Viral Oncology Program, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
J Virol. 2012 May;86(9):5179-91. doi: 10.1128/JVI.00169-12. Epub 2012 Feb 29.
The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein functions in latently infected cells as an essential participant in KSHV genome replication and as a driver of dysregulated cell growth. To identify novel LANA protein-cell protein interactions that could contribute to these activities, we performed a proteomic screen in which purified, adenovirus-expressed Flag-LANA protein was incubated with an array displaying 4,192 nonredundant human proteins. Sixty-one interacting cell proteins were consistently detected. LANA interactions with high-mobility group AT-hook 1 (HMGA1), HMGB1, telomeric repeat binding factor 1 (TRF1), xeroderma pigmentosum complementation group A (XPA), pygopus homolog 2 (PYGO2), protein phosphatase 2A (PP2A)B subunit, Tat-interactive protein 60 (TIP60), replication protein A1 (RPA1), and RPA2 proteins were confirmed in coimmunoprecipitation assays. LANA-associated TIP60 retained acetyltransferase activity and, unlike human papillomavirus E6 and HIV-1 TAT proteins, LANA did not reduce TIP60 stability. The LANA-bound PP2A B subunit was associated with the PP2A A subunit but not the catalytic C subunit, suggesting a disruption of PP2A phosphatase activity. This is reminiscent of the role of simian virus 40 (SV40) small t antigen. Chromatin immunoprecipitation (ChIP) assays showed binding of RPA1 and RPA2 to the KSHV terminal repeats. Interestingly, LANA expression ablated RPA1 and RPA2 binding to the cell telomeric repeats. In U2OS cells that rely on the alternative mechanism for telomere maintenance, LANA expression had minimal effect on telomere length. However, LANA expression in telomerase immortalized endothelial cells resulted in telomere shortening. In KSHV-infected cells, telomere shortening may be one more mechanism by which LANA contributes to the development of malignancy.
卡波西肉瘤相关疱疹病毒（KSHV）的 LANA 蛋白在潜伏感染的细胞中作为 KSHV 基因组复制的必需参与者，以及失调细胞生长的驱动因子发挥作用。为了鉴定可能有助于这些活性的新的 LANA 蛋白-细胞蛋白相互作用，我们进行了蛋白质组筛选实验，其中纯化的腺病毒表达的 Flag-LANA 蛋白与显示 4192 种非冗余人类蛋白质的阵列孵育。一致检测到 61 种相互作用的细胞蛋白。在免疫沉淀实验中，LANA 与高迁移率族 AT 钩 1（HMGA1）、HMGB1、端粒重复结合因子 1（TRF1）、着色性干皮病互补组 A（XPA）、PYGO2 同源物 2（PYGO2）、蛋白磷酸酶 2A（PP2A）B 亚基、Tat 相互作用蛋白 60（TIP60）、复制蛋白 A1（RPA1）和 RPA2 蛋白的相互作用得到了确认。与人类乳头瘤病毒 E6 和 HIV-1 TAT 蛋白不同，LANA 不降低 TIP60 的稳定性，但 LANA 相关的 TIP60 保留了乙酰转移酶活性。与 PP2A A 亚基而非催化 C 亚基相关联的 LANA 结合的 PP2A B 亚基，表明 PP2A 磷酸酶活性受到破坏。这让人联想到猿猴空泡病毒 40（SV40）小 t 抗原的作用。染色质免疫沉淀（ChIP）实验显示 RPA1 和 RPA2 与 KSHV 末端重复序列结合。有趣的是，LANA 表达消除了 RPA1 和 RPA2 与细胞端粒重复序列的结合。在依赖端粒替代维持机制的 U2OS 细胞中，LANA 表达对端粒长度几乎没有影响。然而，在端粒酶永生化的内皮细胞中表达 LANA 导致端粒缩短。在 KSHV 感染的细胞中，端粒缩短可能是 LANA 促进恶性肿瘤发展的另一种机制。
