Construction and Manipulation of a New Kaposi's Sarcoma-Associated Herpesvirus Bacterial Artificial Chromosome Clone
Construction and Manipulation of a New Kaposi's Sarcoma-Associated Herpesvirus Bacterial Artificial Chromosome Clone
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DOI：
10.1128/jvi.01019-12
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发表时间：
2012-09-01
影响因子：
5.4
通讯作者：
Jung, Jae U.
中科院分区：
文献类型：
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作者：
Brulois, Kevin F.;Chang, Heesoon;Jung, Jae U.
疱疹病毒如卡波西肉瘤相关疱疹病毒（KSHV）的有效遗传修饰依赖于细菌人工染色体（BAC）技术。为了促进这种方法，我们产生了一个新的KSHV BAC克隆，称为BAC 16，来自rKSHV.219病毒，其源于KSHV和EB病毒共感染的JSCI原发性渗出性淋巴瘤（PEL）细胞。限制性内切酶和完整测序数据表明，与其他KSHV毒株的完整基因组序列相比，JSCI PEL细胞的KSHV在整个病毒基因组中显示出最低水平的序列变异。BAC 16不仅在大肠杆菌和哺乳动物细胞中稳定增殖，没有明显的遗传重排，而且能够稳健地产生感染性病毒体（类似于5 × 10（7）/ml）。我们还通过产生K3或K5基因的缺失突变体证明了BAC 16的效用，其产物是膜相关RING-CH（MARCH）家族的E3连接酶。虽然以前的研究表明，无论是K3或K5的单独表达的结果在有效的下调主要组织相容性复合物I类（MHC-I）分子的表面表达，我们发现，K5，而不是K3，是KSHV裂解再激活或从头感染后的MHC-I表面表达下调的主要关键因素。本文提供的数据证明了BAC 16用于KSHV敲除和突变重组体的产生和表征的实用性，并进一步强调了除了研究单个基因表达之外，在KSHV基因组背景下病毒基因的功能分析的重要性。
Efficient genetic modification of herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) has come to rely on bacterial artificial chromosome (BAC) technology. In order to facilitate this approach, we generated a new KSHV BAC clone, called BAC16, derived from the rKSHV.219 virus, which stems from KSHV and Epstein-Barr virus-coinfected JSCI primary effusion lymphoma (PEL) cells. Restriction enzyme and complete sequencing data demonstrate that the KSHV of JSCI PEL cells showed a minimal level of sequence variation across the entire viral genome compared to the complete genomic sequence of other KSHV strains. BAC16 not only stably propagated in both Escherichia coli and mammalian cells without apparent genetic rearrangements, but also was capable of robustly producing infectious virions (similar to 5 x 10(7)/ml). We also demonstrated the utility of BAC16 by generating deletion mutants of either the K3 or K5 genes, whose products are E3 ligases of the membrane-associated RING-CH (MARCH) family. While previous studies have shown that individual expression of either K3 or K5 results in efficient downregulation of the surface expression of major histocompatibility complex class I (MHC-I) molecules, we found that K5, but not K3, was the primary factor critical for the downregulation of MHC-I surface expression during KSHV lytic reactivation or following de novo infection. The data presented here demonstrate the utility of BAC16 for the generation and characterization of KSHV knockout and mutant recombinants and further emphasize the importance of functional analysis of viral genes in the context of the KSHV genome besides the study of individual gene expression.