Structure and synthesis of a lipid-containing bacteriophage XIX. Reconstitution of bacteriophage PM2 In Vitro

Abstract

The lipid-containing bacteriophage PM2 was reconstituted stepwise from its separated subunits: the superhelical DNA, the two nucleocapsid proteins, the external coat protein, the spike protein, and the two lipids, phosphatidylglycerol and phosphatidylethanolamine. In the first step the nucleocapsid was reconstituted from the DNA and the two nucleocapsid proteins. Since the yield of nucleocapsid was very low, and since the reconstituted nucleocapsid was identical to that obtained by partial degradation of the virion with urea, we used nucleocapsid obtained from the virion for the further studies on reconstitution. Combination of the nucleocapsid from virions with various mixtures of viral lipids resulted in formation of a lipid-containing nucleocapsid that was not able to bind the remaining coat and spike proteins. The ratio of phosphatidylethanolamine to phosphatidylglycerol in the reconstituted lipid-containing nucleocapsid was identical with the ratio of phospholipids added to the incubation mixture. Combination of the nucleocapsid from virions with the coat and spike proteins in the presence of the viral lipids resulted in the formation of infectious virus. In this case the ratio of phosphatidylethanolamine to phosphatidylglycerol in the reconstituted virus was independent of the ratio of these phospholipids in the incubation mixture and identical with that of the purified bacteriophage. The basis of this effect was demonstrated in two-phase partition experiments. The nucleocapsid proteins do not selectively bind viral phospholipids, whereas the coat protein specifically binds phosphatidylglycerol. The affinity of the coat protein to phosphatidylglycerol, or vice versa , is partially responsible for the assembly in vitro of active virus particles containing the correct ratio of phospholipids.