Title: Functional dissection of LANA binding site LBS3/RE (replication element) in viral genome replication and persistence of Kaposi’s sarcoma-associated herpesvirus
Abstract:
Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) persists in the nucleus as latent episomes in dividing tumor cells. During the latent phase, viral episomes are tethered to host chromatin and segregate to the daughter cells following replication, which critically relies on the latency-associated nuclear antigen (LANA) protein. Deletion mapping of the latent replication origin revealed two distinctive sequence elements: LANA binding sites (LBS1/2) and a 32-bp long GC-rich sequence upstream of LANA binding site 1 (LBS1), referred to as LBS3/RE (replication element), along with LANA binding sites 2 (LBS2), to be sufficient to support replication. LANA has been shown to specifically bind to LANA binding sites (LBS) within the terminal repeat (TR) region. In this study, we dissected the role of the KSHV replication element, LBS3/RE in viral genome replication, maintenance, and latency establishment using a recombinant KSHV (Bacterial Artificial Chromosome). Our data showed that a single copy of the terminal repeat is insufficient to adequately support replication and episome persistence and TR without the LBS3/RE was further impacted for the episome maintenance. Importantly, recombinants with either a single copy of the TR or LBS3 deleted TR were severely defective for the cell’s production of cell-free virions. Analysis of the chromatin architecture at the TR region and the promoters of latent, immediate-early, and late genes showed proportionately similar ratios of active chromatin (H3K4me3) and repressive (H3K27me3) marks on LANA promoter but was slightly altered on the TR and RTA promoter among these recombinants. We used the SingleMolecule Analysis of Replicated DNA (SMARD) approach to analyze the replication initiation sites among these recombinants and found that viruses with single TR or LBS3 deleted TR initiate replication at the other sites. Together, these results suggest that multiple TRs are required for efficient DNA replication and virion production.