA) liposomes and His-tagged poliovirus receptor, and its use in characterizing the early events in poliovirus infection. Receptor-decorated liposomes were able to capture virus and induce a temperature-dependent virus conversion to the 135S particle. Upon conversion, 135S particles became tethered to the liposome independently of receptor by a membrane interaction with the N terminus of VP1. Converted particles had lost VP4, which partitioned with the membrane. The development of a simple model membrane system provides a novel tool for studying poliovirus entry. The liposome system bridges the gap between previous studies using either soluble receptor or whole cells and offers a flexible template which can be extrapolated to electron microscopy experiments that analyze the structural biology of nonenveloped virus entry.

Journal of Virology, 2001

We examined the role of soluble poliovirus receptor on the transition of native poliovirus (160S ... more We examined the role of soluble poliovirus receptor on the transition of native poliovirus (160S or N particle) to an infectious intermediate (135S or A particle). The viral receptor behaves as a classic transition state theory catalyst, facilitating the N-to-A conversion by lowering the activation energy for the process by 50 kcal/mol. In contrast to earlier studies which demonstrated that capsid-binding drugs inhibit thermally mediated N-to-A conversion through entropic stabilization alone, capsid-binding drugs are shown to inhibit receptor-mediated N-to-A conversion through a combination of enthalpic and entropic effects.

Journal of Virology, 2000

Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicat... more Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at ϳ22-Å resolution by cryo-electron microscopy. The 135S and 80S particles are both ϳ4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 Å were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.

Journal of Virology, 2010

Poliovirus infection requires that the particle undergo a series of conformational transitions th... more 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. Using three-dimensional reconstruction methods, we have solved the structure of two distinct forms at subnanometric resolution, and we have built and refined pseudoatomic models into the reconstructions. The reconstructions and the derived models demonstrate that the two structural forms are both slightly expanded, resulting in partial disruption of interprotomer interfaces near their particle 2-fold axes, which may represent the site where RNA is released. The models demonstrate that each of the two 80S structures has undergone a unique set of movements of the capsid proteins, associated with rearrangement of flexible loops and amino-terminal extensions that participate in contacts between protomers, between pentamers, and with the viral RNA.