Rolf Schafer

Structure and synthesis of a lipid-containing bacteriophage. XIX. Reconstitution of bacteriophage PM2 in vitro.

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.

Structure and synthesis of a lipid-containing bacteriophage: in vitro protein synthesis directed by bacteriophage PM2.

Although synthesis of Alteromonas espejiana* DNA stops soon after infection with bacteriophage PM2, the cellular DNA is not degraded and continues to produce cellular RNA throughout the course of infection [ 11. This would imply that cell-specific protein synthesis also occurs to a considerable extent in infected cells. Indeed the overall rate of protein synthesis is the same in control and infected cells, at least until the beginning of cell lysis [2]. This continued synthesis of cellular proteins has made it difficult to investigate PM2directed protein synthesis in vivo [2]. An in vitro study of PM2 DNA-directed protein synthesis is presented here. Twelve polypeptides of -110 000-6000 mol. wt were synthesized in a PM2 DNAdirected cell-free protein synthesizing system from Escherichia coli. When E. coli ribosomes were used in the cell-free system, the 6000 mol. wt polypeptide became dominant. When A. espejiana ribosomes were used, the 6000 mol. wt polypeptide was also synthesized, but comprised a relatively small fraction of the polypeptide spectrum. The polynucleotide-dependent polynucleotide-pyrophosphorylase and endolysine activities, both of which are present in bacteriophage PM2, were also detected as products of the in vitro protein synthesis.

Resistance of the basic membrane proteins of myelin and bacterophage PM2 to proteolytic enzymes.

Basic protein(s) comprise about 30% of the proteins of both peripheral and central nervous system (PNS and CNS) myelin [cf.11 . Although the amino acid sequence of several myelin basic proteins has been determined, the secondary structure is not well understood [ 1,2] . Through our studies on another basic membrane protein, protein II of the lipid-containing bacteriophage PM2 [3], we became interested in a comparison of the properties of the myelin and PM2 basic proteins [4]. For this comparison we have used the basic protein from human or bovine CNS myelin, designated as protein Al [l] . After determining the isoelectric point of human Al by a simple micromethod [3], we studied the susceptibility of Al and PM2 protein II to proteolytic degradation, both in situ and after purification. In the latter case the effect of added acidic lipids on the susceptibility to proteolysis was also investigated. In the case of Al it is known that the acidic lipids form distinct lipoprotein phases with the protein. In the case of the PM2 protein, binding studies have been used to demonstrate a specific interaction between the basic protein and acidic lipids [6] .

Recent studies on the structure of bacteriophage PM2.

Since several reviews of work on lipid-containing bacteriophages have been published recently [8,10] or are now in press [9,14], emphasis will be placed on some of our most recent results, with a brief discussion of earlier work to provide a necessary background and continuity. As the studies were initially conceived, interest was centered on the protein-lipid and protein-protein interactions involved in the formation of the virion. During our studies on in vitro assembly of PM2 it became evident that protein-DNA interactions are of great importance for the assembly process, and these interactions will also be described. Bacteriophage PM2 grows on a marine bacterium, originally identified as the aerobic marine eubacteria Pseudomanas BAL-31 [6]. The recent recognition of a new genus of marine bacteria distinguished from the genus Pseudomonas on the basis of the GC content of the DNA, as well as on the basis of other characteristics, has posed the question as to the proper identity ofPseudomonas BAL-31 [1]. On the basis of the GC concent of 42 to 43% [11], Pseudomonas BAL-31 would appear to belong to the genus Altermonas and this hypothesis is now under investigation (P. Baumann, private communication). PM2 has 72% protein, 14.3% DNA, and 13 to 14% lipid, by weight. The DNA is a double-stranded superhelical DNA of molecular weight 6.3 to 6.4 x 106 [3]. There are four viral proteins, designated proteins I through IV, of molecular weights: 43,640; 27,310; 13,250; and 6,400, respectively [12]. The lipids may de divided into 92 to 93% phospholipids and 7 to 8% neutral lipids. The phospholipid composition may vary somewhat depending on the growth conditions of the bacteriophage, but it is always different from that of the host cell. The host cell has 20 to 22% phosphatidylglycerol (PG), 76 to 78% phosphatidylethanolamine (PE), and 0.3 to 0.6% acyl phosphatidylglycerol (APG) [18]. If PM2 is grown in wild type BAL-31 ceUs in synthetic medium or broth, the phospholipid composition of the virion is 59 to 64% PG, 28 to 39% PE, and less that 1% APG. If PM2 is grown in a fat ty acid auxotroph under conditions where the host cell fatty acid composition is forced to over 90%