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signals, have been replaced by a minimal packaging element located in the 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.