Effect of Fv-1 gene product on synthesis of linear and supercoiled viral DNA in cells infected with murine leukemia virus

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Abstract
Levels of unintegrated viral DNA made in Fv-1b/b (SIM.cntdot.R, JLS-V9) and Fv-1n/n (NIH/3T3) [mouse] cell lines after infection with N- or B-tropic murine leukemia virus (MuLV) were measured. Different forms of viral DNA were sedimented on neutral sucrose or ethidium bromide-CsCl density gradients and detected by hybridization with complementary DNA. The major viral DNA species made in Fv-1 permissive or resistant cells sedimented at 20S on neutral sucrose gradient. Levels of this 20S viral DNA species were not significantly different in both systems. Levels of closed circular (form I) viral DNA separated on ethidium bromide-CsCl gradients were decreased in Fv-1 resistant cells. Various species of viral DNA were also analyzed by the agarose gel-DNA transfer procedure of Southern. The major viral DNA species migrated as a double-stranded linear DNA of 5.7 .times. 106 daltons. The MW of linear viral DNA molecules extracted from Fv-1 permissive or resistant cells appeared to be the same. Levels of this linear viral DNA were almost identical in both systems except in B-tropic MuLV-infected resistant NIH/3T3 cells in which a moderate decrease was measured. Two closed circular viral DNA species were observed by this technique. Their levels were markedly decreased in Fv-1 resistant cells. Apparently the Fv-1 restriction does not grossly affect the formation of linear double-stranded viral DNA but prevents the accumulation of closed circular viral DNA. The Fv-1 gene product could allow the synthesis of a normal linear viral DNA but interfere with the formation of supercoiled viral DNA. It could promote the synthesis of a faulty linear viral DNA whose defect (yet undetected) would prevent its circularization. The Fv-1 restriction mechanism appears to occur before the integration event itself.