The human cytomegalovirus IE2 and UL112-113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein-associated nuclear bodies (PODs or ND10)
The human cytomegalovirus IE2 and UL112-113 proteins accumulate in viral DNA replication compartments that initiate from the periphery of promyelocytic leukemia protein-associated nuclear bodies (PODs or ND10)
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DOI：
10.1128/jvi.73.12.10458-10471.1999
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发表时间：
1999-12-01
影响因子：
5.4
通讯作者：
Hayward, GS
中科院分区：
文献类型：
--
作者：
Ahn, JH;Jang, WJ;Hayward, GS
在人巨细胞病毒（HCMV）感染期间，早幼粒细胞白血病蛋白（PML）相关核体（也称为PML致癌结构域[PODs]或ND10）的周围是输入病毒基因组沉积和立即早期（IE）基因转录的位点。在感染后的早期，IE1蛋白定位并随后破坏pod，而IE2蛋白定位在pod内部或邻近。这一过程似乎是有效的病毒基因表达和DNA复制所必需的。我们通过研究病毒IE蛋白、DNA复制蛋白和PML蛋白在生产感染过程中的定位，研究了病毒DNA复制室形成的起始过程。感染野生型HCMV（Towne）和不能破坏pod的ie1缺失突变型HCMV（CR208）感染的人成纤维细胞（HF细胞）共聚焦显微镜证实，在感染后2至6小时，IE2定位在pod附近。在HCMV（Towne）感染的HE细胞中，在24至48小时，IE2也在新形成的病毒DNA复制室中积累，这些病毒DNA复制室含有聚合酶加工因子（UL44）、单链DNA结合蛋白（SSB; UL57）、UL112-113辅助蛋白和新结合的溴脱氧尿苷（BrdU）。HCMV（CR208）感染的HF细胞的双重标记表明，病毒DNA复制区室的形成始于颗粒结构，颗粒结构从一些pod的外围萌发，随后合并成由pod两侧的更大结构。在瞬时DNA转染实验中，发现IE2第5外显子的N端（密码子136 ~ 290）和C端（密码子379 ~ 579）都是IE2点状分布及其与pod关联所必需的，而不是它们之间的中心区域。与IE2一样，UL112-113辅助复制蛋白也在dna转染和病毒感染的细胞中以pod相关的模式分布，从6小时开始。此外，当UL112-113存在时，所有六种复制核心机械蛋白（聚合酶复合物，SSB和解旋酶-引物酶复合物）一起表达时，它们也在pod相关位点积累。这表明UL112-113蛋白（而不是IE2）可能在将病毒复制叉蛋白募集到pod外围的过程中发挥作用。这些结果表明：(1)在pod的外围积聚之后，IE2与核心蛋白一起被整合到从pod的外围开始的病毒DNA复制室中，然后生长并填充pod组之间的空间；(2)在病毒复制室形成的初始阶段，UL112-113蛋白似乎在组装和招募tore replication machinery蛋白方面发挥了关键作用。
During human cytomegalovirus (HCMV) infection, the periphery of promyelocytic leukemia protein (PML)associated nuclear bodies (also known as PML oncogenic domains [PODs] or ND10) are sites for both input viral genome deposition and immediate-early (IE) gene transcription. At very early times after infection, the IE1 protein localizes to and subsequently disrupts PODs, whereas the IE2 protein Localizes within or adjacent to PODs. This process appears to be required for efficient viral gene expression and DNA replication. We have investigated the initiation of viral DNA replication compartment formation by studying the localization of viral IE proteins, DNA replication proteins, and the PML protein during productive infection. Localization of IE2 adjacent to PODs between 2 and 6 h after infection was confirmed by confocal microscopy of human fibroblasts (HF cells) infected with both wild-type HCMV(Towne) and with an IE1-deletion mutant HCMV(CR208) that fails to disrupt PODs. In HCMV(Towne) -infected HE cells at 24 to 48 h, IE2 also accumulated in newly formed viral DNA replication compartments containing the polymerase processivity factor (UL44), the single-stranded DNA binding protein (SSB; UL57), the UL112-113 accessory protein, and newly incorporated bromodeoxyuridine (BrdU). Double labeling of the HCMV(CR208)-infected HF cells demonstrated that formation of viral DNA replication compartments initiates within granular structures that bud from the periphery of some of the PODs and subsequently coalesce into larger structures that are flanked by PODs. In transient DNA transfection assays, both the N terminus (codons 136 to 290) and the C terminus (codons 379 to 579) of IE2 exon 5, but not the central region between them, were found to be necessary for both the punctate distribution of IE2 and its association with PODs. Like IE2, the UL112-113 accessory replication protein was also distributed in a POD-associated pattern in both DNA-transfected and virus-infected cells beginning at 6 h. Furthermore, when all six replication core machinery proteins (polymerase complex, SSB, and helicase-primase complex) were expressed together in the presence of UL112-113, they also accumulated at POD-associated sites, suggesting that the UL112-113 protein (but not IE2) may play a role in recruitment of viral replication fork proteins into the periphery of PODs. These results show that (i) subsequent to accumulating at the periphery of PODs, IE2 is incorporated together with the core proteins into viral DNA replication compartments that initiate from the periphery of PODs and then grow to fill the space between groups of PODs, and (ii) the UL112-113 protein appears to have a key role in assembling and recruiting the tore replication machinery proteins in the initial stages of viral replication compartment formation.