Study of the Structure of Replicative Intermediates of HSV-1 DNA by Pulsed-Field Gel Electrophoresis

Abstract

DNA from HSV-1-infected cells was separated by pulsed-field gel electrophoresis into two virus-specific bands: one that migrated as the linear monomer genome (152 kb) and another that remained at the origin of the gel. The latter band contained the replicating HSV-1 DNA, as determined by pulse-labeling with [ 3H]thymidine. To investigate the structure of this "gel origin" DNA, we constructed a HSV-1 KOS mutant bearing a unique Pac I restriction site (HSV-1 PAC1DTK). Partial digestion of gel origin PAC1DTK DNA at late times postinfection (24-48 hr) demonstrated the presence of linear concatemers on pulsed-field gel electrophoresis. Within each concatemer, the long (L) regions of adjacent monomer genomes were found in the two possible orientations. In addition, shorter-than-unit-size fragments that corresponded in size to the left end fragments of the viral genome were detected with the U L region in the two possible orientations. At early times postinfection (8-12 hr), digestion with PacI released only a trace of linear fragments, and most of the gel origin DNA did not migrate on pulsed-field gel electrophoresis. Multiple cuts with EcoRI (a restriction enzyme that cuts the HSV-1 KOS genome 12 times) were necessary to release linear fragments that migrated from the origin of the gel. These results indicate that replicative intermediates of HSV-1 DNA are linked in a large network that needs to be unraveled before packaging takes place. This network may be composed of linear molecules linked together by frequent recombination events or of products of a mode of replication other then simple rolling circle (e.g., theta replication).