The Process of Infection with Bacteriophage φX174: I. Evidence for a "Replicative Form"

Procedures are described for the preparation of density- and radioactivity-labelled φX174 particles. The process of bacterial infection with such particles has been studied by density-gradient analysis of the contents of the infected cells prior to normal lysis. Upon entry into a susceptible cell, the single-stranded DNA of φX174 is converted to an altered “replicative form” (RF). The RF, which is infective to bacterial protoplasts, is then multiplied manyfold. Conversion to RF and multiplication of RF can take place in the presence of chloramphenicol. At no time during the infective process is there a pool of free single-stranded DNA. In normal infection single-stranded progeny DNA appears in mature progeny virus particles as early as eight minutes after infection. In chloramphenicol neither progeny virus particles nor single-stranded DNA are formed. Several properties of the RF are similar to those of double-stranded DNA. It is lighter in density than the single-stranded DNA. The density of the RF particles containing the density-labelled parental DNA is approximately that which would be expected if these molecules had been converted to a double-stranded complementary form. Upon heating, the RF appears to denature in a manner similar to double-stranded DNA. The RF is considerably more resistant to inactivation by ultraviolet radiation than is the single-stranded DNA; this resistance can be melted out by heating of the RF