RESUMO
The Corticoviridae is a family of icosahedral, internal-membrane-containing viruses with double-stranded circular DNA genomes of approximately 10 kb. Only one species, Pseudoalteromonas virus PM2, has been recognized. Pseudoalteromonas virus PM2 infects Gram-negative bacteria and was isolated from seawater in 1968. Pseudoalteromonas virus PM2 is the first bacterial virus in which the presence of lipids in the virion has been demonstrated. Viral lipids are acquired selectively during virion assembly from the host cytoplasmic membrane. The outer protein capsid is an icosahedron with a pseudo T=21 symmetry. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Corticoviridae, which is available at www.ictv.global/report/corticoviridae.
Assuntos
Corticoviridae/química , Corticoviridae/classificação , Corticoviridae/genética , Corticoviridae/isolamento & purificação , Genoma Viral , Vírion/química , Vírion/classificação , Vírion/genética , Vírion/isolamento & purificaçãoRESUMO
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.
Assuntos
Corticoviridae/genética , Pseudoalteromonas/virologia , Proteínas do Capsídeo/genética , Corticoviridae/crescimento & desenvolvimento , Corticoviridae/isolamento & purificação , DNA Circular/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/virologia , Vetores Genéticos/genética , Genoma Viral/genética , Modelos Genéticos , Mutagênese Sítio-Dirigida , Mutação , Fenótipo , Plasmídeos/genética , Pseudoalteromonas/genética , RNA de Transferência/genética , Água do Mar/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4 degrees C; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.
Assuntos
Corticoviridae/química , DNA Viral/análise , Endonucleases/metabolismo , Corticoviridae/genética , Corticoviridae/crescimento & desenvolvimento , Corticoviridae/isolamento & purificação , Criopreservação , Crioprotetores/farmacologia , DNA Circular/isolamento & purificação , DNA Circular/metabolismo , DNA Viral/isolamento & purificação , DNA Viral/metabolismo , Genoma Viral , Glicerol/farmacologia , Refrigeração , Trítio/metabolismo , Ensaio de Placa ViralRESUMO
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.
Assuntos
Corticoviridae/química , Corticoviridae/genética , Proteínas de Ligação a DNA , Genoma Viral , Lipídeos/análise , Sequência de Aminoácidos , Sequência de Bases , Corticoviridae/crescimento & desenvolvimento , Corticoviridae/isolamento & purificação , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/fisiologia , Replicação do DNA/genética , Genes Virais/genética , Genes Virais/fisiologia , Bactérias Gram-Negativas/virologia , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Óperon/genética , Plasmídeos/genética , Origem de Replicação/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transativadores/química , Transativadores/genética , Transativadores/fisiologia , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genéticaRESUMO
The marine, icosahedral bacteriophage PM2 was isolated in the late 1960s. It was the first phage for which lipids were firmly demonstrated to be part of the virion structure and it has been classified as the type organism of the Corticoviridae family. The host, Pseudoalteromonas espejiana BAL-31, belongs to a common group of marine bacteria. We developed a purification method producing virions with specific infectivity approximately as high as that of the lipid-containing phages PRD1 and φ6. The sensitivity of the virus to normally used purification media such as those containing sucrose is demonstrated. We also present an alternative host, a pseudoalteromonad, that allows enhanced purification of the virus under reduced salt conditions. We show, using N-terminal amino acid sequencing and comparison with the genomic sequence, that there are at least eight structural proteins in the infectious virus.