1 Department of Medical Microbiology and Immunology, College of Medicine, University of South Florida and the H. Lee Moffitt Cancer Center, MDC Box 10, 12901 Bruce B. Downs Blvd, Tampa, FL 33612-4799, USA
2 Division of Epidemiology, Department of Microbiology, Columbia University School of Public Health, New York, NY 10032, USA
Correspondence
Peter Medveczky
pmedvecz{at}hscprime.hsc.usf.edu
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ABSTRACT |
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Present address: Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary.
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INTRODUCTION |
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Latent replication of EpsteinBarr virus (EBV), a gamma-1 subgroup herpesvirus, provided a model for the maintenance of KSHV episomes at the initial stages of this work. Three components are required for persistence of episomal EBV DNA. EBV nuclear antigen 1 (EBNA-1) interacts specifically with DNA and transactivates two cis-acting elements (Chittenden et al., 1989; Reisman et al., 1985
; Sugden et al., 1985
; Yates et al., 1984
, 1985
). We hypothesized that KSHV encodes cis- and trans-acting elements analogous to those described in EBV.
KSHV does not encode a homologue of EBNA-1 but a nuclear protein designated latency-associated nuclear antigen (LANA), which is expressed in all PEL cells (Kedes et al., 1997; Moore et al., 1996
; Rainbow et al., 1997
). It was reported that LANA co-localizes with viral episomes (Ballestas et al., 1999
; Cotter & Robertson, 1999
), suggesting that LANA may function in episomal maintenance. Ballestas et al. (1999)
also showed that uninfected B cells expressing LANA support episome replication of a cosmid and subclones derived from the left end of the genome containing unique sequences as well as TRs. Most recently, Lim et al. (2002)
demonstrated that LANA supports the transient replication of plasmid DNA containing at least two copies of the TR. These investigators, however, did not extend their search for stable autonomously replicating sequence (ARS) activity and in vivo LANA binding to the entire genome, and left open the possibility that other regions of the viral genome may also be important in the maintenance of latent episomes.
As LANA is thought to be involved in episome maintenance, it is probably a sequence-specific DNA-binding protein. Furthermore, LANA has been shown to interact with different chromatin-associated cellular proteins, such as the retinoblastoma protein, RING3, p53 and mSin3 (Radkov et al., 2000; Platt et al., 1999
; Friborg et al., 1999
; Krithivas et al., 2000
), and may have other functions that require DNA-binding activity. Binding to p53 and the retinoblastoma protein can possibly regulate virus transcription, similar to what has been shown for EBNA-1 (Wysokenski & Yates, 1989
). Ballestas & Kaye (2001)
as well as Garber et al. (2001)
showed in vitro LANATR binding and mapped out minimum interaction sequences using electromobility shift assays with nuclear extracts of cells transfected with LANA and labelled oligonucleotides from the TRs. Thus, these studies did not address the LANATR interaction in living cells, whether it is direct or indirect, and the interaction of LANA with other parts of the viral genome. Since in vitro biochemical approaches do not permit the examination of proteinDNA binding in living cells and can neither reflect the entire specificity nor quantify these interactions, we have set up novel experimental systems for the study of in vivo LANADNA binding.
The work presented here shows that the region of the viral chromosome that binds LANA the most selectively and abundantly in vivo is the TR and this interaction is a direct DNAprotein contact. Consistent with these findings, genetic approaches show that plasmids containing only TRs can replicate stably in cells expressing LANA.
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METHODS |
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To generate the pLANARep expression plasmids, one, three and four TR units were inserted into the unique MluI site of the pBKCMV-LANA expression vector (Fig. 1). Subfragments of the TRs were also cloned into the MluI site of pBKCMV-LANA.
Cell lines, cell culture, transfection methods and drug selection.
BCBL-1, BC-1 and BC-3 cell lines (Cesarman et al., 1995a; Kedes et al., 1997
; Renne et al., 1996b
), and Jurkat, Ramos and 293 cell lines were obtained from the ATCC. Lymphoid cell lines were cultured in RPMI 1640 and 293 cells were cultured in DMEM, both media containing 10 % foetal calf serum and antibiotics. Lymphoid cells (107 cells) were transfected with 10 µg cosmid DNA by electroporation, as described previously (Lund et al., 1997
). Transient and stable transfections into 293 and COS cells were done using lipofection with the GenePORTER reagent (Gene Therapy Systems), according to the manufacturer's protocol.
Episome replication assays.
Episomal DNA from transfected cells was recovered by Hirt extraction (Hirt, 1967) and was digested by either DpnI or MboI. Plasmid DNA amplified in dam methylase-positive Escherichia coli is efficiently digested by DpnI but not by MboI, while DNA replicated in mammalian cells is resistant to DpnI but sensitive to MboI. The product of the digestion reactions was either transformed into E. coli or subjected to electrophoresis and Southern blotting. Positive E. coli transformants were counted and plasmid DNA prepared from these clones was partially sequenced and/or digested with restriction enzymes. Southern blots were probed with vector DNA labelled to a specific activity of approximately 1x109 c.p.m. µg-1. The detection limit of this assay was 0·11·0 molecules per cell.
Another assay was based on the detection of episomes released from intact cells by in situ lysis in the wells of vertical Gardella agarose gels, as described previously (Gardella et al., 1984). Large intact plasmids such as herpesvirus genomes or plasmids larger than about 15 kb have a slower electrophoretic mobility than any linear DNA, including cellular DNA, and form a band between the loading well and the linear cellular DNA band. Specific plasmids were detected on Southern blots, whereas cellular DNA was visualized by ethidium bromide staining. The sensitivity of this assay is 0·11·0 copies per cell.
Chromatin immunoprecipitation (ChIP) assay.
ChIP assays of in vivo formaldehyde or UV cross-linked cells were done essentially as described (Gilmour et al., 1991; Orlando et al., 1997
). Briefly, cells were cross-linked with exposure to either 1 % formaldehyde or UV light and washed. Nuclear extracts were prepared and sonicated to an average chromatin size of 400 bp. To assess the efficiency of LANA immunoprecipitations with a rabbit polyclonal antibody, UK163 (Zhu et al., 1999
), immunoprecipitates were subjected to Western blotting using a human antiserum from a KS patient (a kind gift of K. Nagy, National Institute of Dermato-Venereology, Department of AIDS and Human Retroviruses, Budapest, Hungary). Under the conditions used, 5 µg of rabbit antibody UK163 immunoprecipitated about 50 % of the total LANA protein from cross-linked extracts. For ChIP assays, nuclear extracts were incubated with 5 µg of anti-LANA rabbit antibody at 4 °C overnight. As a negative control, a rabbit polyclonal antibody to herpesvirus saimiri ORF 1 protein has been used (Medveczky et al., 1993
). Immune complexes were collected with protein ASepharose beads and washed under high stringency washes. ProteinDNA complexes were eluted and the cross-links were reversed. DNA was prepared from these samples after proteinase K treatment, phenol/chloroform extraction and ethanol precipitation, labelled with random priming and used as probes for Southern blots.
GSTLANADNA pull-down assay.
Of each cosmid, Z2, Z6, Z8, Z14 and Z15, 2 µg DNA was cut with EcoRI to release the viral DNA insert from the vector. DNA was then sonicated to an average size of 400 bp. Baculovirus-expressed GSTLANA was purified as described earlier (Zhu et al., 1999) and incubated with these DNA fragments in DNA-binding buffer (DBB) (20 mM HEPES pH 7·9, 100 mM KCL, 1 mM MgCl2, 0·1 mM EDTA, 1 mM DTT, 12 % glycerol and 1 mg BSA ml-1) with rotation at room temperature for 1 h. GSTLANA was pulled down with glutathione beads (Sigma) and washed four times with DBB. LANA-bound DNA fragments were eluted from the beads, digested with proteinase K, phenol/chloroform extracted, ethanol precipitated and used on Southern blots as probes.
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RESULTS |
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G418-resistant, transfected BCBL-1 cells were cultured for 2 months and purified episomal DNA was digested with either MboI or DpnI, which digest replicated or non-replicated DNA, respectively. E. coli was transformed with digested DNA and colonies were counted. Table 1 shows that Eco A- or Rep-4- but not Eco B-transfected BCBL-1 cells contain plasmids resistant to DpnI but sensitive to MboI, indicating episome replication in these cells. Transfected BCBL-1 cultures were grown for 9 months (corresponding to 142 cell divisions). Table 1
, experiment 3, shows that several plasmids were recovered from Eco A- and Rep-4- but not from Eco B-transfected cultures.
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Data from two different types of experiments indicated conclusively that plasmids containing TR elements replicated stably and autonomously in BCBL-1 cells.
To evaluate whether any viral protein is required for the ARS activity observed in BCBL-1 cells, various cosmids and plasmids (Fig. 1) were also transfected into the uninfected lymphoblastoid cell lines Jurkat and Ramos or into the human embryonic kidney cell line 293. In five independent experiments after G418 selection, we could not detect any persisting episomes after Gardella gel analysis (data not shown). Thus, virus factor(s) are necessary to achieve episome replication of TRs.
LANA is sufficient to support the stable replication of TRs in 293 cells
As LANA may be involved in the maintenance of KSHV episomes, we investigated whether LANA could support the maintenance of plasmids containing TRs. A vector expressing LANA and also containing various numbers of TRs at a unique MluI site was constructed using the pBKCMV expression vector. The single TR unit was cloned as a NotI fragment and as a Sau3A fragment. Subclones encoding different smaller parts of the TR were also inserted into the LANA expression vector for replication assays. 293 cells were stably transfected with the constructs and controls (pBKCMV-LANA without repeats and empty vector) using G418 selection. Clones resistant to G418 were tested for episome replication of the transfected plasmids. Purified episomal DNA was digested with either DpnI or MboI. Fig. 3(top panel) shows that cells transfected with LANA expression vectors encoding TRs, but not the ones without them, all contained episomal DNA replicated by the mammalian replication machinery. Fig. 3(bottom panel) shows ethidium bromide staining of the gel, indicating equal loading of DNA. LANA expression vectors containing only a single repeat unit replicated efficiently; however, the number of episomal DNA copies was much lower than in cells transfected with vectors containing three or more repeat units. The DNA sequence necessary for LANA-mediated replication was localized further using subclones encoding different parts of the TR to the region between bp 548 and 171 of the repetition unit (data not shown).
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Representative results of ChIP assays using three different PEL cell lines are shown in Fig. 4. First, we compared visually the distribution of hybridization signals (Fig. 4a
c) with the ethidium bromide-stained gel (Fig. 4e
) representing all of the DNA fragments. We concluded that the LANA-specific antibody selectively immunoprecipitated an 801 bp DNA fragment. Several mapping experiments and DNA sequencing showed that the 801 bp fragments correspond to the TRs. These fragments are encoded by cosmids Z2, Z6 and Z14 and present as supermolar bands indicated by the ethidium bromide image of Z2 and Z6 (Fig. 4e
). This apparently selective immunoprecipitation of TR by the LANA antibody was the case with all three different KSHV-harbouring cell lines BCBL-1, BC-1 and BC-3 (Fig. 4a
c). No significant DNA contamination due to the procedure occurred, as we did not observe notable hybridization with probes obtained with immunoprecipitations using a non-specific antibody (Fig. 4a
c). In the control experiment, we used unselected total cellular DNA from BCBL-1 cells as a probe. Fig. 4(c)
shows that repetitive DNA bands hybridized with only slightly stronger binding than those of unique DNA fragments, indicating that the immunoprecipitated DNA contained much higher concentrations of TRs than unique DNA. Taken together, these results suggested significant and specific in vivo LANA binding to the TR.
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Stronger relative signals were obtained not only for TR but also for two other DNA fragments in the Not I/EcoRI Z6 digest. One of them is a truncated TR unit (labelled as tr in Fig. 4a). The other is the leftmost, 6198 bp EcoRI fragment of the unique region of Z6. This fragment is fused to a small part of the TR, even after NotI digestion (labelled as Z6 TRUR junction in Fig. 4a
). Since this part of Z6 representing the TRUR junction was found to hybridize in the anti-LANA chromatin immunoprecipitates, the possibility exists that not only the TR region but also the unique region of Z6 binds LANA in vivo. To address this question, we cut the Z6 cosmid with NotI and then with PstI, EcoRI, BglII and HincII, respectively. Southern blotting with immunoprecipitated DNA was then performed. These enzymes cut at different distances from the TR junction (Fig. 1
). Fig. 4(f)
shows that the immunoprecipitated probe hybridized with the TR fragment and with fragments containing the TR unique region junction (EcoRI and BglII) but not with fragments containing only the unique region of Z6. We observed no hybridization signal using the control antibody (Fig. 4g
). To ensure that the preferential hybridization signals came from the TR only, the same blot was stripped and probed with a gel-purified unit length TR probe (Fig. 4h
). This experiment gave a practically identical pattern of hybridization as the anti-LANA ChIP DNA. Taken together, these ChIP experiments show that LANA binds strongly and specifically to the TR in vivo.
GSTLANA binds DNA in vitro with slight selectivity to the TRs
To assess the in vitro DNA-binding capacity of LANA and to compare it to the in vivo ChIP results, DNA pull-down assays were performed using a baculovirus-expressed GST-tagged LANA, as described in Methods. Purified DNA fragments were labelled and used as probes in Southern blots containing EcoRI/NotI-digested cosmid DNA. Fig. 5 shows the result of a representative set of these assays. GSTLANA bound the DNA fragments effectively (Fig. 5b
). However, this DNA binding was not specific for KSHV DNA, as not only the viral but also the vector DNA bands were readily detectable on the Southern blots. Comparing the results of these blots to those from the ethidium bromide-stained gel (Fig. 5a
), it is obvious that each DNA fragment hybridized with the GSTLANA-bound probes. This DNA-binding activity was not due to the binding of DNA fragments to the GST protein or to the glutathione beads, as baculovirus-expressed GST protein did not show DNA-binding activity in this assay (Fig. 5c
).
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LANA binds TRs in vivo in a direct DNAprotein interaction
Formaldehyde effectively cross-links proteins to DNA, but, besides DNAprotein cross-links, it also creates proteinprotein covalent linkages. Thus, it was not clear if LANA binds DNA directly or indirectly through another bridging protein. To address this question, a ChIP assay with in vivo UV cross-linking has been carried out. The UV method only detects the direct proteinDNA interactions (Ausubel et al., 1995; Gilmour et al., 1991
). Fig. 6
shows the result of such an experiment. Similar to the formaldehyde cross-linking experiments, the major hybridization signal was found with the TR. No signal was detected with the non-specific control immunoprecipitation. Nevertheless, the intensity of the TR signal was less strong than that seen with formaldehyde cross-linking. This weaker reaction is probably due to the fact that UV irradiation is a specific but ineffective method for proteinDNA cross-linking (Ausubel et al., 1995
; Gilmour et al., 1991
). Thus, this experiment has shown that at least a part of the LANADNA interaction is a direct contact.
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Fig. 7 shows a representative set of ChIP experiments performed with formaldehyde (a) and UV (b) cross-linked probes as well as with the control probe (c). Fig. 7(d)
shows hybridization data using the non-specific control antibody and the original ethidium bromide-stained gel is shown in (e). Results showed strong hybridization of the anti-LANA ChIP probe to all SmaI fragments within the TR. Although there were slight differences in the relative intensities of the individual DNA fragments, we could not detect an obvious preferential in vivo LANA-binding site. Results were rather consistent with multiple LANA binding along the whole TR. As found previously, no hybridization occurred with the control probe.
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DISCUSSION |
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Interestingly, ARS activity correlated with the number of repeats present in various plasmids. In 293 cells, we estimated that plasmids containing three or four repeats yielded at least 100 copies of episomes per cell, while the clone containing a single repeat unit yielded only about 10 copies per cell. These data suggest that multiple repeats provide higher copy numbers and are in agreement with data published recently using a transient DNA replication assay (Lim et al., 2002).
In this study, we also mapped and analysed the binding activity of the KSHV LANA protein to the entire viral genome by several methods. In vivo DNA binding of LANA was tested by ChIP assays. Using this assay, we have demonstrated in different KSHV-infected cells that LANA binds to the TRs specifically. Mapping of proteinDNA-binding sites with ChIP assays usually requires the PCR amplification of the immunoprecipitated DNA (Orlando, 2000). Most remarkably, this was not necessary in our case, as the results indicated strong hybridization signals with the TR using the anti-LANA-immunoprecipitated DNA probe directly. Several reasons can explain this fact. The copy number of KSHV in latently infected cells is 50100 and the TR units are present in multiple copies in the viral chromosome. LANA has been shown to multimerize (Schwam et al., 2000
), which gives a further enhancement to the LANA ChIP assay, as formaldehyde treatment makes not only DNAprotein but also proteinprotein cross-links.
No LANA-binding sites were detected in the unique region. This does not necessarily mean that such binding sites do not exist, as their signal could be under the level of detection employed in this study. Nevertheless, the results show a much more prominent in vivo binding of LANA to the TR than to any other part of the viral genome.
Significant non-specific DNA-binding activity of recombinant LANA was detected by in vitro GST pull-down studies. Although recombinant baculovirus-expressed LANA bound the TR more strongly than other fragments, this protein could also bind sequences unrelated to KSHV: for example, DNA of the SuperCos vector. This paradoxical observation can be explained by the effect of other viral or cellular factors that are present in the virus-infected cell but absent in an in vitro LANADNA-binding assay. Indeed, LANA has been shown to bind several other DNA or chromatin-associated cellular proteins (Platt et al., 1999; Krithivas et al., 2000
; Lim et al., 2001
; Friborg et al., 1999
; Radkov et al., 2000
). Alternatively, it is also possible that a complex, chromatin-like packaging of the viral genome in vivo confers greater sequence specificity to LANA. Cotter & Robertson (1999
) demonstrated the association of LANA with various segments of the genome in vitro. This essentially corresponds to our in vitro results showing non-specific DNA binding by LANA and the results collectively underline the necessity of the in vivo ChIP assay. The finding that LANA has a non-specific DNA-binding activity in vitro is not surprising, since this characteristic is a common feature of several proteins, such as p53, that also recognize specific DNA sequences (Bayle et al., 1995
).
UV cross-linking experiments presented here have shown that LANA binds the TR DNA directly. Similar to the formaldehyde cross-linking studies, these experiments also indicated preferential TR binding. Attempts to narrow down the LANA-binding region within the TR using the ChIP assay did not detect any predominant LANA-binding region. There are inherent size limitations of mapping with ChIP, since sonication generates 3500 bp long fragments; nevertheless, results are consistent with at least two or, possibly, multiple LANA-binding sites within the repeats. However, fine mapping of these in vivo-binding sites should be attempted by other more sensitive methods.
Other groups have observed co-localization of LANA to large regions of the viral genome (Ballestas et al., 1999; Cotter & Robertson, 1999
). These regions, which included a minimum of three units of the TR plus about 13 kb of unique sequence to the right of the TR, were also shown to be involved in the maintenance of episomal DNA (Ballestas et al., 1999
; Cotter & Robertson, 1999
). Specific interaction of LANA with the TRs measured by in vitro assays was also reported recently (Ballestas & Kaye, 2001
; Garber et al., 2001
). We comparatively evaluated LANA binding to different regions of the KSHV genome in vivo and characterized the nature of this proteinDNA interaction. Our results, consistent with the above-mentioned data, provide the first evidence that LANA is associated specifically with the TRs in vivo, demonstrate that the interaction is a direct proteinDNA contact and suggest multiple binding sites within the TR, but exclude other parts of the viral genome as major in vivo LANA-binding sites.
Studies on the related tumour virus herpesvirus saimiri also implicate the TRs in episome replication. TRs are essential for the establishment and/or maintenance of herpesvirus saimiri episomes, as demonstrated by construction and analysis of virus mutants lacking intact TRs (Collins et al., 2002). Furthermore, LANA expression vectors encoding TRs replicated as episomes in uninfected cells (Collins et al., 2002
), indicating that the basic molecular mechanisms for the maintenance of these viruses are similar.
Taken together, our findings suggest a model in which multimers of the LANA protein complex with each TR unit at the virus episome during latency so that dozens of LANADNA complexes are concentrated in a very small area. The high local concentration of LANA complexes may be essential for the association of the episome with chromatin structures. These events explain the LANA-mediated tethering observed by microscopy (Ballestas et al., 1999; Cotter & Robertson, 1999
). There is also a likely role for other viral and/or cellular factors in this model. It appears that the LANATR interactions are analogous to those described between EBNA-1 and the family of repeats of EBV (Chittenden et al., 1989
; Reisman et al., 1985
; Sugden et al., 1985
; Yates et al., 1984
, 1985
).
There are still unresolved issues related to the latency of KSHV episomes. Further studies are required to assess the host range of the LANARep plasmids and to understand how TRs promote episome replication. The extremely high G+C-containing repeats are unlikely to provide an origin function, since all known origins have a low G+C content to allow for easy unwinding (Challberg & Kelly, 1989). The comprehensive understanding of the molecular mechanisms involved in the latency of gamma-2 herpesviruses can lead to the development of KSHV-based gene delivery vectors and to specific strategies and drugs against virus factors to destroy latent virus.
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ACKNOWLEDGEMENTS |
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Received 1 November 2002;
accepted 7 February 2003.