RESUMEN
PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.
Asunto(s)
Bacteriófago PRD1/genética , Bacteriófago PRD1/metabolismo , Empaquetamiento del ADN , ADN Viral/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cápside/metabolismo , ADN Viral/química , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutagénesis , Alineación de Secuencia , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8-9 degrees C improved thermal stability and 0.8-0.9 M improved stability for guanidinium chloride were found after 4-5 enrichment cycles. The most stable mutants selected contained either Lys(H)66Arg or Asn(H)52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.
Asunto(s)
Región Variable de Inmunoglobulina/genética , Inmunoglobulinas Estimulantes de la Tiroides/genética , Biblioteca de Péptidos , Animales , Disulfuros/química , Guanidina/química , Calor , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Inmunoglobulinas Estimulantes de la Tiroides/química , Inmunoglobulinas Estimulantes de la Tiroides/inmunología , Ratones , Mutagénesis/genética , Oxidación-Reducción , Mutación Puntual/genética , Desnaturalización Proteica/genética , Pliegue de Proteína , TermodinámicaRESUMEN
Icosahedral double-stranded DNA (dsDNA) bacterial viruses are known to package their genomes into preformed procapsids via a unique portal vertex. Bacteriophage PRD1 differs from the more commonly known icosahedral dsDNA phages in that it contains an internal lipid membrane. The packaging of PRD1 is known to proceed via preformed empty capsids. Now, a unique vertex has been shown to exist in PRD1. We show in this study that this unique vertex extends to the virus internal membrane via two integral membrane proteins, P20 and P22. These small membrane proteins are necessary for the binding of the putative packaging ATPase P9, via another capsid protein, P6, to the virus particle.
Asunto(s)
Cápside/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Ensamble de Virus , Bacteriófago PRD1/genética , Bacteriófago PRD1/metabolismo , Línea Celular , Escherichia coli/virología , Proteínas de la Membrana/genética , Mutación , Salmonella enterica/virología , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo , Virión/metabolismo , Virión/ultraestructuraRESUMEN
Bam35, a 15-kbp double-stranded DNA phage, infects Bacillus thuringiensis. Recently, sequencing of the related Bacillus cereus revealed a 15.1-kbp linear plasmid, pBClin15. We show that pBClin15 closely resembles Bam35 and demonstrate conversion of Bam35 to a prophage. This state is common, as several B. thuringiensis strains release Bam35-related viruses.