Purified membrane-containing procapsids of bacteriophage PRD1 package the viral genome.

Icosahedral-tailed double-stranded DNA (dsDNA) bacteriophages and herpesviruses translocate viral DNA into a preformed procapsid in an ATP-driven reaction by a packaging complex that operates at a portal vertex. A similar packaging system operates in the tailless dsDNA phage PRD1 (Tectiviridae family), except that there is an internal membrane vesicle in the procapsid. The unit-length linear dsDNA genome with covalently linked 5'-terminal proteins enters the procapsid through a unique vertex. Two small integral membrane proteins, P20 and P22, provide a conduit for DNA translocation. The packaging machinery also contains the packaging ATPase P9 and the packaging efficiency factor P6. Here we describe a method used to obtain purified packaging-competent PRD1 procapsids. The optimized in vitro packaging system allowed efficient packaging of defined DNA substrates. We determined that the genome terminal protein P8 is necessary for packaging and provided an estimation of the packaging rate.

Insights into virus evolution and membrane biogenesis from the structure of the marine lipid-containing bacteriophage PM2.

Recent, primarily structural observations indicate that related viruses, harboring no sequence similarity, infect hosts of different domains of life. One such clade of viruses, defined by common capsid architecture and coat protein fold, is the so-called PRD1-adenovirus lineage. Here we report the structure of the marine lipid-containing bacteriophage PM2 determined by crystallographic analyses of the entire approximately 45 MDa virion and of the outer coat proteins P1 and P2, revealing PM2 to be a primeval member of the PRD1-adenovirus lineage with an icosahedral shell and canonical double beta barrel major coat protein. The view of the lipid bilayer, richly decorated with membrane proteins, constitutes a rare visualization of an in vivo membrane. The viral membrane proteins P3 and P6 are organized into a lattice, suggesting a possible assembly pathway to produce the mature virus.

Structure and host-cell interaction of SH1, a membrane-containing, halophilic euryarchaeal virus.

The Archaea, and the viruses that infect them, are the least well understood of all of the three domains of life. They often grow in extreme conditions such as hypersaline lakes and sulfuric hot springs. Only rare glimpses have been gained into the structures of archaeal viruses. Here, we report the subnanometer resolution structure of a recently isolated, hypersalinic, membrane-containing, euryarchaeal virus, SH1, in which different viral proteins can be localized. The results indicate that SH1 has a complex capsid formed from single beta-barrels, an important missing link in hypotheses on viral capsid protein evolution. Unusual, symmetry-mismatched spikes seem to play a role in host adsorption. They are connected to highly organized membrane proteins providing a platform for capsid assembly and potential machinery for host infection.

Selenomethionine labeling of large biological macromolecular complexes: probing the structure of marine bacterial virus PM2.

There is a need for improved tools for labeling protein species within large macromolecular assemblies. Here we describe a method for the efficient selenomethionine labeling of the membrane-containing bacterial virus PM2 for structural studies. By examining potential host cells a strain was found which was auxotrophic for methionine, and by performing a multiparameter search of conditions it was possible to derive a robust protocol which simultaneously minimized the toxic effects of the selenomethionine, so that a reasonable virus yield was maintained, whilst still achieving essentially complete labeling. This has allowed us to fingerprint the protein constituents of the virus in a relatively low resolution electron density map. Such a technique can be adapted to other macromolecule complexes studied by X-ray crystallography.

Genetics for Pseudoalteromonas provides tools to manipulate marine bacterial virus PM2.

The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 (Corticoviridae) has been limited so far. The isolation of an autonomously replicating DNA element of Pseudoalteromonas haloplanktis TAC125 and construction of a shuttle vector replicating in both Escherichia coli and Pseudoalteromonas enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2 sus2 mutant deficient in the structural protein P10. To infect and replicate, PM2 delivers its 10-kbp genome across the cell envelopes of two gram-negative Pseudoalteromonas species. The events leading to the internalization of the circular supercoiled dsDNA are puzzling. In a poorly understood process that follows receptor recognition, the virion capsid disassembles and the internal membrane fuses with the host outer membrane. While beginning to unravel the mechanism of this process, we found that protein P10 plays an essential role in the host cell penetration.

Genome characterization of lipid-containing marine bacteriophage PM2 by transposon insertion mutagenesis.

Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed- and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.

Quantitative dissociation of archaeal virus SH1 reveals distinct capsid proteins and a lipid core.

Viruses infecting archaeal cells are less well understood than those infecting eukaryotic and bacterial cells. Here we study the distribution of the structural proteins between the capsid and the membrane of icosahedral SH1 virus, an archaeal virus infecting extreme halophilic Haloarcula hispanica cells. General features such as morphology, linear dsDNA genome and presence of lipids suggest that it may belong to the recently proposed PRD1-adenovirus lineage of viruses. To investigate this we have initiated structural studies of the virion. Quantitative dissociation of SH1 by 3 M urea or by lowering the salt concentration identified a number of soluble capsid-associated proteins (VP2, VP3, VP4, VP6, VP7 and VP9). These released proteins left behind a particle, or lipid core, containing two major proteins VP10 and VP12 and viral phospholipids. VP1 was released from the lipid core in low ionic strength conditions but not with 3 M urea. Approximately half of the protein VP5 stayed with the lipid core and the other half was released. Analysis of the soluble capsid-associated proteins by their sedimentation and hydrodynamic properties suggests that the most abundant proteins, putative capsomers VP4 and VP7, form an intricate pattern of protein complexes. We also observed large differences in the sizes of the complexes determined by the two different methods suggesting an elongated overall structure for most of the capsid-associated proteins or protein complexes. This work verifies that there is an internal membrane vesicle residing inside the complex icosahedral capsid that is akin to the overall structure of PRD1-like viruses.

SH1: A novel, spherical halovirus isolated from an Australian hypersaline lake.

A novel halovirus, SH1, with a spherical morphology is described. Isolated from a hypersaline lake, SH1 is divalent, producing clear plaques on Haloarcula hispanica and a natural Halorubrum isolate. Single-step growth curves gave a latent period of 5-6 h and a burst size of around 200 PFU/cell. The host can differentiate to form tight clusters of thick cell-walled forms, and these were shown to be resistant to infection. Purified virions had no visible tail, were about 70 nm in diameter, and displayed a fragile outer capsid layer, possibly with an underlying membrane component. The structural proteins of the virion were analyzed by SDS-PAGE and several were found to be cross-linked, forming protein complexes. The genome was linear, dsDNA, of approximately 30 kb in length. This morphology and linear genome are features not observed in any other euryarchaeal viruses, but have properties similar to the bacterial virus PRD1.

Preliminary crystallographic analysis of the major capsid protein P2 of the lipid-containing bacteriophage PM2.

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 P2(1)2(1)2, with two trimers in the asymmetric unit and unit-cell parameters a = 171.1, b = 78.7, c = 130.1 A. The crystals diffract to 4 A 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.

The PM2 virion has a novel organization with an internal membrane and pentameric receptor binding spikes.

Biological membranes are notoriously resistant to structural analysis. Excellent candidates to tackle this problem in situ are membrane-containing viruses where the membrane is constrained by an icosahedral capsid. Cryo-EM and image reconstruction of bacteriophage PM2 revealed a membrane bilayer following the internal surface of the capsid. The viral genome closely interacts with the inner leaflet. The capsid, at a resolution of 8.4 A, reveals 200 trimeric capsomers with a pseudo T = 21 dextro organization. Pentameric receptor-binding spikes protrude from the surface. It is evident from the structure that the PM2 membrane has at least two important roles in the life cycle. First, it acts as a scaffold to nucleate capsid assembly. Second, after host recognition, it fuses with the host outer membrane to promote genome entry. The structure also sheds light on how the viral supercoiled circular double-stranded DNA genome might be packaged and released.

Penetration of membrane-containing double-stranded-DNA bacteriophage PM2 into Pseudoalteromonas hosts.

The icosahedral bacteriophage PM2 has a circular double-stranded DNA (dsDNA) genome and an internal lipid membrane. It is the only representative of the Corticoviridae family. How the circular supercoiled genome residing inside the viral membrane is translocated into the gram-negative marine Pseudoalteromonas host has been an intriguing question. Here we demonstrate that after binding of the virus to an abundant cell surface receptor, the protein coat is most probably dissociated. During the infection process, the host cell outer membrane becomes transiently permeable to lipophilic gramicidin D molecules proposing fusion with the viral membrane. One of the components of the internal viral lipid core particle is the integral membrane protein P7, with muralytic activity that apparently aids the process of peptidoglycan penetration. Entry of the virion also causes a limited depolarization of the cytoplasmic membrane. These phenomena differ considerably from those observed in the entry process of bacteriophage PRD1, a dsDNA virus, which uses its internal membrane to make a cell envelope-penetrating tubular structure.

Bacteriophage PM2 has a protein capsid surrounding a spherical proteinaceous lipid core.

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: