Structure and Synthesis of a Lipid-Containing Bacteriophage. Purification, Chemical Composition, and Partial Sequences of the Structural Proteins

Hinnen, Rosmarie; Chassin, R.; Franklin, Richard M.; Hitz, Hans; Schäfer, Doris

doi:10.1111/j.1432-1033.1976.tb10772.x

Cited by 24 publications

(6 citation statements)

References 36 publications

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“…P1, with a molecular mass of 37.5 kDa, has been identified as the spike protein located at the fivefold vertices, and P2, with a molecular mass of 30.2 kDa, is the major capsid protein. Proteins P3 and P4 are located internally (9,10,22,36,37,38,41,45,46), and P4 has been proposed to be an integral membrane protein interacting with the viral DNA (9,40,42). Dissociation of the PM2 particle has been extensively studied.…”

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confidence: 99%

Bacteriophage PM2 Has a Protein Capsid Surrounding a Spherical Proteinaceous Lipid Core

Kivelä

Kalkkinen

Bamford

2002

J Virol

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The marine double-stranded DNA (dsDNA) bacteriophage PM2, studied since 1968, is the type organism of the family Corticoviridae, infecting two gram-negative Pseudoalteromonas species. The virion contains a membrane underneath an icosahedral protein capsid composed of two structural proteins. The purified major capsid protein, P2, appears as a trimer, and the receptor binding protein, P1, appears as a monomer. The C-terminal part of P1 is distal and is responsible for receptor binding activity. The rest of the structural proteins are associated with the internal phospholipid membrane enclosing the viral genome. This internal particle is designated the lipid core. The overall structural organization of phage PM2 resembles that of dsDNA bacteriophage PRD1, the type organism of the family Tectiviridae.PM2, the only isolate in the family Corticoviridae, is the first bacteriophage for which the presence of lipids in the virion was firmly demonstrated (15,24,26). It is a lytic virus, infecting two Pseudoalteromonas species: marine gram-negative Pseudoalteromonas espejiana BAL-31 (originally Pseudomonas sp. strain BAL-31), isolated along with the virus (25, 32), and Pseudoalteromonas sp. strain ER72M2, obtained from the East River, New York, by Leonard Mindich (38). Originally, considerable interest was directed toward this virus system (about 200 references), but since 1983, only a few studies have been published. Recently, we decided to examine this virus system (38, 39), as our laboratory is focused on lipid-containing bacterial viruses.The mass of the virion (ϳ4.5 ϫ 10 7 Da) is distributed among nucleic acid (14%), lipid (14%), and protein (72%) constituents (15,16,17). The average diameter of the icosahedral particle is ϳ60 nm (24,34,49), and the lipids are located internally (15,34). The sedimentation coefficient (s 20 , w ) of the particle is 293S, and buoyant densities in sucrose and cesium chloride are 1.26 and 1.28 g/cm 3 , respectively (16, 38). The stability of the virion is strongly dependent on sodium and especially on calcium ions, and the virion equilibrated in sucrose is inactive (38). Calcium ions are also essential in the final assembly process during PM2 infection (50).The PM2 genome is a highly supercoiled circular doublestranded DNA (dsDNA) molecule (27) with the highest number of negative supercoils (Ϫ51) observed in a natural molecule (33). The organization of the genes and the transcriptional regulation of operons in the genome have been determined (39; R. H. Männistö, A. M. Grahn, D. H. Bamford, and J. K. H. Bamford, submitted for publication). The 10,079-bp genome replicates via a rolling-circle mechanism (18, 28, 39) while associated with the host cytoplasmic membrane (11).The viral lipids are derived from the host cell membrane during the assembly process (26), and the composition is ϳ64% phosphatidylglycerol, ϳ27% phosphatidylethanolamine, ϳ8% neutral lipids, and small amounts of asylphosphatidylglycerol (15, 53). The proportion of the major phospholipids in the virion is nearly the inverse...

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Bacteriophage PM2 Has a Protein Capsid Surrounding a Spherical Proteinaceous Lipid Core

Kivelä

Kalkkinen

Bamford

2002

J Virol

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“…The apparent isoelectric point of the mutant sp27 is at least 0.5 pH units higher than that of the wild type. Based on the amino acid composition of sp27 (protein II [12]), such a shift is unlikely to be explained by a maximum charge shift substitution of one acidic amino acid for a single basic residue among the 30 lysines and arginines (calculated shift, 0.08 pH). The possibility of a charge shift due to processing of the primary transcript appears unlikely for two reasons.…”

Section: Discussionmentioning

confidence: 99%

Mutant ts1 of bacteriophage PM2 is defective in the major capsid protein and fails to package its DNA

Brewer¹

1983

J Virol

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Infection of Alteromonas espejiana at restrictive temperature with mutant tsl of bacteriophage PM2 resulted in the intracellular accumulation of virus-sized empty-appearing membrane vesicles. The DNA associated with purified vesicles was fully susceptible to digestion with DNase. Sedimentation analysis and electron microscopy suggested a full-length linear form of the normally circular viral genome. A pulse-chase-shift experiment suggested that [3H]thymidinelabeled DNA made under restrictive conditions is assembled into virions after shift to permissive temperature. A defective structural protein in the tsl virion appears to be the cause of a rapid rate of thermal inactivation of infectivity. Analysis of the proteins of tsl by isoelectric focusing indicated a more alkaline isoelectric mobility of the major capsid protein, sp27. Six spontaneous revertants of tsl showed reversion to the wild-type isoelectric form of sp27. These results identify sp27 as the defective gene product of tsi. Taken together, these results suggest that the membrane of PM2 is formed without the aid of an inner core or an outer scaffolding.

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“…From a comparison of the molecular weights of the single polypeptide chains of proteins I1 and I11 determined by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, by gel filtration in the presence of 6 M guanidine . HCl [20] and by amino acid analysis [21] with our determinations in the presence of 4 M and 8 M urea, protein I1 in 4 M urea is a monomer whereas protein I11 in 8 M urea at acidic pH is a dimer (Table 5).…”

Section: O4q2 ( N W 2 )mentioning

confidence: 99%

Structure and Synthesis of a Lipid‐Containing Bacteriophage

Künzler¹,

Lustig²,

Franklin³

1978

European Journal of Biochemistry

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The lipid-containing bacteriophage PM2 was dissociated stepwise in 1 M NaCl (pH 7.2) with increasing urea concentrations. In 2 M urea the following substructures could be identified: (a) a nucleocapsid containing all of the viral lipid, the DNA, proteins I11 and IV, plus a fraction of protein 11, and (b) a second substructure representing particles which contained all viral elements except protein I, the spike protein. In 4 M urea the viral nucleocapsid containing all of proteins I11 and IV, the DNA, plus a fraction of protein IT, was isolated. Upon increasing the urea concentration further, this nucleocapsid is stable up to 8.5 M urea; in 9 M urea protein I11 was partly dissociated from the nucleocapsid. The nucleocapsid in 4 -8.5 M urea is stabilized by the addition of 0.1 -3 M NaCl but dissociates if the NaCl concentration is less than 0.3 M. The nucleocapsid was also dissociated in 4-8.5 M urea at pH 4.5.The nucleocapsid structures and some intermediate morphological subunits have been analysed by physical methods, enabling us to draw some conclusions about the structure and hydration of the virus.Biochemical studies on the lipid-containing bacteriophage PM2, which grows on the marine pseudomonad, Pseudornonas BAL-31, were initiated by Espejo and Canelo [ l~ 31. The individual proteins were localized using stepwise dissociation of bacteriophage PM2 and chemical modifiers [4-71: protein I ( M , = 43000) forms the viral spikes, protein I1 ( M , = 26000 -27 000) the outer icosahedral shell, the hydrophobic protein I11 ( M , = 12500) plus protein IV ( M , = 6500) and the DNA together form the nucleocapsid. The molar ratios of the proteins/virion are 80, 820, 500 and 300 for proteins I, 11, I11 and IV, respectively [7]. The asymmetric phospholipid bilayer with most of the phosphatidylglycerol residues located in the outer lamella and most of the phosphatidylethanolamine residues located in the inner lamella lies between the outer protein shell and the nucleocapsid [S]. In 1 M urea two types of viral subunits were isolated : (a) a nucleocapsid containing viral DNA, lipid, proteins 111 and IV, and a small percentage A detailed study of the dissociation steps of bacteriophage PM2 in the presence of increasing urea concentrations and different NaCl concentrations, as described in the present paper, allowed us to isolate the substructures in good yield and unambiguously correlate all viral proteins with the structure of the virus as understood from electron microscopy [9], X-ray diffraction [lo] and neutron diffraction [lOa]. The physico-chemical properties of the viral substructures have deepened our understanding of the interactions between the various components. MATERIALS AND METHODS GenrrafHighly purified unlabeled bacteriophage PM2 was prepared according to the procedure of Hinnen et al. [4]. The viral proteins were labeled with 3H-containing amino acids and the phospholipid and DNA with [32P]phosphate [4]. The reported pH of the Tris buffers is at 20 "C. Sterile solutions were used for all of the procedures des...

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Structure and Synthesis of a Lipid-Containing Bacteriophage. Purification, Chemical Composition, and Partial Sequences of the Structural Proteins

Cited by 24 publications

References 36 publications

Bacteriophage PM2 Has a Protein Capsid Surrounding a Spherical Proteinaceous Lipid Core

Bacteriophage PM2 Has a Protein Capsid Surrounding a Spherical Proteinaceous Lipid Core

Mutant ts1 of bacteriophage PM2 is defective in the major capsid protein and fails to package its DNA

Structure and Synthesis of a Lipid‐Containing Bacteriophage

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