Abstract
The genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists in latently infected cells as a circular episome. The latency-associated nuclear antigen (LANA) has been shown to tether viral DNA fragments to chromosomes and is proposed to maintain the KSHV genome. In order to identify the in vivo-binding sites for LANA on the whole KSHV genome and to analyse the function of this protein-DNA interaction, different in vivo systems have been developed. Chromatin immunoprecipitation experiments using three different cell lines latently infected with KSHV demonstrated that LANA binds preferentially and directly to the terminal repeats (TRs) but not to other regions of the viral chromosome in vivo. In contrast, in vitro LANA-DNA binding was much less specific. To identify autonomously replicating sequences within the KSHV genome, BCBL-1 cells were transfected with cosmids representing the entire genome. Cosmid Z2, consisting of the right end of the unique region and TRs, persisted as an episome in short-term assays. Long term, stable episome replication was observed with constructs derived from Z2 containing TRs only. LANA expression constructs containing a variable number of TRs replicated stably as episomes in uninfected cells. A 424 bp subfragment of the 801 bp TR could mediate episome replication. These studies show that LANA is a trans-acting protein that binds preferentially to TRs in vivo and these two elements are sufficient for episome replication. These results also suggest that the LANA expression plasmids reported here could be utilized as episomal vectors in a manner similar to Epstein-Barr virus-based vectors.
Original language | English |
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Pages (from-to) | 1451-1462 |
Number of pages | 0 |
Journal | J Gen Virol |
Volume | 84 |
Issue number | 0 |
DOIs | |
Publication status | Published - Jun 2003 |
Keywords
- Antigens
- Viral
- Binding Sites
- Cell Line
- Cosmids
- DNA Replication
- DNA
- Genetic Vectors
- Genome
- Herpesvirus 8
- Human
- Humans
- In Vitro Techniques
- Nuclear Proteins
- Plasmids
- Terminal Repeat Sequences