Angeletti P C, Engler J A
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, 35294-0005, USA.
J Virol. 1998 Apr;72(4):2896-904. doi: 10.1128/JVI.72.4.2896-2904.1998.
Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) via an interaction mediated by the precursor of the terminal protein (pTP). The identities of cellular proteins involved in these complexes have remained obscure. We present evidence that pTP binds to a multifunctional pyrimidine biosynthesis enzyme found at replication domains on the NM. Far-Western blotting identified proteins of 150 and 240 kDa that had pTP binding activity. Amino acid sequencing of the 150-kDa band revealed sequence identity to carbamyl phosphate synthetase I (CPS I) and a high degree of homology to the related trifunctional enzyme known as CAD (for carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase). Western blotting with an antibody directed against CAD detected a 240-kDa band that comigrated with that detected by pTP far-Western blotting. Binding experiments showed that a pTP-CAD complex was immunoprecipitable from cell extracts in which pTP was expressed by a vaccinia virus recombinant. Additionally, in vitro-translated epitope-tagged pTP and CAD were immunoprecipitable as a complex, indicating the occurrence of a protein-protein interaction. Confocal fluorescence microscopy of Ad-infected NM showed that pTP and CAD colocalized in nuclear foci. Both pTP and CAD were confirmed to colocalize with active sites of replication detected by bromodeoxyuridine incorporation. These data support the concept that the pTP-CAD interaction may allow anchorage of Ad replication complexes in the proximity of required cellular factors and may help to segregate replicated and unreplicated viral DNA.
腺病毒（Ad）复制复合体通过末端蛋白前体（pTP）介导的相互作用在核基质（NM）的离散位点形成。参与这些复合体的细胞蛋白的身份一直不清楚。我们提供的证据表明，pTP与在NM上的复制结构域发现的一种多功能嘧啶生物合成酶结合。Far-Western印迹法鉴定出具有pTP结合活性的150 kDa和240 kDa的蛋白质。对150 kDa条带的氨基酸测序显示其与氨甲酰磷酸合成酶I（CPS I）具有序列同一性，并且与相关的三功能酶CAD（氨甲酰磷酸合成酶、天冬氨酸转氨甲酰酶和二氢乳清酸酶）具有高度同源性。用针对CAD的抗体进行的Western印迹检测到一条240 kDa的条带，其迁移位置与pTP Far-Western印迹检测到的条带相同。结合实验表明，pTP-CAD复合体可从痘苗病毒重组体表达pTP的细胞提取物中免疫沉淀。此外，体外翻译的表位标记的pTP和CAD可作为复合体免疫沉淀，表明存在蛋白质-蛋白质相互作用。对Ad感染的NM进行共聚焦荧光显微镜检查显示，pTP和CAD共定位于核灶。pTP和CAD均被证实与通过溴脱氧尿苷掺入检测到的复制活性位点共定位。这些数据支持这样的概念，即pTP-CAD相互作用可能允许Ad复制复合体锚定在所需细胞因子附近，并可能有助于分离复制和未复制的病毒DNA。
