The terminal repeats and latency-associated nuclear antigen of herpesvirus saimiri are essential for episomal persistence of the viral genome
The terminal repeats and latency-associated nuclear antigen of herpesvirus saimiri are essential for episomal persistence of the viral genome
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DOI：
10.1099/0022-1317-83-9-2269
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发表时间：
2002-09-01
影响因子：
3.8
通讯作者：
Medveczky, PG
中科院分区：
文献类型：
--
作者：
Collins, CM;Medveczky, MM;Medveczky, PG
猴疱疹病毒saimiri（HVS）诱导恶性T细胞淋巴瘤，与卡波西肉瘤相关疱疹病毒（KSHV或HHV-8）密切相关。两者都属于γ-2疱疹病毒亚组。HVS的病毒基因组由一个独特的区域（L-DNA）组成，该区域包含侧翼为非编码末端重复序列（H-DNA）的所有病毒基因。在此，我们描述了在基于F'复制子的大肠杆菌中克隆含有致癌HVS株L-DNA的113 kb限制性片段。coli载体。克隆的DNA具有感染性，子代病毒基因组的末端由扩增的载体串联交替重复序列和单个H-DNA单元组成。感染这些病毒的T细胞含有通常在感染后几周发现的线性DNA，但不能形成附加型环状病毒DNA，这是病毒基因组的潜伏形式。产生了具有重建的H-DNA的重组病毒，并且用这些拯救的病毒感染的T细胞含有高拷贝数的附加体DNA。表达潜伏相关核抗原（拉娜）并含有不同数量的H-DNA重复序列的质粒作为附加体稳定复制，但含有三个重复单位的构建体产生最高的拷贝数。这些数据表明，完整的和多个末端重复序列是潜伏感染的T细胞中附加型复制的必要组成部分。此外，拉娜和末端重复序列对于稳定的质粒持久性是足够的。克隆的HVS也可用于HVS的诱变和通过在E.杆菌
The simian herpesvirus saimiri (HVS) induces malignant T cell lymphomas and is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8). Both belong to the gamma-2 herpesvirus subgroup. The viral genome of HVS consists of a unique region (L-DNA) that contains all of the viral genes flanked by non-coding terminal repeats (H-DNA). Here we describe the cloning of a 113 kb restriction fragment containing the L-DNA of an oncogenic HVS strain in an F' replicon-based E. coli vector. Cloned DNA was infectious and the ends of the progeny viral genome consisted of amplified tandem alternating repeats of vector and a single H-DNA unit. T cells infected with these viruses contained the linear DNA typically found a few weeks after infection, but were unable to form episomal circular viral DNA, which is the latent form of the viral genome. Recombinant viruses with reconstructed H-DNA were generated and T cells infected with these rescued viruses contained high copy numbers of episomal DNA. Plasmids expressing the latency-associated nuclear antigen (LANA) and containing various numbers of H-DNA repeats stably replicated as episomes, but constructs containing three repeat units produced the highest copy numbers. These data show that intact and multiple terminal repeats are essential components for episomal replication in latently infected T cells. Moreover, LANA and terminal repeats are sufficient for stable plasmid persistence. Cloned HVS can also be utilized for mutagenesis of HVS and for the expression of foreign genes through efficient manipulation of plasmids in E. coli.