Signal Transduction Research Group, Molecular Mechanisms of Growth Control Research Group, University of Alberta, 327C Heritage Medical Research Center, Edmonton, Alberta T6G 2S2, Canada.
J Virol. 2010 Feb;84(4):1920-33. doi: 10.1128/JVI.01934-09. Epub 2009 Dec 9.
The genomes of herpes simplex virus type 1 (HSV-1) are regularly chromatinized during latency such that their digestion with micrococcal nuclease (MCN) releases nucleosome-sized DNA fragments. In lytically infected cells, in contrast, MCN releases HSV-1 DNA in primarily heterogeneously sized fragments. Consistently, only a small percentage of this HSV-1 DNA coimmunoprecipitates with histones. Most current models propose that histones associate with HSV-1 DNA during lytic infections at low occupancy. However, histone modification or occupation is also proposed to regulate HSV-1 transcription. It remains unclear how the histones associated with a small percentage of HSV-1 DNA may regulate transcription globally. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. We evaluated the HSV-1 DNA-containing complexes at 5 h after (lytic) infection by biochemical fractionations. Nuclear HSV-1 DNA did not fractionate as protein-free HSV-1 DNA but as DNA in cellular nucleosomes. Moreover, MCN released HSV-1 DNA in complexes that fractionate as cellular mono- and dinucleosomes by centrifugation followed by sucrose gradients and size-exclusion chromatography. The HSV-1 DNA in such complexes was protected to heterogeneous sizes and was more accessible to MCN than DNA in most cellular chromatin. Using a modified MCN digestion to trap unstable digestion intermediates, HSV-1 DNA was quantitatively recovered in discrete mono- to polynucleosome sizes in complexes fractionating as cellular mono- to polynucleosomes. The HSV-1 DNAs in complexes fractionating as mono- to dinucleosomes were stabilized by cross-linking. Therefore, most HSV-1 DNA forms particularly unstable nucleosome-like complexes at 5 h of lytic infection.
单纯疱疹病毒 1 型（HSV-1）的基因组在潜伏期经常被染色质化，使得用微球菌核酸酶（MCN）消化会释放出核小体大小的 DNA 片段。相比之下，在裂解感染的细胞中，MCN 释放的 HSV-1 DNA 主要是异质大小的片段。一致地，只有一小部分 HSV-1 DNA 与组蛋白共免疫沉淀。目前大多数模型提出，在裂解感染时，组蛋白以低占有率与 HSV-1 DNA 结合。然而，组蛋白修饰或占有率也被提议用来调节 HSV-1 转录。仍不清楚与一小部分 HSV-1 DNA 结合的组蛋白如何全局调节转录。此外，含有组蛋白和 HSV-1 DNA 的复合物的物理性质尚不清楚。我们通过生化分级分离评估了 5 小时后（裂解）感染的 HSV-1 DNA 复合物。核 HSV-1 DNA 没有作为无蛋白的 HSV-1 DNA 分级分离，而是作为细胞核小体中的 DNA 分级分离。此外，MCN 释放的 HSV-1 DNA 与通过离心、蔗糖梯度和大小排阻层析分级分离为细胞单和二核小体的复合物结合。这些复合物中的 HSV-1 DNA 受到保护，大小不均一，并且比大多数细胞染色质中的 DNA 更易受 MCN 消化。使用改良的 MCN 消化来捕获不稳定的消化中间产物，在分级分离为细胞单到多核小体大小的复合物中，HSV-1 DNA 以离散的单到多核小体大小被定量回收。在分级分离为单核到二核小体复合物的复合物中，HSV-1 DNA 通过交联而稳定。因此，在裂解感染 5 小时时，大多数 HSV-1 DNA 形成特别不稳定的核小体样复合物。
