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Infectivity of Molecular Forms of Simian Virus 40 DNA

Division of Biochemical Virology, Baylor College of Medicine, Houston, Texas 77025

The conversion of superhelical (Form I) SV40 DNA to nicked molecular forms was studied by incubating SV40 [³H]DNA for to 2 hr with monolayer cultures of monkey, human, or mouse cells in the presence of DEAE-dextran. The superhelical and the nicked DNA forms were then extracted from the cultures and separated by equilibrium centrifugation in caesium chloride-ethidium bromide (CsCl-EtBr) density gradients. About 66 and 42% of the Form I [³H]DNA was converted to nicked DNA during 2 hr of adsorption to monkey and to human cells, respectively. Form I DNA was adsorbed less by mouse kidney cells than by monkey cells, and only about 25% of the input DNA was converted to nicked forms in 2 hr. The specific infectivities (p.f.u./counts/min.) of the SV40 DNA extracted from the cultures were about the same as those of the input DNA, despite the fact that as much as 69% of the extracted DNA represented nicked molecular forms.

Cultures of monkey cells infected with SV40 were synchronized by treatment with 1--D-arabinofuranosylcytosine (ara-C) from 2 to 24 hr after infection and pulse-labelled with [³H]thymidine at 37 to 39 hr after infection. The pulse-labelled Form I and nicked forms of SV40 [³H]DNA were extracted from the cells, purified by CsCl-EtBr equilibrium centrifugation and velocity sedimentation in sucrose gradients, and assayed for infectivity. The nicked (Form II) SV40 DNA obtained from infected cells was about half as infective as superhelical (Form I) SV40 DNA.

When SV40 [³H]DNA was incubated with monkey kidney cells for 6 to 24 hr, 90 to 97% of the Form I DNA was converted to nicked molecular forms and specific infectivities were reduced by 85 to 98%, respectively.

These results suggest that minimally nicked SV40 DNA was about as infectious as superhelical (Form I) DNA in the SV40 DNA+DEAE-dextran plaque assay but that multiply nicked SV40 DNA was much less infectious.

Received 19 August 1970; accepted 30 October 1970.