BRANCHED STRUCTURES IN THE INTRACELLULAR DNA OF HERPES-SIMPLEX VIRUS TYPE-1

Herpes simplex virus type 1 (HSV-1) replication produces large intrace llular DNA molecules that appear to be in a head-to-tail concatemeric arrangement. We have previously suggested (A. Severini, A.R. Morgan, D .R. Tovell, and D.L.J. Tyrrell, Virology 200:428-435, 1994) that these DNA species may have a complex branched structure. We now provide dir ect evidence for the presence of branches in the high-molecular-weight DNA produced during HSV-1 replication. On neutral agarose two-dimensi onal gel electrophoresis, a technique that allows separation of branch ed restriction fragments from Linear fragments, intracellular HSV-1 DN A produces arches characteristic of Y junctions (such as replication f orks) and X junctions (such as merging replication forks or recombinat ion intermediates). Branched structures were resolved by T7 phage endo nuclease I (gene 3 endonuclease), an enzyme that specifically lineariz es Y and X structures. Resolution was detected by the disappearance of the arches on two-dimensional gel electrophoresis. Branched structure s were also visualized by electron microscopy. Molecules with a single Y junction were observed, as well as large tangles containing two or more consecutive Y junctions. We had previously shown that a restricti on enzyme which cuts the HSV-1 genome once does not resolve the large structure of HSV-1 intracellular DNA on pulsed-field gel electrophores is. We have confirmed that result by using sucrose gradient sedimentat ion, in which both undigested and digested replicative intermediates s ediment to the bottom of the gradient. Taken together, our experiments show that the intracellular HSV-1 DNA is held together in a large com plex by frequent branches that create a network of replicating molecul es. The fact that most of these branches are Y structures suggests tha t the network is held together by frequent replication forks and that it resembles the replicative intermediates of bacteriophage T4. Our fi ndings add complexity to the simple model of rolling-circle DNA replic ation, and they pose interesting questions as to how the network is fo rmed and how it is resolved for packaging into progeny virions.