Collins Christopher M, Medveczky Maria M, Lund Troy, Medveczky Peter G
The H. Lee Moffitt Cancer Center, Tampa, Florida 33612-4799, USA2.
Department of Medical Microbiology and Immunology, College of Medicine, University of South Florida, MDC Box 10, 12901 Bruce B. Downs Blvd, Tampa, Florida 33612-4799, USA1.
J Gen Virol. 2002 Sep;83(Pt 9):2269-2278. doi: 10.1099/0022-1317-83-9-2269.
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.
猴疱疹病毒西尼罗河病毒（HVS）可诱发恶性T细胞淋巴瘤，且与卡波西肉瘤相关疱疹病毒（KSHV或HHV - 8）密切相关。二者均属于γ-2疱疹病毒亚组。HVS的病毒基因组由一个独特区域（L-DNA）组成，该区域包含所有病毒基因，两侧为非编码末端重复序列（H-DNA）。在此，我们描述了在基于F'复制子的大肠杆菌载体中克隆一个包含致癌性HVS毒株L-DNA的113 kb限制性片段。克隆的DNA具有感染性，子代病毒基因组的末端由载体的扩增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细胞含有感染后几周通常可发现的线性DNA，但无法形成游离的环状病毒DNA，而游离环状病毒DNA是病毒基因组的潜伏形式。产生了具有重建H-DNA的重组病毒，感染这些拯救病毒的T细胞含有高拷贝数的游离DNA。表达潜伏相关核抗原（LANA）并含有不同数量H-DNA重复序列的质粒作为游离体稳定复制，但含有三个重复单元的构建体产生的拷贝数最高。这些数据表明，完整且多个末端重复序列是潜伏感染T细胞中游离体复制的必需成分。此外，LANA和末端重复序列足以使质粒稳定存在。克隆的HVS还可用于HVS的诱变以及通过在大肠杆菌中高效操作质粒来表达外源基因。
