| Budget Amount *help |
¥15,100,000 (Direct Cost: ¥15,100,000)
Fiscal Year 2005: ¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 2004: ¥10,400,000 (Direct Cost: ¥10,400,000)
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| Research Abstract |
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
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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
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