Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Immune Disease Institute, Boston, Massachusetts, USA.
mBio. 2012 Feb 7;3(1). doi: 10.1128/mBio.00301-11. Print 2012.
Drastic reorganization of the nucleus is a hallmark of herpesvirus replication. This reorganization includes the formation of viral replication compartments, the subnuclear structures in which the viral DNA genome is replicated. The architecture of replication compartments is poorly understood. However, recent work with human cytomegalovirus (HCMV) showed that the viral DNA polymerase subunit UL44 concentrates and viral DNA synthesis occurs at the periphery of these compartments. Any cellular factors involved in replication compartment architecture are largely unknown. Previously, we found that nucleolin, a major protein component of nucleoli, associates with HCMV UL44 in infected cells and is required for efficient viral DNA synthesis. Here, we show that nucleolin binds to purified UL44. Confocal immunofluorescence analysis demonstrated colocalization of nucleolin with UL44 at the periphery of replication compartments. Pharmacological inhibition of viral DNA synthesis prevented the formation of replication compartments but did not abrogate association of UL44 and nucleolin. Thus, association of UL44 and nucleolin is unlikely to be a nonspecific effect related to development of replication compartments. No detectable colocalization of 5-ethynyl-2'-deoxyuridine (EdU)-labeled viral DNA with nucleolin was observed, suggesting that nucleolin is not directly involved in viral DNA synthesis. Small interfering RNA (siRNA)-mediated knockdown of nucleolin caused improper localization of UL44 and a defect in EdU incorporation into viral DNA. We propose a model in which nucleolin anchors UL44 at the periphery of replication compartments to maintain their architecture and promote viral DNA synthesis.
Human cytomegalovirus (HCMV) is an important human pathogen. HCMV infection causes considerable rearrangement of the structure of the nucleus, largely due to the formation of viral replication compartments within the nucleus. Within these compartments, the virus replicates its DNA genome. We previously demonstrated that nucleolin is required for efficient viral DNA synthesis and now find that the nucleolar protein nucleolin interacts with a subunit of the viral DNA polymerase, UL44, specifically at the periphery of replication compartments. Moreover, we find that nucleolin is required to properly localize UL44 at this region. Nucleolin is, therefore, involved in the organization of proteins within replication compartments. This, to our knowledge, is the first report identifying a cellular protein required for maintaining replication compartment architecture.
疱疹病毒复制的一个标志是细胞核的剧烈重组。这种重组包括病毒复制隔室的形成，即病毒 DNA 基因组复制的亚核结构。复制隔室的结构知之甚少。然而，最近对人类巨细胞病毒（HCMV）的研究表明，病毒 DNA 聚合酶亚基 UL44 浓缩，病毒 DNA 合成发生在这些隔室的外围。涉及复制隔室结构的任何细胞因子在很大程度上尚不清楚。此前，我们发现核仁蛋白成分核仁素在受感染的细胞中与 HCMV UL44 结合，并需要有效的病毒 DNA 合成。在这里，我们表明核仁素与纯化的 UL44 结合。共聚焦免疫荧光分析表明核仁素与 UL44 在复制隔室的外围共定位。病毒 DNA 合成的药理学抑制阻止了复制隔室的形成，但没有消除 UL44 和核仁素的结合。因此，UL44 和核仁素的结合不太可能是与复制隔室形成相关的非特异性效应。未观察到 5-乙炔基-2'-脱氧尿苷（EdU）标记的病毒 DNA 与核仁素的明显共定位，表明核仁素不直接参与病毒 DNA 合成。核仁素的小干扰 RNA（siRNA）介导的敲低导致 UL44 的定位不当和 EdU 掺入病毒 DNA 的缺陷。我们提出了一个模型，即核仁素将 UL44 锚定在复制隔室的外围，以维持其结构并促进病毒 DNA 合成。
人巨细胞病毒（HCMV）是一种重要的人类病原体。HCMV 感染导致细胞核结构的大量重排，主要是由于细胞核内病毒复制隔室的形成。在这些隔室中，病毒复制其 DNA 基因组。我们之前证明核仁素是有效病毒 DNA 合成所必需的，现在发现核仁蛋白核仁素与病毒 DNA 聚合酶的亚基 UL44 特异性相互作用，特别是在复制隔室的外围。此外，我们发现核仁素需要正确地将 UL44 定位在该区域。因此，核仁素参与复制隔室中蛋白质的组织。据我们所知，这是第一个鉴定维持复制隔室结构所需的细胞蛋白的报告。
