Evaluation of the factors controlling the time-dependent inactivation rate coefficients of bacteriophage MS2 and PRD1.

Static and dynamic batch experiments were conducted to study the effects of temperature and the presence of sand on the inactivation of bacteriophage MS2 and PRD1. The experimental data suggested that the inactivation process can be satisfactorily represented by a pseudo-first-order expression with time-dependent rate coefficients. The time-dependent rate coefficients were used to determine pertinent thermodynamic properties required for the analysis of the molecular processes involved in the inactivation of each bacteriophage. A combination of high temperature and the presence of sand appears to produce the greatest disruption to the surrounding protein coat of MS2. However, the lower activation energies for PRD1 indicate a weaker dependence of the inactivation rate on temperature. Instead, the presence of air-liquid and air-solid interfaces appears to produce the greatest damage to specific viral components that are related to infection. These results indicate the importance of using thermodynamic parameters based on the time-dependent inactivation model to better predict the inactivation of viruses in groundwater.

Integral membrane protein P16 of bacteriophage PRD1 stabilizes the adsorption vertex structure.

The icosahedral membrane-containing double-stranded DNA bacteriophage PRD1 has a labile receptor binding spike complex at the vertices. This complex, which is analogous to that of adenovirus, is formed of the penton protein P31, the spike protein P5, and the receptor binding protein P2. Upon infection, the internal phage membrane transforms into a tubular structure that protrudes through a vertex and penetrates the cell envelope for DNA injection. We describe here a new class of PRD1 mutants lacking virion-associated integral membrane protein P16. P16 links the spike complex to the viral membrane and is necessary for spike stability. We also show that the unique vertex used for DNA packaging is intact in the P16-deficient particle, indicating that the 11 adsorption vertices and the 1 portal vertex are functionally and structurally distinct.

Comparative analysis of bacterial viruses Bam35, infecting a gram-positive host, and PRD1, infecting gram-negative hosts, demonstrates a viral lineage.

Extra- and intracellular viruses in the biosphere outnumber their cellular hosts by at least one order of magnitude. How is this enormous domain of viruses organized? Sampling of the virosphere has been scarce and focused on viruses infecting humans, cultivated plants, and animals as well as those infecting well-studied bacteria. It has been relatively easy to cluster closely related viruses based on their genome sequences. However, it has been impossible to establish long-range evolutionary relationships as sequence homology diminishes. Recent advances in the evaluation of virus architecture by high-resolution structural analysis and elucidation of viral functions have allowed new opportunities for establishment of possible long-range phylogenic relationships-virus lineages. Here, we use a genomic approach to investigate a proposed virus lineage formed by bacteriophage PRD1, infecting gram-negative bacteria, and human adenovirus. The new member of this proposed lineage, bacteriophage Bam35, is morphologically indistinguishable from PRD1. It infects gram-positive hosts that evolutionarily separated from gram-negative bacteria more than one billion years ago. For example, it can be inferred from structural analysis of the coat protein sequence that the fold is very similar to that of PRD1. This and other observations made here support the idea that a common early ancestor for Bam35, PRD1, and adenoviruses existed.

Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane.

DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double-stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build-up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.

Field and laboratory investigations of inactivation of viruses (PRD1 and MS2) attached to iron oxide-coated quartz sand.

Field and laboratory experiments were conducted to investigate inactivation of viruses attached to mineral surfaces. In a natural gradient transport field experiment, bacteriophage PRD1, radiolabeled with 32P, was injected into a ferric oxyhydroxide-coated sand aquifer with bromide and linear alkylbenzene sulfonates. In a zone of the aquifer contaminated by secondary sewage infiltration, small fractions of infective and 32P-labeled PRD1 broke through with the bromide tracer,followed bythe slow release of 84% of the 32P activity and only 0.011% of the infective PRD1. In the laboratory experiments, the inactivation of PRD1, labeled with 35S (protein capsid), and MS2, dual radiolabeled with 35S (protein capsid) and 32P (nucleic acid), was monitored in the presence of groundwater and sediment from the contaminated zone of the field site. Release of infective viruses decreased at a much faster rate than release of the radiolabels, indicating that attached viruses were undergoing surface inactivation. Disparities between 32P and 35S release suggest that the inactivated viruses were released in a disintegrated state. Comparison of estimated solution and surface inactivation rates indicates solution inactivation is approximately 3 times as fast as surface inactivation. The actual rate of surface inactivation may be substantially underestimated owing to slow release of inactivated viruses.

The small viral membrane-associated protein P32 is involved in bacteriophage PRD1 DNA entry.

The lipid-containing bacteriophage PRD1 infects a variety of gram-negative cells by injecting its linear double-stranded DNA genome into the host cell cytoplasm, while the protein capsid is left outside. The virus membrane and several structural proteins are involved in phage DNA entry. In this work we identified a new infectivity protein of PRD1. Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. The absence of the protein P32 did not compromise the particle assembly but led to a defect in phage DNA injection. In P32-deficient particles the phage membrane is unable to undergo a structural transformation from a spherical to a tubular form. Since P32(-) particles are able to increase the permeability of the host cell envelope to a degree comparable to that found with wild-type particles, we suggest that the tail-tube formation is needed to eject the DNA from the phage particle rather than to reach the host cell interior.