RESUMEN
The connector, the structure located between the bacteriophage capsid and tail, is interesting from several points of view. The connector is in many cases involved in the initiation of the capsid assembly process, functions as a gate for DNA transport in and out of the capsid, and is, as implied by the name, the structure connecting a tail to the capsid. Occupying a position on a 5-fold axis in the capsid and connected to a coaxial 6-fold tail, it mediates a symmetry mismatch between the two. To understand how the connector is capable of all these interactions its structure needs to be worked out. We have focused on the bacteriophage P2/P4 connector, and here we report an image reconstruction based on 2D crystalline layers of connector protein expressed from a plasmid in the absence of other phage proteins. The overall design of the connector complies well with that of other phage connectors, being a toroid structure having a conspicuous central channel. Our data suggests a 12-fold symmetry, i.e., 12 protrusions emerge from the more compact central part of the structure. However, rotational analysis of single particles suggests that there are both 12- and 13-mers present in the crude sample. The connectors used in this image reconstruction work differ from connectors in virions by having retained the amino-terminal 26 amino acids normally cleaved off during the morphogenetic process. We have used different late gene mutants to demonstrate that this processing occurs during DNA packaging, since only mutants in gene P, coding for the large terminase subunit, accumulate uncleaved connector protein. The suggestion that the cleavage might be intimately involved in the DNA packaging process is substantiated by the fact that the fragment cleaved off is highly basic and is homologous to known DNA binding sequences.
Asunto(s)
Bacteriófago P2/ultraestructura , Cápside/ultraestructura , Bacteriófago P2/fisiología , Cápside/fisiología , Ensamble de VirusRESUMEN
The sequences of two previously defined tail genes, V and J, of the temperate bacteriophage P2, and those of two new essential tail genes, W and I, were determined. Their order is the late gene promoter, VWJI, followed by the tail fiber genes H and G, and a transcription terminator. The V gene product is the small spike at the tip of the tail, and the J gene product lies at the edge of the baseplate. The W gene product may be homologous to the product of gene 25 of T4 phage, which is part of the T4 baseplate. A temperature-sensitive mutation in gene V affects satellite phage P4 production more than it affects the production of P2 helper phage. P4 mutations that partially compensate for this defect of gene V lie in the P4 capsid size determination gene, sid.
Asunto(s)
Bacteriófago P2/genética , Genes Virales/genética , Genoma Viral , Proteínas Estructurales Virales/genética , Ensamble de Virus/fisiología , Secuencia de Aminoácidos , Animales , Bacteriófago P2/fisiología , Bacteriófago P2/ultraestructura , Secuencia de Bases , Cápside/genética , Cápside/ultraestructura , Clonación Molecular , Cartilla de ADN/química , ADN Viral/análisis , Electroforesis en Gel de Poliacrilamida , Regulación Viral de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Conejos , Proteínas Recombinantes , Transcripción Genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The portal structure has been implicated in several aspects of the bacteriophage life cycle, including capsid assembly initiation and DNA packaging. Here we present evidence that P2 gene Q codes for the P2 and P4 portal protein. First, microsequencing shows that capsid protein h6 is derived from gpQ, most probably by proteolytic cleavage. Second, antibodies against gpQ bind to the portal structure in disrupted P2 phage virions, as observed by electron microscopy. Third, gpQ partially purified from an overexpressing plasmid assembles into portal-like structures. We also show by microsequencing that capsid protein h7 is encoded by the P2 scaffold gene, O, and is probably derived from gpO by proteolytic cleavage. Previous work has demonstrated processing of the major capsid protein. Thus, all essential capsid proteins of P2 and P4 are proteolytically cleaved during the morphogenetic process.
Asunto(s)
Bacteriófago P2/crecimiento & desarrollo , Proteínas de la Cápside , Cápside/metabolismo , Colifagos/crecimiento & desarrollo , Virus Satélites/crecimiento & desarrollo , Secuencia de Aminoácidos , Bacteriófago P2/genética , Bacteriófago P2/ultraestructura , Cápside/genética , Cápside/ultraestructura , Colifagos/genética , Colifagos/ultraestructura , Datos de Secuencia Molecular , Morfogénesis , Procesamiento Proteico-Postraduccional , Virus Satélites/genética , Virus Satélites/ultraestructura , Análisis de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
We show that the product of the polarity suppression (psu) gene from bacteriophage P4 associates with P4 capsids. This association can occur when Psu is (i) provided in vivo from the P4 genome or from a plasmid or (ii) provided in vitro by mixing viable phage particles with Psu protein. Psu is unable to associate with the larger capsid of P4's helper phage P2. Discrimination of the P4 and P2 capsids by Psu appears to be independent of the presence of the P4 genome in the capsid, since P2 size capsids filled with P4 DNA cannot accommodate Psu association. P4 psu particles devoid of Psu are less stable than P4 particles carrying Psu. These results indicate that, in addition to its antitermination activity at Rho-dependent terminators, Psu is also a decoration protein that stabilizes the P4 capsids.
Asunto(s)
Proteínas de la Cápside , Cápside/ultraestructura , Colifagos/genética , Virus Satélites/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cápside/genética , Colifagos/crecimiento & desarrollo , Colifagos/ultraestructura , ADN Viral/genética , Genes Virales , Datos de Secuencia Molecular , Mutación , Oligodesoxirribonucleótidos/química , Proteínas Estructurales Virales/genética , Replicación ViralRESUMEN
An interesting feature of the bacteriophage P2-P4 system is the switch in size between a large P2 (60 nm) and a small P4 (45 nm) capsid. We have investigated whether the protein processing reactions cleaving the primary translation product gpN to several capsid proteins (h1, h2, and N*) are involved in this switch. Using antibodies specific against gpN and its derivatives we have identified all the structural components of mature P2 and P4 particles that are derived from gpN. Our estimate of the relative amounts of gpN derivatives suggests that the previously identified minor capsid proteins h1 and h2 can only be essential structural components of the P4, and not the P2, capsid. Nevertheless, the relative amounts are similar in vivo during a P2 and a P4 infection. This indicates that the switch in head size is not caused by the presence of elevated amounts of h1 and h2 during P4 morphogenesis. We have also identified the sites where gpN is cleaved to its derivatives h1, h2, and N*, ascertaining that the cleavage sites are the same in P2 and P4. Our results indicate that the processing reactions are not directly involved in the head size determination mechanism.