Analysis of unselected HSV-1 McKrae/HSV-2 HG52 recombinants demonstrates preferential recombination between intact genomes and restriction endonuclease fragments containing an origin of replication

Summary

To identify viral genes involved in reactivation of herpes simplex virus from latency, intertypic HSV-1 strain McKrae/HSV-2 strain HG 52 recombinants were selected following cotransfection of intact McKrae DNA and XbaI or Hpal cleaved HG 52 DNA. Eleven separately obtained recombinants containing HG 52 inserts between 0.35–0.56 and/or 0.82–1.0 map units (mu) were isolated. It was noted that with HpaI digested HG 52 DNA, only recombinants containing type 2 inserts from Hpald (0.35–0.57) and/or containing an intact type 2 [S] region were isolated. Similarly with XbaI cleaved HG 52 DNA only recombinants containing type 2 sequences from XbaIc (0–0.45) were isolated. In effect, the type 2 insert always contained one or both origins of replication (ORI_L/ORI_S). In reciprocal experients isolation of two recombinants from cotransfection of HpaI cleaved McKrae DNA with intact HG 52 DNA confirmed this finding; one contained both copies of ORI_S and the intervening short region sequences of McKrae, the other contained approximately 3 kb of McKrae in which ORI_L is located. These results indicate that either (a) the presence of an origin of replication in a RE fragment amplifies the fragment thereby increasing its concentration and hence recombination potential with intact genomes; and/or (b) recombination and replication may be correlated. In either case isolation of recombinants containing ORI_L and ORI_S from the restricted DNA parent strongly suggests that both origins are functional in vitro.