Circularization of the herpes simplex virus type 1 genome upon lytic infection

For many years, the generally accepted model for the replication of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1) incorporated initial circularization of linear molecules in the cell nucleus. Ensuing DNA synthesis resulted in the generation of head-to-tail concatemers which were subsequently cleaved into monomeric units and packaged into the nascent viral capsid. Recently, however, it has been proposed that circularization of HSV-1 genomes does not occur at the onset of lytic infection and moreover that this event is specifically inhibited by the HSV-1 transcriptional transactivator, ICP0 (S.A. Jackson and N.A. DeLuca, Proc. Natl. Acad. Sci. USA 100:7871-7876, 2003). To further investigate genome circularization, we have generated HSV-1 derivatives in which the viral a sequences, which contain the cleavage-packaging signals, have been replaced by a minimal packaging element located in the thymidine kinase gene. In contrast to wild-type HSV-1, fusion of the genomic termini of these viruses produces a novel fragment in circular or concatemeric DNA which can be detected by Southern blot hybridization. Utilizing these viruses, we demonstrate that fusion of the genomic termini occurred rapidly upon infection and in the presence of inhibitors of viral DNA or protein synthesis. We provide evidence indicating that the end joining represented circularization rather than concatemerization of input molecules and that circularized molecules functioned as templates for replication. Since the termini of these viruses lack direct repeats, our findings indicate that circularization can be mediated by direct end-to-end ligation of linear input genomes.