THE REPLICATING INTERMEDIATES OF HERPES-SIMPLEX VIRUS TYPE-1 DNA ARE RELATIVELY SHORT

Herpes simplex virus type 1 (HSV-1) replication is thought to occur via a rolling circle type of mechanism, generating large DNA concatemers from which unit length genomes are subsequently cleaved. In this report, we have employed field inversion gel electrophoresis (FIGE), Southern blot hybridization, and endonuclease digestion, to identify and characterize these DNAs. Two species of HSV-1 DNA: (1) genome-length and (2) DNA that remained at the electrophoresis origin (referred to as well-associated DNA) were detected. To ascertain that the latter was large in size and not virion DNA trapped at the origin with high molecular weight cellular DNA, the infected cell DNA was digested with a restriction enzyme that does not cut the viral DNA. In order to do this HSV-1 strain 1702, lacking any XbaI sites in its genome, was utilized. After digestion of samples with XbaI, and FIGE, cellular DNA was seen to migrate into the gel; however, the viral DNA remained in the sample wells. Pulse labeling experiments showed that this large DNA was processed to 150 kb genome lengths. Endonuclease digestion of the well-associated DNA revealed that it contained a greater ratio of joint to terminal fragments than virion DNA - a characteristic of long concatemers. Quantitation of the terminal fragments revealed mainly L termini. Surprisingly, the ratio of joint to terminal fragments was 2.5 suggesting that the lengths of concatemers were short (in the order of 1-2 genome lengths) and that the well association was due to conformation rather than concatemeric length. Because one of these genome lengths is present as the replication intermediate, the growing tail must be less than genome length. Thus genome lengths must be processed from the replication intermediate soon after they are completed.