Structure and Synthesis of a Lipid‐Containing Bacteriophage

Abstract

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...

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

“…It was previously proposed that infectious viral particles can be reconstituted from viral subunits (43,44,54). We repeated systematically these experiments and extended the analysis by using more defined purified constituents.…”

“…Dissociation of the PM2 virion to separate subunits was previously reported (36,47). The subviral particles were used in reconstitution experiments as described by Schäfer and Franklin (43,44). We repeated these experiments with the PM2 subunits defined in this investigation.…”

“…Reconstitution of viral particles was carried out in principle as described by Schäfer and Franklin (43,44). Viral particles in PM2 buffer with 20 mM 2-mercaptoethanol were disrupted with 4.5 M urea (0.7 mg of protein/ml).…”

“…5B). Dissociation and the following reconstitution were carried out in the presence of 2-mercaptoethanol as described by Schäfer and Franklin (43,44). The addition of 2-mercaptoethanol to a final concentration of 20 mM, either to virions in PM2 buffer or to 4.5 M urea-treated samples, did not have a measurable effect on the infectivity or the outcome of the disruption experiments.…”

“…Previous literature describes different subviral particles as well as infectious virions reconstituted from the separated viral components (36,43,44,47,54). It has also been proposed that no lipid-free nucleocapsids exist, as the dissociation products still contain the viral phospholipids (1,41).…”