[Show abstract][Hide abstract] ABSTRACT: Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate the viral fusion and nucleocapsid penetration into the cytoplasm. However, the dynamic nature of the fusogenic trimer has made the determination of its structure a challenge. Here we have used Fab fragments of the neutralizing antibody DV2-E104 to stop the conformational change of Dengue virus at an intermediate stage of the fusion process. Using cryo-electron microscopy, we show that in this intermediate stage the E glycoproteins form 60 trimers that are similar to the predicted "open" fusogenic trimer.
[Show abstract][Hide abstract] ABSTRACT: Chloroviruses infect their hosts by specifically binding to and degrading the cell wall of their algal hosts at the site of attachment, using an intrinsic digesting enzyme(s). Chlorovirus PBCV-1 stored as a lysate survived longer than virus alone, suggesting virus attachment to cellular debris may be reversible. Ghost cells (algal cells extracted with methanol) were used as a model to study reversibility of PBCV-1 attachment because ghost cells are as susceptible to attachment and wall digestion as are live cells. Reversibility of attachment to ghost cells was examined by releasing attached virions with a cell wall degrading enzyme extract. The majority of the released virions retained infectivity even after re-incubating the released virions with ghost cells two times. Thus the chloroviruses appear to have a dynamic attachment strategy that may be beneficial in indigenous environments where cell wall debris can act as a refuge until appropriate host cells are available.
[Show abstract][Hide abstract] ABSTRACT: The bacteriophage T4 baseplate is the control center of the virus, where the recognition of an E. coli host by the long tail fibers is translated into a signal to initiate infection. The short tail fibers unfold from the baseplate for firm attachment to the host, followed by shrinkage of the tail sheath that causes the tail tube to enter and cross the periplasmic space ending with injection of the genome into the host. During this process, the 6.5 MDa baseplate changes its structure from a "dome" shape to a "star" shape. An in vitro assembled hubless baseplate has been crystalized. It consists of six copies of the recombinantly expressed trimeric gene product (gp) 10, monomeric gp7, dimeric gp8, dimeric gp6 and monomeric gp53. The diffraction pattern extends, at most, to 4.0 Å resolution. The known partial structures of gp10, gp8, and gp6 and their relative position in the baseplate derived from earlier electron microscopy studies were used for molecular replacement. An electron density map has been calculated based on molecular replacement, single isomorphous replacement with anomalous dispersion data and 2-fold non-crystallographic symmetry averaging between two baseplate wedges in the crystallographic asymmetric unit. The current electron density map indicates that there are structural changes in the gp6, gp8, and gp10 oligomers compared to their structures when separately crystallized. Additional density is also visible corresponding to gp7, gp53 and the unknown parts of gp10 and gp6.
[Show abstract][Hide abstract] ABSTRACT: Although the φX174 DNA pilot protein H is monomeric during procapsid assembly, it forms an oligomeric tube on the surface of host cells. Reminiscent of a double-stranded DNA phage tails in form and function, the H-tube transports the single-stranded φX174 genome across the E. coli cell wall. The 2.4 Å resolution, H-tube crystal structure suggests functional and energetic mechanisms, which may be common features of DNA transport through virally encoded conduits.
[Show abstract][Hide abstract] ABSTRACT: Abstract Nucleocytoplasmic large dsDNA viruses (NCLDVs) encompass an ever-increasing group of large eukaryotic viruses, infecting a wide variety of organisms. The set of core genes shared by all these viruses includes a major capsid protein with a double jelly-roll fold forming an icosahedral capsid, which surrounds a double layer membrane that contains the viral genome. Furthermore, some of these viruses, such as the members of the Mimiviridae and Phycodnaviridae have a unique vertex that is used during infection to transport DNA into the host.
[Show abstract][Hide abstract] ABSTRACT: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, <1.5 PFU) from SA14-14-2MCV (LD50, >1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.
[Show abstract][Hide abstract] ABSTRACT: Alphaviruses can be serious, sometimes lethal human pathogens that belong to the family Togaviridae. Structures of human Venezuelan equine encephalitis virus (VEEV), an alphavirus, in complex with two strongly neutralizing antibody Fab fragments (F5 and 3B4C-4) have been determined using a combination of cryo-electron microscopy (cryo-EM) and homology modeling. Here we characterize these monoclonal antibody Fab fragments known to abrogate VEEV infectivity by binding to the E2 (envelope) surface glycoprotein. Both these antibody Fab fragments cross-link the surface E2 glycoproteins and, therefore, probably inhibit infectivity by blocking the conformational changes that are required for making the virus fusogenic. The F5 Fab fragment cross-links E2 proteins within one trimeric spike, whereas the 3B4C-4 Fab fragment cross-links E2 proteins from neighboring spikes. Furthermore, F5 probably blocks the receptor-binding site, whereas 3B4C-4 sterically hinders the exposure of the fusion loop at the end of the E2 B-domain.
[Show abstract][Hide abstract] ABSTRACT: A brief history is given of how X-ray diffraction data from crystals have been recorded. Today there are new possibilities, spawned by the availability of free electron lasers that produce powerful femtosecond long X-ray pulses.
[Show abstract][Hide abstract] ABSTRACT: Antibodies were prepared by immunizing mice with empty, immature particles of human enterovirus 71 (EV71), a picornavirus that causes severe neurological disease in young children. The capsid structure of these empty particles is different from that of the mature virus and is similar to "A" particles encountered when picornaviruses recognize a potential host cell before genome release. The monoclonal antibody E18, generated by this immunization, induced a conformational change when incubated at temperatures between 4 °C and 37 °C with mature virus, transforming infectious virions into A particles. The resultant loss of genome that was observed by cryo-EM and a fluorescent SYBR Green dye assay inactivated the virus, establishing the mechanism by which the virus is inactivated and demonstrating that the E18 antibody has potential as an anti-EV71 therapy. The antibody-mediated virus neutralization by the induction of genome release has not been previously demonstrated. Furthermore, the present results indicate that antibodies with genome-release activity could also be produced for other picornaviruses by immunization with immature particles.
Proceedings of the National Academy of Sciences 01/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tailed double-stranded DNA bacteriophages, or Caudovirales, constitute ~96% of all the known phages. Although these phages come in a great variety of sizes and morphology, their virions are mainly constructed of similar molecular building blocks via similar assembly pathways. Here we review the structure of tailed double-stranded DNA bacteriophages at a molecular level, emphasizing the structural similarity and common evolutionary origin of proteins that constitute these virions.
[Show abstract][Hide abstract] ABSTRACT: Prokaryotic viruses have evolved various mechanisms to transport their genomes across bacterial cell walls. Many bacteriophages use a tail to perform this function, whereas tail-less phages rely on host organelles. However, the tail-less, icosahedral, single-stranded DNA ΦX174-like coliphages do not fall into these well-defined infection processes. For these phages, DNA delivery requires a DNA pilot protein. Here we show that the ΦX174 pilot protein H oligomerizes to form a tube whose function is most probably to deliver the DNA genome across the host's periplasmic space to the cytoplasm. The 2.4 Å resolution crystal structure of the in vitro assembled H protein's central domain consists of a 170 Å-long α-helical barrel. The tube is constructed of ten α-helices with their amino termini arrayed in a right-handed super-helical coiled-coil and their carboxy termini arrayed in a left-handed super-helical coiled-coil. Genetic and biochemical studies demonstrate that the tube is essential for infectivity but does not affect in vivo virus assembly. Cryo-electron tomograms show that tubes span the periplasmic space and are present while the genome is being delivered into the host cell's cytoplasm. Both ends of the H protein contain transmembrane domains, which anchor the assembled tubes into the inner and outer cell membranes. The central channel of the H-protein tube is lined with amide and guanidinium side chains. This may be a general property of viral DNA conduits and is likely to be critical for efficient genome translocation into the host.
[Show abstract][Hide abstract] ABSTRACT: Rubella virus (RV) is a leading cause of birth defects due to infectious agents. When contracted during pregnancy, RV infection leads to severe damage in fetuses. Despite its medical importance, compared with the related alphaviruses, very little is known about the structure of RV. The RV capsid protein is an essential structural component of virions as well as a key factor in virus-host interactions. Here we describe three crystal structures of the structural domain of the RV capsid protein. The polypeptide fold of the RV capsid protomer has not been observed previously. Combining the atomic structure of the RV capsid protein with the cryoelectron tomograms of RV particles established a low-resolution structure of the virion. Mutational studies based on this structure confirmed the role of amino acid residues in the capsid that function in the assembly of infectious virions.
Proceedings of the National Academy of Sciences 11/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flaviviruses, such as dengue, West Nile, and yellow fever viruses, assemble as fusion-incompetent particles and subsequently undergo a large reorganization of their glycoprotein envelope resulting in formation of mature infectious virions. Here we used a combination of three-dimensional cryo-electron tomography and two-dimensional image analysis to study pleiomorphic maturation intermediates of dengue virus 2. Icosahedral symmetries of immature and mature regions within one particle were mismatched relative to each other. Furthermore, the orientation of the two regions relative to each other differed among particles. Therefore, there cannot be a specific pathway determining the maturation of all particles. Instead, the region with mature structure expands when glycoproteins on its boundary acquire suitable orientation and conformation to allow them to become a stable part of the mature region. This type of maturation is possible because the envelope glycoproteins are anchored to the phospholipid bilayer that is a part of flavivirus virions and are thus restricted to movement on the two-dimensional surface of the particle. Therefore, compounds that limit movement of the glycoproteins within the virus membrane might be used as inhibitors of flavivirus maturation.
Journal of Structural Biology 11/2013; · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special five-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1 and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the E. coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and fit the dodecamer into the cryoEM density of the phage portal vertex. The core structure, like that from other phages, is cone-shaped with the wider end containing the "wing" and "crown" domains inside the phage head. A long "stem" encloses a central channel, and a narrow "stalk" protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and portal. The "tunnel" loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging.
Journal of Molecular Biology 10/2013; · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 3.5 Å resolution X-ray crystal structure of mature cricket parvovirus (Acheta domesticus densovirus, AdDNV) has been determined. Structural comparisons show that vertebrate and invertebrate parvoviruses have evolved independently, although there are common structural features among all parvovirus capsid proteins. It was shown that raising the temperature of the AdDNV particles caused a loss of their genomes. The structure of these emptied particles was determined by cryo-electron microscopy to 5.5 Å resolution and found to have the same capsid structure as the full, mature virus except for the absence of the three ordered nucleotides observed in the crystal structure. The viral protein 1 (VP1) amino termini could be externalized without significant damage to the capsid. In vitro, this externalization of the VP1 amino termini is accompanied by the release of the viral genome.
[Show abstract][Hide abstract] ABSTRACT: Amoeba infected with mimivirus were disrupted at sequential stages of virus production and visualized by atomic force microscopy. The development of virus factories proceeded over 3 to 4 hours post infection and resulted from the coalescence of 0.5 to 2 μm vesicles, possibly bearing nucleic acid, derived from either the nuclear membrane or closely associated rough endoplasmic reticulum. Virus factories actively producing virus capsids on their surfaces were imaged and this allowed the morphogenesis of the capsids to be delineated. The first feature to appear on a virus factory surface when a new capsid is born is the center of a stargate, which is a pentameric protein oligomer. As the arms of the stargate grow from the pentamer, a rough disk the diameter of a capsid thickens about it. This marks the initial emergence of a protein coated membrane vesicle. The capsid self assembles on the vesicle. Hillocks capped by different pentameric proteins spontaneously appear on the emerging vesicle at positions that are ultimately occupied by five-fold icosahedral vertices. A lattice of coat protein nucleates at each of the fivefold verticies, but not at the stargate, and then spreads outward from the vertices over the surface, merging seamlessly to complete the icosahedral capsid. Filling with DNA and associated proteins occurs by transfer of nucleic acid from the interior of the virus factory into the nearly completed capsids. The portal, through which the DNA enters, is sealed by a plug of protein having diameter about 40 nm. A layer of integument protein that anchors the surface fibers is acquired by passage of capsids through a membrane enriched in the protein. The coating of surface fibers is similarly acquired when the integument protein coated capsids pass through a second membrane that has a forest of surface fibers embedded on one side.
[Show abstract][Hide abstract] ABSTRACT: The 2H2 monoclonal antibody recognizes the precursor peptide on the immature dengue virus and might, therefore, be a useful tool for investigating the conformational change that occurs when the immature virus enters an acidic environment. During dengue virus maturation, the spiky, immature, non-infectious virions change their structure to smooth-surfaced particles in the slightly acid environment of the trans-Golgi network, thereby allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions become fully infectious when they release the cleaved precursor peptide on reaching the neutral pH environment of the extracellular space. Here we report on the cryo-electron microscopy structures of the immature virus complexed with the 2H2 antigen binding fragments (Fab) at different concentrations and varied pH conditions. At neutral pH and high concentration of the Fab molecules, three Fab molecules bind to three precursor-membrane proteins on each spike of the immature virus. However, at a low concentration of the Fab molecules and at pH 7.0, only two Fab molecules bind to each spike. Changing to slightly acidic pH caused no detectable change of structure for the high Fab concentration sample, but caused severe structural damage to the low concentration sample. Therefore, the 2H2 Fab inhibits the maturation process of immature dengue virus when the Fab molecules are at high concentration, because the three Fab molecules on each spike hold the precursor-membrane molecules together, thereby inhibiting the normal conformational change that occurs during maturation.
[Show abstract][Hide abstract] ABSTRACT: This review is a partially personal account of the discovery of virus structure and its implication for virus function. Although I have endeavored to cover all aspects of structural virology and to acknowledge relevant individuals, I know that I have favored taking examples from my own experience in telling this story. I am anxious to apologize to all those who I might have unintentionally offended by omitting their work. The first knowledge of virus structure was a result of Stanley's studies of tobacco mosaic virus (TMV) and the subsequent X-ray fiber diffraction analysis by Bernal and Fankuchen in the 1930s. At about the same time it became apparent that crystals of small RNA plant and animal viruses could diffract X-rays, demonstrating that viruses must have distinct and unique structures. More advances were made in the 1950s with the realization by Watson and Crick that viruses might have icosahedral symmetry. With the improvement of experimental and computational techniques in the 1970s, it became possible to determine the three-dimensional, near-atomic resolution structures of some small icosahedral plant and animal RNA viruses. It was a great surprise that the protecting capsids of the first virus structures to be determined had the same architecture. The capsid proteins of these viruses all had a 'jelly-roll' fold and, furthermore, the organization of the capsid protein in the virus were similar, suggesting a common ancestral virus from which many of today's viruses have evolved. By this time a more detailed structure of TMV had also been established, but both the architecture and capsid protein fold were quite different to that of the icosahedral viruses. The small icosahedral RNA virus structures were also informative of how and where cellular receptors, anti-viral compounds, and neutralizing antibodies bound to these viruses. However, larger lipid membrane enveloped viruses did not form sufficiently ordered crystals to obtain good X-ray diffraction. Starting in the 1990s, these enveloped viruses were studied by combining cryo-electron microscopy of the whole virus with X-ray crystallography of their protein components. These structures gave information on virus assembly, virus neutralization by antibodies, and virus fusion with and entry into the host cell. The same techniques were also employed in the study of complex bacteriophages that were too large to crystallize. Nevertheless, there still remained many pleomorphic, highly pathogenic viruses that lacked the icosahedral symmetry and homogeneity that had made the earlier structural investigations possible. Currently some of these viruses are starting to be studied by combining X-ray crystallography with cryo-electron tomography.
Quarterly Reviews of Biophysics 05/2013; 46(2):133-80. · 11.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Coordinated interplay between membrane proteins and the lipid bilayer is required for such processes as transporter function and the entrance of enveloped viruses into host cells. In this study, three-dimensional cryo-electron microscopy density maps of mature and immature flaviviruses were analyzed to assess the curvature of the membrane leaflets and its relation to membrane-bound viral glycoproteins. The overall morphology of the viral membrane is determined by icosahedral scaffolding composed of envelope (E) and membrane (M) proteins through interaction of the proteins' stem-anchor regions with the membrane. In localized regions, small membrane regions exhibit convex, concave, flat or saddle-shaped surfaces that are constrained by the specific protein organization within each membrane leaflet. These results suggest that the organization of membrane proteins in small enveloped viruses mediate the formation of membrane curvature.
Journal of Structural Biology 04/2013; · 3.36 Impact Factor