Synthesis of infectious ΦX-174 bacteriophage in vitro

Abstract

STUDY of virus assembly has indicated the involvement of a diverse spectrum of protein–protein and protein–nucleic acid interactions (for review see ref. 1). In Escherichia coli cells infected with bacteriophage ΦX-174, synthesis of single-stranded circular viral DNA requires the functions of at least seven phage-coded genes which include genes specifying viral structural proteins F, G and H non-virion proteins A, B, C and D. The only known phage gene that is not involved in single-stranded DNA synthesis is gene E (lysis) (for review see ref. 2). It seems that synthesis of single-stranded circular viral DNA of ΦX-174 is very tightly coupled with the phage morphogenetic pathway. The synthesis of ΦX-174 viral DNA involves a replicative form (RF) DNA with an extended tail of single-stranded viral DNA up to one genome in length (‘rolling circle’ or a structure DNA)^3–7. Fujisawa and Hayashi^8,9 found that σ DNA in infected cells is associated with a number of phage and host proteins as a 50S complex which is a precursor of mature phage particles. They have proposed that synthesis and packaging of ΦX-174 single-stranded DNA occur in the 50S complex. Formation of the 50S complex requires the association of a functional RF DNA template with a 108S capsomeric structure containing the protein products of genes D, F, G and H (ref. 10). The functions of genes B and C are also required for formation of the 50S complex. The gene B protein is necessary to catalyse assembly of gene F and gene G proteins¹¹ to form a precursor of the 108S structure^10–12. In the absence of gene C protein, the 108S structure is produced but the 50S complex is not formed¹⁰. Apparently, RF DNA cannot act as a functional template for formation of the SOS complex and, consequently, for synthesis of single-stranded DNA if the gene C protein is absent. To elucidate the function of ΦX-174-coded proteins during phage morphogenesis and single-stranded DNA synthesis, we have developed an in vitro system that synthesizes circular, single-stranded viral DNA¹³. The system contained an extract prepared from cells infected with a lysis-defective mutant of ΦX-174. We show here that synthesis of circular viral DNA in the system does depend on the functions of the proteins encoded by ΦX-174 genes B and C. The circular single-stranded viral DNA synthesised in vitro is contained in infectious particles whose appearance is very much like that of mature ΦX-174 phage particles when examined by electron microscopy.