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

我们分析了为什么病毒基因的表达在潜伏期非常有限，以及病毒基因组是如何根据宿主细胞周期复制并保持相同拷贝数的。我们在巴氏杆菌中克隆了卡波西肉瘤相关疱疹病毒(KSHV)的几乎全长末端重复序列(TR)，并测试了基因组是否在有或没有病毒潜伏期特异性基因产物的细胞中复制和维持。我们发现，只有在LANA存在的情况下，bacmid才能复制，并且只有在G418的选择压力下才能保持相同的拷贝数，G418是载体中编码的抗性基因。这说明在病毒基因组的复制过程中，仅用tr和LANA就足够了，但对病毒基因组的维持来说还不够。那么，除了tr外，基因组中是否还有其他元件可以维持病毒基因组呢？我们尝试了在巴氏杆菌中克隆的完整病毒基因组是否保持不变。The Results…更多的结果表明，没有负责病毒基因组维护的因素。在这种情况下，选择压力，在这种情况下，潮霉素的选择，总是需要维持。因此，可能还有其他原因导致KSHV基因组在感染细胞中保持相同的拷贝数，特别是在起源于原发渗出性淋巴瘤的细胞系中。潜伏期只有LANA、v-cyc、v-flip和kaposin四个基因表达。这些基因并排聚集在一起。我们发现LANA与一种称为SUV39H1的细胞基因相互作用，该基因是一种组蛋白H3甲基转移酶，并招募异染色质因子，如Hp1α。染色质免疫沉淀分析(CHIP)显示，SUV39H1和Hp1α定位于Tr和LANA上。因此，LANA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。LANA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。我们把重点放在TRR上，并试图通过制备TRDNA柱来用穷举的方法鉴定TRR结合蛋白。我们鉴定了几种与该柱结合的蛋白质，其中几种是感染细胞特异性的，另一种仍然是TRDNA特异性的，但不是感染细胞特异性的。其中一个是聚腺苷二磷酸核糖聚合酶1(PARP1)。我们对这个因子进行了表征，发现它与TR内的特定序列结合在离LANA结合位点非常近的地方。免疫沉淀分析证实了PARP1与LANA的直接相互作用，但LANA被PARP1证明是ADP核糖化的。PARP1活性受某些药物的调节。低浓度的羟基脲提高PARP1活性，3-氨基苯甲酰胺(3-ABA)降低PARP1活性。结果表明，LANA的核糖化程度越高，病毒基因组的拷贝数越低，而核糖化程度越低，感染细胞的拷贝数越高。较少

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