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Bacteriophage PRD1 is an icosahedral dsDNA virus with a diameter of 740 Å and an outer protein shell composed of 720 copies of major coat protein P3. Spike complexes at the vertices are composed of a pentameric base (protein P31) and a spike structure (proteins P5 and P2) where the N-terminal region of the trimeric P5 is associated with the base and the C-terminal region of P5 is associated with receptor-binding protein P2. The functionality of proteins P3 and P5 was investigated using insertions and deletions. It was observed that P3 did not tolerate changes whereas P5 tolerated changes much more freely. These properties support the hypothesis that viruses have core structures and functions, which remain stable over time, as well as other elements, responsible for host interactions, which are evolutionally more fluid. The insertional probe used was the apex of exposed loop 4 of group B meningococcal outer membrane protein PorA, a medically important subunit vaccine candidate. It was demonstrated that the epitope could be displayed on the virus surface as part of spike protein P5.

Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of the P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES.

Assembly of the broad-host-range bacteriophage PRD1 involves translocation of the virus-specific membrane to the inside of the icosahedral protein shell formed of trimeric coat proteins. The formation of PRD1 particles is, in addition to the virus-encoded assembly factors P10 and P17, dependent on GroEL/GroES chaperonins. The chaperonins assist in the folding of the capsid proteins P3 and P5 and in the assembly of viral membrane proteins.

Four new specificity class MAbs against PRD1 proteins (P6, P7/14, P11, P18) and polyclonal antiserum against the minor capsid protein P5 were produced. The antibodies were used to analyze the phage protein distribution inside the host cell during infection as well as in the virion. The minor component of the capsid, P5, was shown to be located on the surface of the virion. The proteins responsible for particle infectivity were localized to the membrane fraction of the host cells. In addition, by detection with MAbs, genes encoding proteins P14 and P18 were positively localized on the PRD1 genome.

PRD1 is a dsDNA virus that infects Escherichia coli and Salmonella typhimurium. The genome is a linear molecule with 5' covalently linked terminal protein. The virus has a lipid membrane inside the protein coat. We describe the large-scale purification of the virus using a zonal rotor and the yields and quality of the virus and its subviral assemblies for subsequent biophysical measurements.

Bacteriophage PRD1 infecting Escherichia coli and Salmonella typhimurium translocates its membrane from the host plasma membrane to the virus particle. One obligatory component in this process is the major capsid protein. In this investigation we describe characteristics of the homomultimeric major and minor capsid proteins including the sequences of the corresponding genes. The minor capsid protein was found to contain a short collagen-like region (Gly-X-Y)6. This is the first time this motif has been reported for a prokaryotic protein.

Bacteriophage PM2 was isolated from the Pacific Ocean off the coast of Chile in the late 1960s. It was a new virus type, later classified as Corticoviridae, and also the first bacterial virus for which it was demonstrated that lipids are part of the virion structure. Here we report the determination and analysis of the 10,079-bp circular dsDNA genome sequence. Noteworthy discoveries are the replication initiation system, which is related to the rolling circle mechanism described for phages such as φX174 and P2, and a 1.2-kb sequence that is similar to the maintenance region of a plasmid found in a marine Pseudoalteromonas sp. strain A28.

Trimeric capsomeres of protein P3 (395 aa) are the main component of the phage PRD1 capsid, which encloses a lipid protein vesicle containing the vital dsDNA genome. In this study characterize a panel of monoclonal antibodies (MAb) against P3. The epitopes recognized by the MAbs are analyzed by immunoprecipitation of intact virions or of released P3 trimers, and by Western blotting using a series of C-terminally truncated P3 molecules. Nine of the MAbs recognize epitopes on the virion surface, whereas five require unmasking of epitopes by disruption of the virions. Several of the MAbs are capable of neutralizing the virus; this is most probably due to virus aggregation by the antibodies. Analysis of the C-terminal truncations (the 6 Western blot-positive MAbs were used) delineates three major antigenic regions of the protein. The epitope of MAb 3T74 is included in the 66 N-terminal amino acids, and is not accessible on the virion surface, suggesting that the N-terminus is internally located in the capsid. MAbs 3N81 and 3R2 recognize epitopes In the region of amino acids 159-168, which is part of the first predicted β-barrel structure of P3. The third antigenic region is in the second predicted β-barrel, between amine acids 217-242, where the epitopes of 3N180, 3P4, and 3T5 map. The trimerization of P3 was found to be independent of the non-structural assembly factor proteins P10 and P17. Functional studies of the truncated proteins reveal that molecules comprising of 294 or more residues from the P3 N-terminus are capable of trimer formation.

We have determined the nucleotide sequence of the late region (11 kbp) of the lipid-containing bacteriophage PRD1. Gene localization was carried out by complementing nonsense phage mutants with genomic clones containing specific reading frames. The localization was confirmed by sequencing the N-termini of isolated gene products as well as sequencing the N-termini of tryptic fragments of the phage membrane-associated proteins. This, with the previously obtained sequence of the early regions, allowed us to organize most of the phage genes in the phage genome.

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.