Further observations on the structure of the lipid-containing bacteriophage PM2

Schaefer, Rolf; Hinnen, Rosmarie; Franklin, Richard M.

doi:10.1038/248681a0

Cited by 20 publications

(8 citation statements)

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“…It was previously shown that the outer protein layer of the icosahedral PM2 virion is composed of major coat protein P2 and cell attachment protein P1 located at the fivefold vertices (9,34,36,46). The protease treatment experiments indicated that the N-terminal portion of spike protein P1 is associated with the capsid vertex.…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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 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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Section: Discussionmentioning

confidence: 99%

mentioning

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 outer coat protein, on the other hand, was not modified. We assume that free amino groups of lysine residues of protein I1 interact with the acidic phosphatidylglycerol [4,5,23]. Thus the amino groups of proteins I and I1 either may not be available for reaction with amino-group-specific bifunctional reagents or the separation of the amino groups of these proteins are larger than the separation of the reactive groups of the bifunctional reagents, so that only monovalent reactions take place.…”

Section: Discussionmentioning

confidence: 99%

“…The bilayer is asymmetric; most of the phosphatidylglycerol residues form the outer lipid layer, most of the phosphatidylethanolamine residues the inner lipid layer [4]. Electrostatic interactions between phosphatidylglycerol and protein 11, which is basic (PI 12.3), play a major role in the structural integrity, as well as in the assembly, of the bacteriophage [4,5]. To study the protein-lipid interactions further, the virus was modified with cross-linking reagents, resulting in either protein-lipid or protein-protein crosslinking.…”

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Structure and Synthesis of a Lipid‐Containing Bacteriophage

Huber

Franklin

Seelig

1975

European Journal of Biochemistry

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The nucleocapsid proteins of bacteriophage PM2 and the inner lamella of the lipid bilayer, containing most of the phosphatidylethanolamine residues, were selectively cross-linked in the presence of 0.1 -0.5 % glutaraldehyde, 5 mM dimethylsuberimidate, or 0.05 % toluene 2,4-diisocyanate. The biological activity (p.f.u.) of PM2 modified by these reagents decreased 106-fold in all cases. The spike and coat proteins were selectively cross-linked in the presence of 7.5 mM N,N'-p-phenylenedimaleimide. The biological activity of virus modified by this reagent was unaffected. The electron paramagnetic resonance spectra of fatty acid spin labels incorporated into native and chemically modified viral membranes were qualitatively similar but show quantitative differences. Fixation with glutaraldehyde increased the rigidity of the membrane while Triton X-100 induced a more flexible structure. There was no change in the electron paramagnetic resonance spectrum of virus treated with N,N'-pphenylenedimaleimide, however.Physical interactions between lipids and proteins stabilize natural membrane structures [l]. The lipidcontaining bacteriophage PM2 represents a simple model to study such physical interactions in one type of biological membrane [2]. The bacteriophage contains superhelical DNA, a lipid bilayer, and four structural proteins. Protein I forms the spikes, protein I1 the outer icosahedral shell, and protein I11 the inner icosahedral shell around the DNA [3]. Protein IV is closely associated with the DNA [3]. The phospholipid bilayer lies between the two protein shells. The bilayer is asymmetric; most of the phosphatidylglycerol residues form the outer lipid layer, most of the phosphatidylethanolamine residues the inner lipid layer [4]. Electrostatic interactions between phosphatidylglycerol and protein 11, which is basic (PI 12.3), play a major role in the structural integrity, as well as in the assembly, of the bacteriophage [4,5]. To study the protein-lipid interactions further, the virus was modified with cross-linking reagents, resulting in either protein-lipid or protein-protein crosslinking. Electron spin resonance was then used to investigate the movement of the hydrocarbon chains in the bilayer of such modified viruses. Several stearic This is paper number XXI in the series.Abbreviations. EPR, electron paramagnetic resonance; p.f.u., plaque-forming units.acid analogs with the nitroxide group at different positions were used as spin label in these experiments ~~7 1 . MATERIALS AND METHODS GeneralThe preparation of highly purified bacteriophage PM2 was described by Hinnen et al. [3]. Standard techniques such as analytical polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, quantitative protein determination, plaque test and determination of the radioactivity by liquid scintillation counting have been described earlier [3,4]. Virus labeled with [G-3H]glycerol was prepared and purified as described by Tsukagoshi and Franklin [8]. Lipid was extracted from the virus as described by Frankli...

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“…There was a striking difference, reconstituted from nucleocapsid (e), protein-IV . DNA complex (t), dissociated DNA and protein IV (8) and dissociated nucleocapsid (11). PtdGro = phosphotidylglycerol, PtdEtn = phosphatidylcthanolamine.…”

Section: Discussionmentioning

confidence: 99%

Structure and Synthesis of a Lipid‐Containing Bacteriophage

Franklin

1978

European Journal of Biochemistry

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The lipid-containing bacteriophage PM2 was reconstituted stepwise from its purified denatured subunits. In the first step the nucleocapsid was reconstituted from the DNA and the two nucleocapsid proteins. Slight biochemical differences between reconstituted nucleocapsids and those isolated from native virus were seen. Combination of reconstituted nucleocapsid or nucleocapsid from virions with the coat and spike proteins in the presence of the viral lipids resulted in the formation of infectious virus in both cases. The reconstituted particles contained amounts of viral components similar to those in native virus, except for the lipid content. The amount of lipids present in the reconstituted particles was twice as high as the lipid content of native bacteriophage.The molecular biology of the lipid-containing bacteriophage PM2, which grows on a marine pseudomonad, Pseudonzonus BAL-31, was initiated by Espejo and Canelo [1,2]. Stepwise dissociation of the bacteriophage allowed us to localize the individual proteins [3,4]: protein 1 ( M I = 43000) forms the viral spikes, protein I1 ( M , = 26000-27000) the outer shell, and protein I11 ( M I = 12500) the inner capsid shell around the superhelical DNA ( M , = 6.4 x lo6) which is closely associated with protein IV (MI = 6500). The molar ratios of the proteins/virion are: protein I/ II/lII/IV = 80/820/500/300 [5]. The asymmetric phospholipid bilayer, with most of the phosphatidylglycerol residues forming the outer lamella and most of the phosphatidylethanolamine residues forming the inner lamella, lies between the two protein shells [4]. In the preceding paper we described further details of the dissociation of bacteriophage PM2 as a function of urea concentration and ionic strength. In the presence of 1-2 M urea and 1 M NaCl (at pH 7.2) spikeless virions and lipid-containing nucleocapsids were isolated. In the presence of 4-8.5 M urea and 0.1 -3 M NaCl (at pH 7.2) a nucleocapsid without any lipids can be isolated. This nucleocapsid could be partly dissociated in 9 M urea and 1 M NaCl (at pH 7.2) and completely dissociated either in 4-8.5 M urea and NaCl concentrations below 0.1 M (at pH 7.2) or at 4-8.5 M urea and acidic pH (3.6). Protein IV interacts specifically with DNA at low ionic strength, as could be demonstrated by using a This is paper number XXVIII in the series reagent which crosslinks DNA and protein [3] and by binding of PM2 DNA and protein IV in vitro [7].Bacteriophage PM2 has been partially reconstituted in two steps [8]. In the first step the nucleocapsid was reconstituted from the DNA and the two nucleocapsid proteins. Since the yield of nucleocapsid was low, nucleocapsid obtained from the virion was used for further reconstitution. In the second step the combination of nucleocapsid from virions with the viral coat and spike proteins in the presence of the viral lipids resulted in the formation of infectious virus. This allowed us to study the effect of different fatty acid residues in the viral lipids on reconstitution of infectious virus [9]. All th...

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Further observations on the structure of the lipid-containing bacteriophage PM2

Cited by 20 publications

References 9 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

Structure and Synthesis of a Lipid‐Containing Bacteriophage

Structure and Synthesis of a Lipid‐Containing Bacteriophage

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