Analysis of the infection dynamics and the host gene expression by the viral latency specific genes

本研究分析了卡波西肉瘤相关疱疹病毒（Kaposi's sarcoma-associated herpesvirus，KSHV）基因在潜伏期内表达极其局限于少数基因的原因，以及病毒基因组是如何随着宿主细胞周期的变化而复制并维持在相同拷贝数的。并测试基因组是否在有或没有病毒潜伏期特异性基因产物如拉娜（ORF 73）的细胞中复制和维持。我们发现杆粒仅在拉娜存在下复制，并且仅在使用G418的选择压力下保持相同的拷贝数，G418是载体中编码的抗性基因。这表明TR和拉娜对于病毒基因组复制是足够的，但对于病毒基因组维持是不够的。那么，除了TR之外，基因组中是否还有其他元件用于病毒基因组的维持？我们尝试了克隆在杆粒中的完整病毒基因组是否被保留。结果 ...更多信息 表明没有负责病毒基因组维持的元件。选择压力，在这种情况下是潮霉素选择，总是需要维持的。因此，KSHV基因组在感染细胞中，尤其是在原发性渗出性淋巴瘤细胞系中保持相同的拷贝数可能有其他原因。潜伏期表达的基因只有拉娜、v-cyc、v-FLIP和Kaposin四个。这些基因并排聚集在一起。我们发现，拉娜与一种名为Suv 39 H1的细胞基因相互作用，Suv 39 H1是一种组蛋白H3甲基转移酶，并招募异染色质因子，如HP 1 α。染色质免疫沉淀分析（ChIP）显示Suv 39 H1和HP 1 α定位于TR和拉娜。因此，拉娜可以使病毒基因组处于异染色质状态，这可能是病毒基因表达仅限于几个基因的机制之一，TR和拉娜在病毒复制和基因组维持中可能起重要作用。拉娜与TR内的特定序列结合，执行病毒的复制和维持，并在潜伏期内调节病毒基因的表达。我们专注于TR，并试图通过制备TR DNA柱来鉴定TR结合蛋白。我们鉴定了几种与柱结合的蛋白质，其中几种是感染细胞特异性的，另一些仍然是TR DNA特异性的，但不是感染细胞特异性的。其中之一是聚（ADP-核糖）聚合酶1（PARP 1）。我们对该因子进行了表征，发现其与TR内非常靠近拉娜结合位点的特定序列结合。免疫沉淀分析证明PARP 1与拉娜之间直接相互作用，而拉娜被PARP 1修饰为ADP-核糖基化。PARP 1活性受某些药物调节。低浓度的羟基脲可提高PARP 1的活性，3-氨基苯甲酰胺（3-阿坝）可降低PARP 1的活性。使用这些药物，结果表明，在感染的细胞中，拉娜的较高核糖基化导致病毒基因组的较低拷贝数，而较低核糖基化导致较高拷贝数。少

We analyzed why the viral gene expression was extremely limited to a few genes in the latency, and how the viral genome replicated and was maintained at the same copy number according to the host cell cycles.We cloned the almost full length of terminal repeats (TR) of the Kaposi's sarcoma-associated herpesvirus (KSHV) in the bacmid, and tested whether the genome replicated and was maintained in the cells with or without viral latency specific gene products such as LANA (ORF73). We found that the bacmid replicated only in the presence of LANA and was maintained at the same copy number only under the selective pressure using G418, the resistant gene against which is encoded in the vector. This suggested that TR and LANA should be enough for the viral genome replication but not for the viral genome maintenance. Then, are there elements in the genome other than TR for the viral genome maintenance? We tried whether the full viral genome cloned in a bacmid was maintained or not. The results … More showed that there was no element responsible for the viral genome maintenance. The selective pressure, hygromycin selection in this case, was always required for the maintenance. Thus, there could be other reason why the KSHV genome was maintained at the same copy number in the infected cells, especially in the primary effusion lymphoma originated cell lines.KSHV gene expression is quite limited in the latency. There are only four genes such as LANA, v-cyc, v-FLIP and kaposin expressing in the latency. These genes are clustered side by side. We found that LANA interacted with a cellular gene called Suv39H1, which is a histone H3 methyl transferase and recruits heterochromatin factor such as HP1α. Chromatin immunoprecipitation analysis (ChIP) showed that Suv39H1 and HP1α were located on the TR as well as LANA. Thus, the viral genome could be in the heterochromatin state by LANA and this could be one of the mechanisms why the viral gene expression was quite limited to the several genes.TR and LANA seem to have an important role for the viral replication and genome maintenance. LANA binds with a specific sequence within TR and executes the viral replication and maintenance and regulates the viral gene expression in the latency. We focused on the TR and tried to identify TR binding proteins with an exhaustive approach by preparing a TR DNA column. We identified several proteins binding to the column, several of which were infected cell specific and the other were still TR DNA specific but not infected cell specific. One of them was poly (ADP-ribose) polymerase 1 (PARP1). We characterized this factor and found that this bound to the specific sequence within TR very near the LANA binding site. The direct interaction between PARP1 and LANA with the immunoprecipitaion analysis but LANA was proved to be ADP-ribosylated by PARP1. PARP1 activity is modulated by some drugs. Hydroxy Urea at the low concentration increases PARP1 activity and 3-aminobenzamide (3-ABA) decreases the activity. Using these drugs, it was shown the higher ribosylation of LANA resulted in the lower copy number of the viral genome and the lower ribosylation did in the higher copy number in the infected cells. Less