This work demonstrates that LANA1 acts in trans on cis-acting KSHV TR DNA to mediate episome persistence and that LANA1 specifically binds KSHV TR DNA. Plasmids containing one or more TR units persisted as episomes in F-LANA1-expressing cells but not in cells lacking F-LANA1. Immunoprecipitated F-LANA1 but not control immune precipitates specifically bound certain TR restriction fragments. Specific TR oligonucleotides were gel shifted by in vitro-translated F-LANA1, and a 20-nt LANA1 binding site (TR-13) was defined. Nuclear extracts from BCBL-1 and BC-1 PEL cells specifically gel shifted TR-13, and the presence of LANA1 in these complexes was confirmed by supershift analyses. Z6-A DNA contains truncated TR sequence but lacks the LANA1 binding site defined here (TR-13) and did not persist as an episome in F-LANA1-expressing cells. Medveczky and colleagues have also shown that LANA1 binds to KSHV TR sequence (Medveczky et al., Third International Workshop on KSHV and Related Agents, abstr. 21, 2000). These data support a model in which LANA1 directly binds KSHV episomes via TR DNA to efficiently mediate episome persistence.
The high TR copy number (~40 copies) (28
) in genomic KSHV may enhance LANA1-mediated episome persistence. The EBV oriP contains 24 EBNA1 binding sites distributed among 20 tandem repeats and a dyad symmetry element (29
). More than one EBNA1 binding site is required for efficient EBNA1 mediated episome retention (11
). Plasmids lacking the full complement of EBNA1 binding sites may dimerize or form tetramers in EBNA1-expressing cells, increasing the number of EBNA1 binding sites per episome (11
). Although LANA1 may bind to additional sites in the TR unit, the tandemly repeated TR elements in genomic KSHV provide a large number of LANA1 binding sites. Multimerization of input plasmid DNA and TR duplication (Fig. and data not shown) in artificial KSHV episomes increases the number of LANA1 binding sites and suggests that this increase improves the efficiency of LANA1-mediated episome maintenance.
It is likely that tandemly repeated TR elements mediate the focal concentration of LANA1 to dots seen by immunofluorescence microscopy in KSHV-infected cells (23
). LANA1 localizes to dots in BJAB/F-LANA1 cells with artificial KSHV episomes containing tandemly repeated TR elements but is diffusely distributed in the nucleus in the absence of KSHV episomal DNA (4
). Localization of LANA1 to dots was also observed in cells with episomal Z6-1TR, Z6-2TR, Z6-3TRA, or Z6-BE DNA (data not shown). LANA1 concentration to dots in cells with these artificial episomes may be dependent on increased numbers of LANA1 binding sites from TR duplication and multimerization of input plasmids, as occurred when cells proliferated over time. Also consistent with the idea that DNA mediates the focal LANA1 concentration, DNase but not RNase treatment of BCBL-1 nuclei eliminated the punctate nuclear immunofluorescent staining pattern characteristic of LANA1 in PEL cells (49
). LANA1 forms dimers in the absence of KSHV DNA (44
) and may also dimerize and oligomerize at binding sites, as suggested by the presence of three different F-LANA1 gel-shifted TR-13 complexes in EMSA experiments (Fig. A, lane 1). By analogy, EBNA1 binds to its cognate DNA sequence as a dimer, and dimerization is required for binding (6
In order to persist, episomes must replicate at least once per cell cycle in addition to efficiently segregating to progeny cells. DNA replication initiates at or near the EBNA1 binding region of dyad symmetry in the EBV oriP and at other sites in the EBV genome during latency (16
). The dyad symmetry element, which contains four EBNA1 binding sites, is also a site of termination of DNA replication (14
). Although EBNA1 is necessary for long-term persistence of EBV oriP episomes, its role in EBV DNA replication remains controversial (1
). Whether LANA1 plays a role in KSHV DNA replication and how the TR functions as an origin of replication await further investigation.
Open reading frame 73 genes of other gamma-2 herpesviruses may also act on cognate TR DNA to mediate episome persistence. A cis
-acting oriP sequence has been defined for herpesvirus saimiri (HVS), although the trans
-acting factor has not been described (27
). Despite the HVS oriP location within the long unique region of the genome, it is likely that HVS has an additional region(s) involved in episome maintenance, perhaps in the TR elements, since a strain of HVS lacking the described oriP sequence persists as an episome (27
). Similar to KSHV, HVS and other gamma-2 herpesviruses also have high TR copy numbers, which may reflect a TR role in episome persistence.
The model of LANA1 tethering TR DNA to chromosomes to mediate episome persistence requires simultaneous association of LANA1 with episomes and chromosomes, and this work demonstrates that LANA1 directly binds KSHV TR DNA. With respect to the association of LANA1 with chromosomes, LANA1 may bind chromosomal DNA or chromosome-associated proteins to mediate episome persistence. In this regard, LANA1 binds to histone H1, RING3, p53, members of the mSin3 corepressor complex, and retinoblastoma protein (12
), and it is possible that any of these proteins may mediate its attachment to chromosomes. The DNA binding domain of the EBV EBNA1 protein is different from the domain involved in chromosome association (2
). Further investigation should elucidate the functional domains of LANA1 and how they coordinate to mediate episome maintenance.