Structure and Synthesis of a Lipid‐Containing Bacteriophage Preparation of Virus and Localization of the Structural Proteins

Abstract

The four structural proteins of bacteriophage PM2 have been related to the morphological structures in the virion. Protein I can be selectively removed from the virus by degradation with bromelain, a proteolytic enzyme. From electron microscopic studies and from experiments showing that the virus cannot attach to its host cell after bromelain treatment, we conclude that protein I forms the outer spikes at the vertices of the icosahedron. Protein I1 can be labeled with [35S]sulfanilic acid diazonium salt. As shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, proteins I and I1 can be removed if the virus is treated with 1 M urea. From the above studies and from packing considerations, we conclude that protein I1 forms the outer shell of the virus.The dissociation of the virus in 1 M urea leads to: (a) a nucleocapsid core containing viral lipid, DNA, protein 111 and IV, (b) a second fraction which consists of the same elements obtained by treatment of the virus with bromelain. Both particles have been identified by electron microscopy.The dissociation of the virus in 1 M urea plus 0.5 Triton X-100, a nonionic detergent, leads to a third fraction representing an empty shell which contains all of the lipid, proteins I1 and 111, and about 5076 of protein IV, but no DNA. We have also been able to isolate a second type of nucleocapsid core by dissociation of the virus in the presence of 4.5 M urea. This core, which has an approximate sz0,.+ = 133 S and which has been identified in the electron microscope as a core with icosahedral shape, contains proteins 111 and IV, as well as the DNA, but not phospholipid.In the presence of 4.5 M urea this core is resistant to Triton X-100 concentrations up to 0.1 %.Higher concentrations of the nonionic detergent dissociate the core into DNA and the solubilized proteins I11 and IV. In the absence or in the presence of low concentrations of urea (1 M) the core is resistant to TritonX-100 concentrations up to 0.5%. The DNA in the core is digestible by pancreatic DNAase I and the core structure is broken down after DNAase treatment. Protein IV interacts with the DNA as shown by transformation of non-covalent interactions between DNA and protein into covalent bonds induced by a nucleophilic reagent, In conclusion, protein I forms the spikes, protein I1 the outer icosahedral shell and protein I11 the inner icosahedral shell around the DNA, which is closely associated with protein IV. The phospholipid bilayer is sandwiched between these two shells. Protein I11 may also form the protein bridges to protein I which connect the inner and outer shells through the bilayer.Extensive studies on the molecular biology of the lipid-containing bacteriophage PM2 were initiated by Espejo and Canelo [1,2]. This bacteriophage was isolated from the Pacific Ocean and the host organism is a marine pseudomonad, Pseudomonas BAL-31 [3]. Since PM2 is readily dissociated by ethyl ether or ionic detergents [l], it is reasonable to propose that the integrity of the virus depends o...

Structure and Synthesis of a Lipid‐Containing Bacteriophage.

“…After dialysis, larger aggregates were removed by centrifugation (10000 rev./min, 10 min, 4 "C). Reconstituted particles were purified from the supernatant by centrifuging in a SW52 rotor (40000 rev./min, 3 h, 10 "C) either on a 10-60% linear sucrose gradient or a 20-40% linear sucrose gradient on a 60% sucrose cushion made up in buffer 1 [2,7]. The gradients were fractionated and a distinct band was measured at 260 nm at almost the same position as native virus.…”

“…Bacteriophage PM2 was purified from Pseudomonas BAL-31 grown in nutrient broth [2]. Radioactive virus was prepared from Pseudomonas BAL-3 1 grown in amino acid medium containing a 3H-labeled amino acid mixture as described by Hinnen et al [2].…”

“…The adsorption of virus and particles to host cells and the bromelain treatment of particles were carried out using 3H-labeled virus [2].…”

“…Radioactive virus was prepared from Pseudomonas BAL-3 1 grown in amino acid medium containing a 3H-labeled amino acid mixture as described by Hinnen et al [2]. An unsaturated fatty acid auxotroph, strain UFA, was grown in amino acid medium supplemented with either cis or trans-palmitoleic acid at a concentration of 0.01 % [5,6].…”

“…The lipids of bacteriophage PM2 are arranged as a bilayer between the nucleocapsid and outer shell [l]. There are four polypeptides in the virion: proteins 1 and I1 form spikes and outer shell, respectively; protein I11 is the nucleocapsid protein; protein IV is associated with the viral DNA [2]. Protein I1 has a high isoelectric point (12.3) and binds the acidic phospholipid, phosphatidylglycerol, through electrostatic interaction [3].…”