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

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 II can be labeled with [ 35 S]sulfanilic acid diazonium salt. As shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, proteins I and II can be removed if the virus is treated with 1 M urea. From the above studies and from packing considerations, we conclude that protein II 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 III 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 II and III, and about 50% 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 s 20,w , = 133 S and which has been identified in the electron microscope as a core with icosahedral shape, contains proteins III 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 111 and IV. In the absence or in the presence of low concentrations of urea (1 M) the core is resistant to Triton X‐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 II the outer icosahedral shell and protein III the inner icosahedral shell around the DNA, which is closely associated with protein IV. The phospholipid bilayer is sandwiched between these two shells. Protein III may also form the protein bridges to protein I which connect the inner and outer shells through the bilayer.