REPLICATION OF TRANSFECTED PLASMID DNA BY CELLS INFECTED WITH AFRICANSWINE FEVER VIRUS

Recombinant plasmids containing African swine fever virus (ASFV) DNA f ragments covering all the virus genome were transfected into infected cells in order to detect viral origins of DNA replication. Plasmid rep lication was monitored by sensitivity to Mbol, which cleaves only repl icated, unmethylated DNA, and resistance to Dpnl, which cleaves only t he same methylated sequence. All the recombinants replicated to a simi lar extent, indicating that ASFV does not use a preferred origin for D NA replication. Circular plasmids without viral inserts were also repl icated, but linearized plasmids or lambda bacteriophage DNA were not r eplicated. Replicated plasmid DNA began to accumulate with a time cour se similar to viral DNA, starting between 6 and 12 hr p.i. and increas ing steadily for about 18 hr. This apparent dependence on Viral functi ons was confirmed by the sensitivity of plasmid replication to phospho noacetic acid and resistance to aphidicolin and by the reduction of re plication in cells infected with a mutant defective in DNA replication . Replicated plasmid DNA was present as unit length circles and as lar ge dimension forms, probably head-to-tail concatemers. The results of two-dimensional electrophoresis (neutral/alkaline) favor a rolling-cir cle mechanism for plasmid DNA replication. (C) Press Academic Press, I nc.