Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma and several lymphoproliferative diseases including primary effusion lymphoma, also called body-cavity based lymphoma, and some cases of multicentric Castleman's disease. The latency-associated nuclear antigen (LANA) of KSHV has been implicated in the maintenance of the viral genome during latent infection. The C-terminal DNA-binding domain of LANA interacts with sequences located in terminal repeats (TRs) of viral genome, and the N-terminal chromosome-binding sequence (CBS) of LANA associates with host chromosomes. LANA co-localizes with KSHV genome on the host chromosome, suggesting that LANA tethers viral genome to host chromosome for its persistence in infected cells. This chromosome-tethering model seems to be conserved among DNA viruses such as papillomavirus and Epstein-Barr virus (EBV), which possess extra-chromosomal genome during their latent infection. Using a long-term replication assay, it has been previously shown that KSHV TR and LANA act as cis- and trans-elements for the persistence of viral genome, respectively. In this study, we established a transient replication assay with a methylation-sensitive restriction enzyme, Dpnl, and confirmed that LANA also actively participates in the replication of TR-containing plasmid. Using this assay system, we found that 293, 293T, BJAB, C33A, HCT116, and COS-1, but not NIH/3T3 cell lines are permissive for the replication of KSHV TR-containing plasmid by LANA, and further characterized viral cis- and trans-acting elements of KSHV latent replication. Transient reporter assay and transient replication assay disclosed that the orientation and basal transcriptional activity of TR constructs did not significantly affect the efficiency of replication. However, at least two TR units were necessary for efficient replication. The N-terminal 22 amino acids comprising the CBS of LANA were necessary and sufficient for the mediation of its C-terminal DNA-binding and dimerization domain to support the transient replication of KSHV TR-containing plasmid, although the C-terminus alone can bind sequences within TR and repress the TR-dependent transcription in electrophoretic mobility shift assay and transient reporter assay, respectively. We generated several point mutations on sequences within LANA CBS and tested their functional activities including sub-cellular localization, mitotic chromosome association, biochemical fractionation, inhibition of TR-dependent transcription, and transient replication of KSHV TR-containing plasmid. Our data indicate that chromosome-binding activity of LANA is prerequisite to its replication activity and the latent DNA replication of KSHV genome seems to be tightly coupled to its equal segregation and nuclear retention, while papillomavirus and EBV have evolved independent mechanisms for DNA replication and partition of viral genome. LANA interacted with components of the origin recognition complexes (CRCs), similarly to Epstein-Barr virus nuclear antigen-1 (EBNA-1), implicating that LANA may recruit ORCs to KSHV TR for mimicking cellular origins of replication. Unexpectedly, the N-terminus of LANA displayed the general inhibitory activity of viral replication systems in transiently transfected 293 and 293T cells, but the C-terminus of LANA specifically acted as a dominant-negative mutant for the replication of KSHV TR-containing plasmid in different cell lines. Transiently overexpressed catalytic subunit of PKA and temporal treatment of PMA but not n-butyrate inhibited the replication of KSHV TR-containing plasmid in 293T cells. In addition, we found that LANA associates with DNAPK/Ku complex and contains multiple DNA-PK phosphorylation sites on its N-terminus. Preliminary transient replication assays indicated that DNA-PK/Ku complex would also negatively regulate the replication of KSHV TR-containing plasmid by LANA. Taken together, these data suggest that the latent DNA replication of KSHV can be regulated by specific signal pathway involving protein kcascade cascades.
카포시 육종 허피스 바이러스는 카포시 육종 및 임파종 질환의 원인균으로 알려져 있다. 이 바이러스의 LANA 단백질은 잠복기 감염시, 바이러스의 지놈을 숙주 세포의 염색체에 고정 시킴으로써 복제에 중요한 역할을 수행한다고 알려져 있다. 본 연구에서는, methylation-sensitive 제한 효소를 이용한 DNA 복제 분석법을 확립하였으며 여러가지 세포주에 대하여 이 바이러스의 DNA 복제여부를 조사하였다. 그 결과 사람이나 원숭이로부터 유래한 세포주에서는 이 바이러스의 DNA 복제가 일어났으나, 쥐로부터 유래한 세포주에서는 그러한 결과를 관찰 할 수 없었다. 바이러스 지놈의 양 말단에 존재하는 terminal repeat (TR) sequence 에 LANA 단백질이 결합하여 DNA 복제가 일어나는데, 효과적인 복제를 위해서는 적어도 두 개 이상의 TR sequence 를 필요로 하였다. 또한 여러가지 변형된 형태의 LANA 단백질을 제조하여 그것의 DNA 복제 활성을 측정한 결과, LANA 단백질의 DNA 결합 부위와 더불어 숙주 세포의 염색체에 고정되는 부위가 필요하였다. LANA 단백질의 DNA 결합 부위만으로도 TR sequence 에 결합하여 전사활성을 저해하며, DNA 복제에 필요한 세포 내 복제 인자들과 결합할 수 있는 바, 이와 같은 결과는 염색체에 결합되어 있는 세포 내 인자가 LANA 단백질의 DNA 복제 활성에 중요한 역할을 수행한다는 사실을 시사한다. 카포시 육종 허피스 바이러스와 유사하게 바이러스 지놈을 숙주 세포의 염색체에 고정 시킴으로써 그것의 지속성을 유지하는 파필로마바이러스나 Epstein-Barr 바이러스의 경우, DNA 복제와 염색체 고정화의 과정이 서로 독립적으로 일어난다. 따라서 LANA 단백질을 통한 카포시 육종 허피스 바이러스의 DNA 복제와 염색체 고정화의 연결은, 감염 세포 내에서 바이러스 지놈의 유지를 위한 이 바이러스의 독특한 메커니즘으로 사료된다. 이와 더불어, 카포시 육종 허피스 바이러스의 DNA 복제에 관여하고 이를 조절할 수 있는 바이러스 혹은 세포 내 인자를 동정하였으며 그 기작을 실험적으로 증명하거나 고찰하였다.