Severini A, Scraba D G, Tyrrell D L
GlaxoWellcome Heritage Research Insititute, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Canada.
J Virol. 1996 May;70(5):3169-75. doi: 10.1128/JVI.70.5.3169-3175.1996.
Herpes simplex virus type 1 (HSV-1) replication produces large intracellular 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 direct evidence for the presence of branches in the high-molecular-weight DNA produced during HSV-1 replication. On neutral agarose two-dimensional gel electrophoresis, a technique that allows separation of branched restriction fragments from linear fragments, intracellular HSV-1 DNA produces arches characteristic of Y junctions (such as replication forks) and X junctions (such as merging replication forks or recombination intermediates). Branched structures were resolved by T7 phage endonuclease I (gene 3 endonuclease), an enzyme that specifically linearizes Y and X structures. Resolution was detected by the disappearance of the arches on two-dimensional gel electrophoresis. Branched structures 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 restriction enzyme which cuts the HSV-1 genome once does not resolve the large structure of HSV-1 intracellular DNA on pulsed-field gel electrophoresis. We have confirmed that result by using sucrose gradient sedimentation, in which both undigested and digested replicative intermediates sediment to the bottom of the gradient. Taken together, our experiments show that the intracellular HSV-1 DNA is held together in a large complex by frequent branches that create a network of replicating molecules. The fact that most of these branches are Y structures suggests that the network is held together by frequent replication forks and that it resembles the replicative intermediates of bacteriophage T4. Our findings add complexity to the simple model of rolling-circle DNA replication, and they pose interesting questions as to how the network is formed and how it is resolved for packaging into progeny virions.
1型单纯疱疹病毒（HSV-1）复制会产生大的细胞内DNA分子，这些分子似乎呈头尾串联排列。我们之前曾提出（A. 塞韦里尼、A.R. 摩根、D.R. 托维尔和D.L.J. 泰勒尔，《病毒学》200:428 - 435，1994年）这些DNA种类可能具有复杂的分支结构。我们现在为HSV-1复制过程中产生的高分子量DNA中存在分支提供了直接证据。在中性琼脂糖二维凝胶电泳中，这是一种能将分支限制片段与线性片段分离的技术，细胞内HSV-1 DNA产生了Y型连接（如复制叉）和X型连接（如合并的复制叉或重组中间体）特征性的弓形。分支结构通过T7噬菌体核酸内切酶I（基因3核酸内切酶）得以解析，该酶能特异性地使Y型和X型结构线性化。通过二维凝胶电泳上弓形的消失检测到了解析。分支结构也通过电子显微镜得以可视化。观察到了具有单个Y型连接的分子，以及包含两个或更多连续Y型连接的大缠结。我们之前曾表明，一种在HSV-1基因组上切割一次的限制酶在脉冲场凝胶电泳中无法解析HSV-1细胞内DNA的大结构。我们通过蔗糖梯度沉降证实了这一结果，其中未消化和消化后的复制中间体都沉降到了梯度底部。综合来看，我们的实验表明细胞内HSV-1 DNA通过频繁的分支聚集在一个大的复合体中，这些分支形成了一个复制分子网络。这些分支大多是Y型结构这一事实表明，该网络由频繁的复制叉维系在一起，并且它类似于噬菌体T4的复制中间体。我们的发现为滚环DNA复制的简单模型增添了复杂性，并且就该网络如何形成以及如何解析以包装进子代病毒粒子提出了有趣的问题。
