Disruption of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen leads to abortive episome persistence
Disruption of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen leads to abortive episome persistence
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DOI：
10.1128/jvi.78.20.11121-11129.2004
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发表时间：
2004-10-01
影响因子：
5.4
通讯作者：
Gao, SJ
中科院分区：
文献类型：
--
作者：
Ye, FC;Zhou, FC;Gao, SJ
潜伏核抗原（LNA）与卡波济肉瘤相关疱疹病毒（KSHV）附加体持久性有关。LNA与KSHV附加体在中期共定位于染色体上，并维持含有KSHV末端重复序列的质粒的稳定性和复制。在这项研究中，我们研究了LNA的功能，在附加体持久性的背景下，全长KSHV基因组的突变分析。我们产生了KSHV突变体，BAC 36-DeltaLNA，其中LNA通过以KSHV BAC克隆BAC 36作为模板的基于转座子的诱变而被破坏。免疫荧光抗体染色显示，插入一个转座子盒到LNA破坏其表达，但对两个相邻的基因，vCyclin和vFLIP基因的表达没有影响。使用绿色荧光蛋白（GFP）盒作为KSHV附加体的跟踪标记，我们发现在通过转染将附加体递送到293细胞中后的早期阶段，BAC 36-DeltaLNA培养物中的GFP阳性细胞比野生型BAC 36培养物中的GFP阳性细胞少8.7倍，这可以通过与LNA表达质粒而不是对照质粒共转染而部分挽救。基于GFP表达和Gardella凝胶分析以及用于检测KSHV基因组的定量PCR测定，携带BAC 36-DeltaLNA（有或没有瞬时互补）的细胞在培养2周后迅速丢失附加体并变得无病毒。相反，BAC 36附加体在同一时期稳定维持。通过对BAC 36的潮霉素选择而不是对BAC 36-DeltaLNA的潮霉素选择，容易建立具有接近100%的携带KSHV附加体的细胞的稳定培养物。这些结果最终表明LNA对于KSHV附加体在哺乳动物细胞中的建立和持久性是必不可少的。
Latent nuclear antigen (LNA) is implicated in Kaposi's sarcoma-associated herpesvirus (KSHV) episome persistence. LNA colocalizes with KSHV episomes on chromosomes in metaphase, and it maintains the stability and replication of KSHV terminal repeat-containing plasmids. In this study, we examined the function of LNA in episome persistence in the context of full-length KSHV genome by mutagenesis analysis. We generated a KSHV mutant, BAC36-DeltaLNA, with LNA disrupted by transposon-based mutagenesis with a KSHV BAC clone, BAC36, as a template. Immunofluorescence antibody staining revealed that the insertion of a transposon cassette into LNA disrupted its expression but had no effect on the expression of two adjacent genes, the vCyclin and vFLIP genes. Using a green fluorescent protein (GFP) cassette as a tracking marker for the KSHV episome, we found 8.7-fold-fewer GFP-positive cells in BAC36-DeltaLNA cultures than in wild-type BAC36 cultures at the early stage following episome delivery into 293 cells by transfection, which could be partially rescued by cotransfection with a LNA expression plasmid but not a control plasmid. Cells harboring BAC36-DeltaLNA with or without transient complementation rapidly lost episomes and became virus-free after 2 weeks of culture based on GFP expression and Gardella gel analysis and quantitative PCR assays for detecting KSHV genomes. In contrast, BAC36 episomes were stably maintained during the same period. Stable cultures with close to 100% of cells harboring KSHV episomes were readily established by hygromycin selection for BAC36 but not for BAC36-DeltaLNA. These results conclusively indicate that LNA is essential for the establishment and persistence of KSHV episomes in mammalian cells.