Alan A. Simpson

The structure and evolution of the major capsid protein of a large, lipid-containing DNA virus

Paramecium bursaria Chlorella virus type 1 (PBCV-1) is a very large, icosahedral virus containing an internal membrane enclosed within a glycoprotein coat consisting of pseudohexagonal arrays of trimeric capsomers. Each capsomer is composed of three molecules of the major capsid protein, Vp54, the 2.0-Å resolution structure of which is reported here. Four N-linked and two O-linked glycosylation sites were identified. The N-linked sites are associated with nonstandard amino acid motifs as a result of glycosylation by virus-encoded enzymes. Each monomer of the trimeric structure consists of two eight-stranded, antiparallel β-barrel, “jelly-roll” domains related by a pseudo-sixfold rotation. The fold of the monomer and the pseudo-sixfold symmetry of the capsomer resembles that of the major coat proteins in the double-stranded DNA bacteriophage PRD1 and the double-stranded DNA human adenoviruses, as well as the viral proteins VP2-VP3 of picornaviruses. The structural similarities among these diverse groups of viruses, whose hosts include bacteria, unicellular eukaryotes, plants, and mammals, make it probable that their capsid proteins have evolved from a common ancestor that had already acquired a pseudo-sixfold organization. The trimeric capsid protein structure was used to produce a quasi-atomic model of the 1,900-Å diameter PBCV-1 outer shell, based on fitting of the Vp54 crystal structure into a three-dimensional cryoelectron microscopy image reconstruction of the virus.

The structure of an insect parvovirus (Galleria mellonella densovirus) at 3.7 Å resolution

BACKGROUND Parvoviruses infect vertebrates, insects and crustaceans. Many arthropod parvoviruses (densoviruses) are highly pathogenic and kill approximately 90% of the host larvae within days, making them potentially effective as selective pesticides. Improved understanding of densoviral structure and function is therefore desirable. There are four different initiation sites for translation of the densovirus capsid protein mRNA, giving rise to the viral proteins VP1 to VP4. Sixty copies of the common, C-terminal domain make up the ordered part of the icosahedral capsid. RESULTS The Galleria mellonella densovirus (GMDNV) capsid protein consists of a core beta-barrel motif, similar to that found in many other viral capsid proteins. The structure most closely resembles that of the vertebrate parvoviruses, but it has diverged beyond recognition in many of the long loop regions that constitute the surface features and intersubunit contacts. The N termini of twofold-related subunits have swapped their positions relative to those of the vertebrate parvoviruses. Unlike in the vertebrate parvoviruses, in GmDNV there is no continuous electron density in the channels running along the fivefold axes of the virus. Electron density corresponding to some of the single-stranded DNA genome is visible in the crystal structure, but it is not as well defined as in the vertebrate parvoviruses. CONCLUSIONS The sequence of the glycine-rich motif, which occupies each of the channels along the fivefold axes in vertebrate viruses, is conserved between mammalian and insect parvoviruses. This motif may serve to externalize the N-terminal region of the single VP1 subunit per particle. The domain swapping of the N termini between insect and vertebrate parvoviruses may have the effect of increasing capsid stability in GmDNV.

Structure determination of the head–tail connector of bacteriophage φ29

The head-tail connector of bacteriophage phi29 is composed of 12 36 kDa subunits with 12-fold symmetry. It is the central component of a rotary motor that packages the genomic dsDNA into preformed proheads. This motor consists of the head-tail connector, surrounded by a phi29-encoded, 174-base, RNA and a viral ATPase protein, both of which have fivefold symmetry in three-dimensional cryo-electron microscopy reconstructions. DNA is translocated into the prohead through a 36 A diameter pore in the center of the connector, where the DNA takes the role of a motor spindle. The helical nature of the DNA allows the rotational action of the connector to be transformed into a linear translation of the DNA. The crystal structure determination of connector crystals in space group C2 was initiated by molecular replacement, using an approximately 20 A resolution model derived from cryo-electron microscopy. The model phases were extended to 3.5 A resolution using 12-fold non-crystallographic symmetry averaging and solvent flattening. Although this electron density was not interpretable, the phases were adequate to locate the position of 24 mercury sites of a thimerosal heavy-atom derivative. The resultant 3.2 A single isomorphous replacement phases were improved using density modification, producing an interpretable electron-density map. The crystallographically refined structure was used as a molecular-replacement model to solve the structures of two other crystal forms of the connector molecule. One of these was in the same space group and almost isomorphous, whereas the other was in space group P2(1)2(1)2. The structural differences between the oligomeric connector molecules in the three crystal forms and between different monomers within each crystal show that the structure is relatively flexible, particularly in the protruding domain at the wide end of the connector. This domain probably acts as a bearing, allowing the connector to rotate within the pentagonal portal of the prohead during DNA packaging.

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

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

Erratum: Detailed architecture of a DNA translocating machine: The high-resolution structure of the bacteriophage φ29 connector particle (Journal of Molecular Biology (2002))

The recent paper by Guasch et al.1 describing the structure of the ϕ29 bacteriophage head–tail connector at 2.1 A resolution (PDB code 1h5w), makes reference to the original 3.2 A resolution structure (PDB code 1fou) published by Simpson et al.2 and used by Guasch et al. to solve the 1h5w structure by molecular replacement. The Guasch paper states that there are marked differences between the 1h5w and 1fou structures in the chain tracing and the side-chain positioning in the wide domain. As these and a number of other statements in the Guasch paper may have led to the impression that these differences are larger than they really are, we give here Tables summarizing the similarities and differences (Tables 1 and ​and22). Table 1 Statistical comparison of the ϕ29 connector wide domains in different structures Table 2 Superposition of the entire monomers in the four reported structures Another paper by Simpson et al.,3 which was accidentally omitted from the text of the Guasch et al. paper, reports two additional structures of the ϕ29 connector: at 3.2 A resolution and at 2.9 A resolution (PDB codes 1jnb and 1ijg, respectively). The 2.9 A resolution structure shows no significant differences from the structure subsequently reported by Guasch et al. (Figure 1; Tables 1 and ​and22). Figure 1 Stereo diagram showing the superposition of the Cα back-bone of monomer A in the 2.1 A resolution structure 1h5w (green) and the 2.9 A resolution 1ijg structure (red).

Structure of the bacteriophage ϕ29 DNA packaging motor

In a digital communication system in which the information is transmitted by successions of bits termed packets, stations may be called upon to perform the function of a relay between other stations. The stations which may be called upon to perform the function of a relay comprise: a checking device which merely checks the address code of a packet received; an eliminating and switching device which either destroys the packet (doubtful address code) or orients the packet toward receiving means pertaining to the station (address code identical to the code of the station) or toward the station corresponding to the address code by passing through a transit memory; and information producing means for producing packets within the station. The contents of the transit memory are transmitted in priority by the station. The packets produced within a station are transmitted in the gaps between the packets coming from the transit memory and in an order which is a function of a classification between the priorities allocated to the information producing means of the considered station.