Studies on viral capsid architectures and coat protein folds have revealed the evolutionary lineages of viruses branching to all three domains of life. A widespread group of icosahedral tailless viruses, the PRD1-adenovirus lineage, was the first to be established. A double β-barrel fold for a single major capsid protein is characteristic of these viruses. Similar viruses carrying genes coding for two major capsid proteins with a more complex structure, such as Thermus phage P23-77 and haloarchaeal virus SH1, have been isolated. Here, we studied the host range, life cycle, biochemical composition, and genomic sequence of a new isolate, Haloarcula hispanica icosahedral virus 2 (HHIV-2), which resembles SH1 despite being isolated from a different location. Comparative analysis of these viruses revealed that their overall architectures are very similar except that the genes for the receptor recognition vertex complexes are unrelated even though these viruses infect the same hosts.
The viral capsid protein P2 of bacteriophage PM2 has been crystallized. Preliminary X-ray analysis demonstrates the position and orientation of the two trimers in the asymmetric unit.
PM2 (Corticoviridae) is a dsDNA bacteriophage which contains a lipid membrane beneath its icosahedral capsid. In this respect it resembles bacteriophage PRD1 (Tectiviridae), although it is not known whether the similarity extends to the detailed molecular architecture of the virus, for instance the fold of the major coat protein P2. Structural analysis of PM2 has been initiated and virus-derived P2 has been crystallized by sitting-nanodrop vapour diffusion. Crystals of P2 have been obtained in space group P21212, with two trimers in the asymmetric unit and unit-cell parameters a = 171.1, b = 78.7, c = 130.1 Å. The crystals diffract to 4 Å resolution at the ESRF BM14 beamline (Grenoble, France) and the orientation of the non-crystallographic threefold axes, the spatial relationship between the two trimers and the packing of the trimers within the unit cell have been determined. The trimers form tightly packed layers consistent with the crystal morphology, possibly recapitulating aspects of the arrangement of subunits in the virus.
virus crystallography; lipid-containing bacteriophages; PRD1-adenoviral lineage
The morphogenesis of poxviruses such as vaccinia virus (VACV) sees the virion shape mature from spherical to brick-shaped. Trimeric capsomers of the VACV D13 protein form a transitory, stabilizing lattice on the surface of the initial spherical immature virus particle. The crystal structure of D13 reveals that this major scaffolding protein comprises a double β barrel “jelly-roll” subunit arranged as pseudo-hexagonal trimers. These structural features are characteristic of the major capsid proteins of a lineage of large icosahedral double-stranded DNA viruses including human adenovirus and the bacteriophages PRD1 and PM2. Structure-based phylogenetic analysis confirms that VACV belongs to this lineage, suggesting that (analogously to higher organism embryogenesis) early poxvirus morphogenesis reflects their evolution from a lineage of viruses sharing a common icosahedral ancestor.
► Poxvirus D13 acts as a scaffold for the morphogenesis of spherical immature virions ► D13 has a double “jelly-roll” structure, like other large DNA virus capsid proteins ► Structure-based phylogenetics places D13 into an icosahedral viral lineage ► Poxvirus morphogenesis reflects Vaccinia virus evolution from an icosahedral ancestor
We have sequenced the genome and identified the structural proteins and lipids of the novel membrane-containing, icosahedral virus P23-77 of Thermus thermophilus. P23-77 has an ∼17-kb circular double-stranded DNA genome, which was annotated to contain 37 putative genes. Virions were subjected to dissociation analysis, and five protein species were shown to associate with the internal viral membrane, while three were constituents of the protein capsid. Analysis of the bacteriophage genome revealed it to be evolutionarily related to another Thermus phage (IN93), archaeal Halobacterium plasmid (pHH205), a genetic element integrated into Haloarcula genome (designated here as IHP for integrated Haloarcula provirus), and the Haloarcula virus SH1. These genetic elements share two major capsid proteins and a putative packaging ATPase. The ATPase is similar with the ATPases found in the PRD1-type viruses, thus providing an evolutionary link to these viruses and furthering our knowledge on the origin of viruses.
Bombyx mori densovirus 1 (BmDNV-1), a major pathogen of silkworms, causes significant losses to the silk industry. The structure of the recombinant BmDNV-1 virus-like particle has been determined at 3.1-Å resolution using X-ray crystallography. It is the first near-atomic-resolution structure of a virus-like particle within the genus Iteravirus. The particles consist of 60 copies of the 55-kDa VP3 coat protein. The capsid protein has a β-barrel “jelly roll” fold similar to that found in many diverse icosahedral viruses, including archaeal, bacterial, plant, and animal viruses, as well as other parvoviruses. Most of the surface loops have little structural resemblance to other known parvovirus capsid proteins. In contrast to vertebrate parvoviruses, the N-terminal β-strand of BmDNV-1 VP3 is positioned relative to the neighboring 2-fold related subunit in a “domain-swapped” conformation, similar to findings for other invertebrate parvoviruses, suggesting domain swapping is an evolutionarily conserved structural feature of the Densovirinae.
Crystals of the portal protein from Staphylococcus epidermidis bacteriophage CNPH82, diffracting to ∼4.2 Å resolution, have been obtained. The protein is a 13-subunit oligomer both in solution and in the crystal.
The portal protein cn3 of bacteriophage CNPH82 is predicted to serve as a gateway for translocation of viral genome into preformed pro-capsid, like portal proteins from other double-stranded DNA tailed bacteriophages. The host of bacteriophage CNPH82 is the opportunistic human pathogenic bacterium Staphylococcus epidermidis, a major cause of nosocomial infections. The portal protein of this phage has been cloned, overexpressed and purified. Size-exclusion chromatography–multi-angle laser light scattering analysis has indicated that the portal protein contains ∼13 subunits. Crystals of the portal protein, diffracting to 4.2 Å, have been obtained. These crystals belong to the space group C2221 with the unit-cell parameters of a = 252.4, b = 367.0, c = 175.5 Å. The self-rotation function revealed the presence of a single 13-subunit oligomer in the asymmetric unit.
portal protein; DNA translocation; bacteriophage CNPH82; oligomeric state
Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven’t been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J. Mol. Biol. 339, 379–394, Figure 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. These density fit well with the characteristic beta-barrel domains of OMPs, thus may be due to the two host proteins. Locations of these density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.
virus assembly; bacteriophage; cementing protein; Shigella; outer membrane protein; OmpA; OmpC
It has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. However, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. Bacteriophage P23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. We have solved multiple crystal structures of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. They fit the 14 Å resolution cryo-electron microscopy reconstruction of the entire virus exquisitely well, allowing us to propose a model for both the capsid architecture and viral assembly, quite different from previously published models. The structures of the capsid proteins and their mode of association to form the viral capsid suggest that the P23-77-like and adeno-PRD1 lineages of viruses share an extremely ancient common ancestor.
•High-resolution structures of the two major capsid proteins of bacteriophage P23-77•P23-77 capsid proteins exhibit a conserved single β-barrel core fold•P23-77 is an ancient relative of the double β-barrel lineage of viruses•Capsid model illustrates that P23-77 uses a novel method of organization
Rissanen et al. propose a model for the architecture and assembly of bacteriophage P23-77 quite different from those previously published. The capsid proteins and their mode of association to form the virus particle suggest that P23-77 share a common evolutionary origin with the PRD1/Adenovirus lineage.
Members of the Ku superfamily are DNA-end-binding proteins involved in non-homologous end-joining (NHEJ) DNA repair. The published crystal structure of human Ku-DNA complex reveals a heterodimer that forms a ring around dsDNA by means of the Ku core modules. These modules contain a highly conserved seven-stranded β-barrel, which in turn contains an insertion, termed the bridge-region, between its second and third β-strands. The bridge-region adopts an unusual β-strand-rich structure critical for dsDNA-binding and Ku function, but its provenance remains unclear. Here, we demonstrate that the bridge-region of Ku is a novel member of the diverse Zn-ribbon fold group. Sequence analysis reveals that Ku from several Gram-positive bacteria and bacteriophages retain metal-chelating motifs, whereas they have been lost in the versions from most other organisms. Structural comparisons suggest that the Zn-ribbon from Ku bridge-region is the first example of a circularly permuted, segment-swapped Zn-ribbon. This finding helps explain how Ku is likely to bind DNA as an obligate dimer. Further, we hypothesize that retention of the unusual conformation of the turns of the Zn-ribbons, despite loss of the Zn-binding sites, provides clues regarding the mechanism by which the Ku bridge-regions sense the DNA state.
NHEJ; zinc finger; zinc ribbon; protein flexibility; segment-swapping
The marine double-stranded DNA (dsDNA) bacteriophage PM2, studied since 1968, is the type organism of the family Corticoviridae, infecting two gram-negative Pseudoalteromonas species. The virion contains a membrane underneath an icosahedral protein capsid composed of two structural proteins. The purified major capsid protein, P2, appears as a trimer, and the receptor binding protein, P1, appears as a monomer. The C-terminal part of P1 is distal and is responsible for receptor binding activity. The rest of the structural proteins are associated with the internal phospholipid membrane enclosing the viral genome. This internal particle is designated the lipid core. The overall structural organization of phage PM2 resembles that of dsDNA bacteriophage PRD1, the type organism of the family Tectiviridae.
Many supramolecular complexes form crystals that have lattice constants of the order of 1000 Å. An optimized method for data collection and processing is described.
Studies of icosahedral virus capsids provide insights into the function of supramolecular machines. Virus capsid crystals have exceptionally large unit cells; as a result, they diffract weakly compared with protein crystals. HK97 is a dsDNA lambda-like bacteriophage whose 13 MDa capsid expands from 550 Å to 650 Å with large subunit conformational changes during virus maturation. The HK97 penultimate maturation intermediate was crystallized in a tetragonal unit cell that has lattice constants of 1010 Å × 1010 Å × 730 Å. The crystals could be cryoprotected, but diffracted to a modest resolution of 5 Å at a bending-magnet beamline. When these crystals were optimally exposed with two orders-of-magnitude more photons from a new insertion-device beamline, data extending to better than 3.8 Å resolution were obtained. Here, the strategies to collect and process such data are described. These strategies can be adapted for other crystals with large unit cells and for microcrystals.
virus crystals; bacteriophage HK97; insertion-device beamlines
Viruses utilize a diverse array of mechanisms to deliver their genomes into hosts. While great strides have been made in understanding the genome delivery of eukaryotic and prokaryotic viruses, little is known about archaeal virus genome delivery and the associated particle changes. The Sulfolobus turreted icosahedral virus (STIV) is a double-stranded DNA (dsDNA) archaeal virus that contains a host-derived membrane sandwiched between the genome and the proteinaceous capsid shell. Using cryo-electron microscopy (cryo-EM) and different biochemical treatments, we identified three viral morphologies that may correspond to biochemical disassembly states of STIV. One of these morphologies was subtly different from the previously published 27-Å-resolution electron density that was interpreted with the crystal structure of the major capsid protein (MCP). However, these particles could be analyzed at 12.5-Å resolution by cryo-EM. Comparing these two structures, we identified the location of multiple proteins forming the large turret-like appendages at the icosahedral vertices, observed heterogeneous glycosylation of the capsid shell, and identified mobile MCP C-terminal arms responsible for tethering and releasing the underlying viral membrane to and from the capsid shell. Collectively, our studies allow us to propose a fusogenic mechanism of genome delivery by STIV, in which the dismantled capsid shell allows for the fusion of the viral and host membranes and the internalization of the viral genome.
The putative small terminase protein from the thermostable bacteriophage G20C has been produced, purified and crystallized.
The assembly of double-stranded DNA bacteriophages is dependent on a small terminase protein that normally plays two important roles. Firstly, the small terminase protein specifically recognizes viral DNA and recruits the large terminase protein, which makes the initial cut in the dsDNA. Secondly, once the complex of the small terminase, the large terminase and the DNA has docked to the portal protein, and DNA translocation into a preformed empty procapsid has begun, the small terminase modulates the ATPase activity of the large terminase. Here, the putative small terminase protein from the thermostable bacteriophage G20C, which infects the Gram-negative eubacterium Thermus thermophilus, has been produced, purified and crystallized. Size-exclusion chromatography–multi-angle laser light scattering data indicate that the protein forms oligomers containing nine subunits. Crystals diffracting to 2.8 Å resolution have been obtained. These belonged to space group P212121, with unit-cell parameters a = 94.31, b = 125.6, c = 162.8 Å. The self-rotation function and Matthews coefficient calculations are consistent with the presence of a nine-subunit oligomer in the asymmetric unit.
putative small terminase; Thermus thermophilus; bacteriophage G20C
Proper assembly of viruses must occur through specific interactions between capsid proteins. Many double-stranded DNA viruses and bacteriophages require internal scaffolding proteins to assemble their coat proteins into icosahedral capsids. The 303 amino acid bacteriophage P22 scaffolding protein is mostly helical, and its C-terminal helix-turn-helix (HTH) domain binds to the coat protein during virion assembly, directing the formation of an intermediate structure called the procapsid. The interaction between coat and scaffolding protein HTH domain is electrostatic, but the amino acids that form the protein-protein interface have yet to be described. In the present study, we used alanine scanning mutagenesis of charged surface residues of the C-terminal HTH domain of scaffolding protein. We have determined that P22 scaffolding protein residues R293 and K296 are crucial for binding to coat protein and that the neighboring charges are not essential but do modulate the affinity between the two proteins.
virus assembly; ankyrin repeat; electrostatic interactions; recombineering; scaffolding protein; procapsid
The virophage Sputnik is a satellite virus of the giant mimivirus and is the only satellite virus reported to date whose propagation adversely affects its host virus' production. Genome sequence analysis showed that Sputnik has genes related to viruses infecting all three domains of life. Here, we report structural studies of Sputnik, which show that it is about 740 Å in diameter, has a T=27 icosahedral capsid, and has a lipid membrane inside the protein shell. Structural analyses suggest that the major capsid protein of Sputnik is likely to have a double jelly-roll fold, although sequence alignments do not show any detectable similarity with other viral double jelly-roll capsid proteins. Hence, the origin of Sputnik's capsid might have been derived from other viruses prior to its association with mimivirus.
The predicted eight-stranded β-barrel outer membrane protein TTC0834 from T. thermophilus HB27 was overexpressed in T. thermophilus HB8 and crystallized. Diffraction data were collected to a maximal resolution of 3.2 Å.
The cell envelope of the thermophilic bacterium Thermus thermophilus is multilayered and includes an outer membrane with integral outer membrane proteins that are not well characterized. The hypothetical protein TTC0834 from T. thermophilus HB27 was identified as a 22 kDa outer membrane protein containing eight predicted β-strands. TTC0834 was expressed with an N-terminal His tag in T. thermophilus HB8 and detected in the S-layer/outer membrane envelope fraction. His-TTC0834 was purified and crystallized under various conditions. Native data sets were collected to 3.2 Å resolution and the best diffracting crystals belonged to space group P3121 or P3221, with unit-cell parameters a = b = 166.67, c = 97.53 Å.
Thermus thermophilus; outer membrane protein; TTC0834
K. Fukuyama, S. S. Abdel-Meguid, J. E. Johnson, and M. G. Rossmann (J. Mol. Biol. 167:873-984, 1983) reported the structure of alfalfa mosaic virus assembled from the capsid protein as a T=1 icosahedral empty particle at 4.5-A resolution. The information contained in the structure included the particle size, protein shell thickness, presence of wide holes at the icosahedral fivefold axes, and a proposal that the capsid protein adopts a beta-barrel structure. In the present work, the X-ray diffraction data of Fukuyama et al. as well as the data subsequently collected by I. Fita, Y. Hata, and M. G. Rossmann (unpublished) were reprocessed to 4.0-A resolution, and the structure was solved by molecular replacement. The current structure allowed the tracing of the polypeptide chain of the capsid protein confirming the beta-sandwich fold and provides information on intersubunit interactions in the particle. However, it was not possible to definitively assign the amino acid sequence to the side chain density at 4-A resolution. The particle structure was also determined by cryoelectron microscopy and image reconstruction methods and found to be in excellent agreement with the X-ray model.
Bacteriophages are involved in many aspects of the spread and establishment of virulence factors in Staphylococcus aureus, including the mobilization of genetic elements known as pathogenicity islands (SaPIs), which carry genes for superantigen toxins and other virulence factors. SaPIs are packaged into phage-like transducing particles using proteins supplied by the helper phage. We have used cryo-electron microscopy and icosahedral reconstruction to determine the structure of the procapsid and the mature capsid of 80α, a bacteriophage that can mobilize several different SaPIs. The 80α capsid has T = 7 icosahedral symmetry with the capsid protein organized into pentameric and hexameric clusters that interact via prominent trimeric densities. The 80α capsid protein was modeled based on the capsid protein fold of bacteriophage HK97, and fitted into the 80α reconstructions. The models show that the trivalent interactions are mediated primarily by a 22-residue β hairpin structure called the P loop that is not found in HK97. Capsid expansion is associated with a conformational switch in the spine helix that is propagated throughout the subunit, unlike the domain rotation mechanism in phages HK97 or P22.
procapsid; structure; assembly; three-dimensional reconstruction; pathogenicity island
In contrast to most enveloped viruses, poxviruses produce infectious particles that do not acquire their internal lipid membrane by budding through cellular compartments. Instead, poxvirus immature particles are generated from atypical crescent-shaped precursors whose architecture and composition remain contentious. Here we describe the 2.6 Å crystal structure of vaccinia virus D13, a key structural component of the outer scaffold of viral crescents. D13 folds into two jellyrolls decorated by a head domain of novel fold. It assembles into trimers that are homologous to the double-barrel capsid proteins of adenovirus and lipid-containing icosahedral viruses. We show that, when tethered onto artificial membranes, D13 forms a honeycomb lattice and assembly products structurally similar to the viral crescents and immature particles. The architecture of the D13 honeycomb lattice and the lipid-remodeling abilities of D13 support a model of assembly that exhibits similarities with the giant mimivirus. Overall, these findings establish that the first committed step of poxvirus morphogenesis utilizes an ancestral lipid-remodeling strategy common to icosahedral DNA viruses infecting all kingdoms of life. Furthermore, D13 is the target of rifampicin and its structure will aid the development of poxvirus assembly inhibitors.
Poxviruses are arguably the largest viruses infecting humans. The unique brick-shape architecture of their infectious virus particles sets them apart from any other viral family in the virosphere. The infectious particles are produced through a series of assembly steps where intermediates of distinct composition and architecture can be identified. In particular, atypical crescent-shaped precursors of immature particles have generated much controversy regarding their structure and the origin of their lipidic membrane. Here, we used a combination of X-ray crystallography and electron microscopy to investigate the role of a crucial structural component of viral crescents called D13. Our atomic structure of D13 firmly establishes an evolutionary link between poxviruses and a group of large DNA viruses. In addition, we show that, when tethered to artificial membranes, this protein assembles into a scaffold analogous to that in immature particles. The resulting pseudo-atomic model of the honeycomb lattice reveals similarities to the mimivirus, which suggests that giant viral shells use common assembly principles. Overall, our findings reveal that poxviruses utilize an ancestral lipid-remodeling strategy common to DNA viruses infecting all kingdoms of life. They also provide a basis for structure-based design of assembly inhibitors against poxvirus pathogens.
Double-stranded DNA (dsDNA) viruses such as herpesviruses and
bacteriophages infect by delivering their genetic material into cells, a task
mediated by a DNA channel called “portal protein.” We have used
electron cryomicroscopy to determine the structure of bacteriophage P22 portal
protein in both the procapsid and mature capsid conformations. We find that,
just as the viral capsid undergoes major conformational changes during virus
maturation, the portal protein switches conformation from a procapsid to a
mature phage state upon binding of gp4, the factor that initiates tail assembly.
This dramatic conformational change traverses the entire length of the DNA
channel, from the outside of the virus to the inner shell, and erects a large
dome domain directly above the DNA channel that binds dsDNA inside the capsid.
We hypothesize that this conformational change primes dsDNA for injection and
directly couples completion of virus morphogenesis to a new cycle of
The bacteriophage T4 capsid is an elongated icosahedron, 120 nm long and 86 nm wide, and is built with three essential proteins; gp23*, which forms the hexagonal capsid lattice, gp24*, which forms pentamers at eleven of the twelve vertices, and gp20, which forms the unique dodecameric portal vertex through which DNA enters during packaging and exits during infection. The past twenty years of research has greatly elevated the understanding of phage T4 head assembly and DNA packaging. The atomic structure of gp24 has been determined. A structural model built for gp23 using its similarity to gp24 showed that the phage T4 major capsid protein has the same fold as that found in phage HK97 and several other icosahedral bacteriophages. Folding of gp23 requires the assistance of two chaperones, the E. coli chaperone GroEL and the phage coded gp23-specific chaperone, gp31. The capsid also contains two non-essential outer capsid proteins, Hoc and Soc, which decorate the capsid surface. The structure of Soc shows two capsid binding sites which, through binding to adjacent gp23 subunits, reinforce the capsid structure. Hoc and Soc have been extensively used in bipartite peptide display libraries and to display pathogen antigens including those from HIV, Neisseria meningitides, Bacillus anthracis, and FMDV. The structure of Ip1*, one of the components of the core, has been determined, which provided insights on how IPs protect T4 genome against the E. coli nucleases that degrade hydroxymethylated and glycosylated T4 DNA. Extensive mutagenesis combined with the atomic structures of the DNA packaging/terminase proteins gp16 and gp17 elucidated the ATPase and nuclease functional motifs involved in DNA translocation and headful DNA cutting. Cryo-EM structure of the T4 packaging machine showed a pentameric motor assembled with gp17 subunits on the portal vertex. Single molecule optical tweezers and fluorescence studies showed that the T4 motor packages DNA at a rate of up to 2000 bp/sec, the fastest reported to date of any packaging motor. FRET-FCS studies indicate that the DNA gets compressed during the translocation process. The current evidence suggests a mechanism in which electrostatic forces generated by ATP hydrolysis drive the DNA translocation by alternating the motor between tensed and relaxed states.
Sulfolobus turreted icosahedral virus (STIV) was the first icosahedral virus characterized from an archaeal host. It infects Sulfolobus species that thrive in the acidic hot springs (pH 2.9 to 3.9 and 72 to 92°C) of Yellowstone National Park. The overall capsid architecture and the structure of its major capsid protein are very similar to those of the bacteriophage PRD1 and eukaryotic viruses Paramecium bursaria Chlorella virus 1 and adenovirus, suggesting a viral lineage that predates the three domains of life. The 17,663-base-pair, circular, double-stranded DNA genome contains 36 potential open reading frames, whose sequences generally show little similarity to other genes in the sequence databases. However, functional and evolutionary information may be suggested by a protein's three-dimensional structure. To this end, we have undertaken structural studies of the STIV proteome. Here we report our work on A197, the product of an STIV open reading frame. The structure of A197 reveals a GT-A fold that is common to many members of the glycosyltransferase superfamily. A197 possesses a canonical DXD motif and a putative catalytic base that are hallmarks of this family of enzymes, strongly suggesting a glycosyltransferase activity for A197. Potential roles for the putative glycosyltransferase activity of A197 and their evolutionary implications are discussed.
Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.
Many members of the Omp85 family of proteins form essential β-barrel outer membrane protein (OMP) biogenesis machinery in Gram-negative bacteria, chloroplasts, and mitochondria. In Escherichia coli, BamA, a member of the Omp85 family, folds into an outer membrane-embedded β-barrel domain and a soluble periplasmic polypeptide-transport-associated (POTRA) domain. Although the high-resolution structures of only the BamA POTRA domain of E. coli are available, the crystal structure of FhaC, an Omp85 family member and a component of the two-partner secretion system in Bordetella pertussis, suggests that the BamA β-barrel likely folds into a 16-stranded β-barrel. The FhaC β-barrel is occluded by an N-terminal α-helix and a large β-barrel loop, L6, which carries residues that are highly conserved among the Omp85 family members. Deletion of L6 in FhaC did not affect its biogenesis but abolished its secretion function. In this study, we tested the hypothesis that the conserved residues of the putative L6 loop, which presumably folds back into the lumen of the BamA β-barrel like the FhaC counterpart, play an important role in OMP and/or BamA biogenesis. The conserved 641RGF643 residues of L6 were either deleted or replaced with alanine in various permutations. Phenotypic and biochemical characterization of various BamA L6 mutants revealed that the conserved RGF residues are critical for OMP biogenesis. Moreover, three BamA L6 alterations, ΔRGF, AAA, and AGA, produced a conditional lethal phenotype, concomitant with severely reduced BamA levels and folding defects. Thus, the conserved 641RGF643 residues of the BamA L6 loop are important for BamA folding and biogenesis.
The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported.
Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Å resolution using synchrotron radiation and belong to the orthorhombic space group P212121, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Å. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.
adeno-associated virus serotype 5; gene therapy