University of Edinburgh, Centre for Infectious Diseases, R(D)SVS, Summerhall, Edinburgh EH9 1QH, UK
Correspondence
Simon J. Talbot
stalbot{at}ed.ac.uk
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ABSTRACT |
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INTRODUCTION |
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LANA, identified as a 226234 kDa doublet by SDS-PAGE (Gao et al., 1996a, 1999
), is a versatile protein with multiple functions. It tethers viral episomes to host chromatin during mitosis, allowing delivery of viral progeny to all daughter cells (Ballestas et al., 1999
). LANA binds to two short motifs within the terminal repeat of the KSHV genome through a region in its C-terminal domain (Ballestas & Kaye, 2001
; Garber et al., 2001
, 2002
). Interaction with host cell mitotic chromosomes is mediated through a 32 aa domain at the N terminus (Piolot et al., 2001
). It is thought that LANA acts as a bridge between chromosomal and viral episomal DNA.
Similar to the oncogenes of other DNA tumour viruses, LANA binds to and interferes with the functions of the tumour suppressors p53 and retinoblastoma protein (Friborg et al., 1999; Radkov et al., 2000
), and transforms primary rat embryo cells with Hras (Radkov et al., 2000
). Recently, it was reported that LANA also exploits the Wnt-
-catenin pathway to activate genes that can promote cell growth (Fujimuro et al., 2003
).
Through a domain in its C terminus, LANA activates and/or represses certain cellular and viral promoters, probably through interaction with cellular transcription factors such as CREB, CBP and Sp1 (An et al., 2002; Krithivas et al., 2000
; Lim et al., 2000
; Radkov et al., 2000
). A family of nuclear factors typified by RING3 has also been shown to interact with the C terminus of LANA resulting in the phosphorylation of serine and threonine residues between aa 951 and 1107 of LANA (Mattsson et al., 2002
; Platt et al., 1999
). The functional significance of this phosphorylation is not yet understood.
This report describes the identification of an isoform of LANA truncated by 76 aa at the C terminus. We have investigated the DNA binding and subcellular localization of this protein in PEL cells.
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METHODS |
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Plasmids.
The C-terminal coding region of LANA (nt 28053488) and LANA-76 (nucleotides 28053258) were PCR amplified with the primers GATCAGATCTCCCATAATCTTGCACGGGTC and GCATTCTAGATTATGTCATTTCCTGTGGAGAGTC (LANA) or GATCAGATCTCCCATAATCTTGCACGGGTC and GCATTCTAGATTAGGACACGGGGCCTGCCT (LANA-
76). These PCR products were cloned into the BglII/XbaI restriction sites of the plasmid pEGFP-C1 (Clontech). All inserts were fully sequenced and found to contain no mutations.
The poly(A) site identified in the LANA coding sequence was mutated from AGTAAA to TGTACA using a Quick-change mutagenesis kit (Stratagene) and oligonucleotides CTTCCAGTTTGGAGGTGTACAGGCAGGCCCCGTGTC and GACACGGGGCCTGCCTGTACACCTCCAAACTGGAAG. The mutated sequence introduced a BsrGI restriction site into the LANA sequence.
Transfection of cells.
HEK293 (5x104 cells per well) or BCP-1 cells (1x105 cells per well) were seeded in 24-well trays and incubated overnight. The cells were then transfected with 0·5 µg of DNA and 1·5 µl Transfast transfection reagent per well according to the manufacturer's instructions (Promega).
Immunofluorescence analysis of transfected cells.
Transfected cells were fixed with 4 % (w/v) paraformaldehyde for 20 min at room temperature and air-dried onto microscope slides. BCP-1 cells were stained with the LANA-specific monoclonal antibody LN53 (ABI) and goat anti-rat antibody conjugated with TRITC (Sigma). LN53 recognizes the epitope EQEQE present in the central repeat region of LANA (Kellam et al., 1999). Fluorescence images were obtained with a confocal microscope.
Northern blots.
Poly(A)+ RNA was isolated (Micro-FastTrack mRNA isolation system; Invitrogen) from 1x107 BCP-1, BC-3 or BJAB cells. The mRNA (equivalent to 5x106 cells per lane) was separated on formaldehyde/agarose gels as described (Sambrook & Russell, 2001) and transferred onto Genescreen-plus membrane by capillary blotting according to the manufacturer's instructions (NEN). The filters were baked at 80 °C for 2 h and then hybridized to radioactive probes. Filters were prehybridized for 20 min and hybridized for 60 min at 60 °C in QuickHyb solution (Stratagene). Blots were washed twice for 15 min at 22 °C in 2x SSC, 0·1 % (w/v) SDS and once for 30 min at 60 °C in 0·1x SSC, 0·1 % SDS, before exposure to X-ray film (X-OMAT-AR; Kodak) at 80 °C with an intensifying screen.
RNA protection assay (RPA).
Poly(A)+ RNA was isolated (Micro-FastTrack mRNA isolation system) from BCP-1 and HEK293 cells and hybridized to an oligonucleotide complementary to the 3' terminus of LANA-76 mRNA with a 20 nt 5' T sequence (T20AGGACACGGGGCCTGCCTTT). The oligonucleotide was labelled at the 3' end using terminal transferase (NEB) and [
-32P]dideoxy-ATP (Amersham), according to the manufacturer's instructions (NEB). The use of dideoxy-ATP ensured that only one residue was added to the end of each oligonucleotide probe. The RNA-protection assay was performed using the Multi-NPA kit from Ambion.
Electrophoretic mobility shift assay (EMSA).
LANA-EGFP, LANA-76-EGFP fusion proteins and EGFP were prepared using the TNT in vitro transcriptiontranslation system (Promega). The correct expression of the proteins was confirmed by Western blot analysis using an anti-GFP antibody (Clontech). The complementary oligonucleotides TR-1 (GATCTCCCGCCCGGGCATGGGGCC) and TR-2 (GATCGGCCCCATGCCCGGGCGGGA) were annealed and the ends filled using Klenow polymerase and dGTP, dATP, dTTP and [
-32P]CTP. Approximately 0·510 µl of the protein preparation was mixed with 50 000 c.p.m. 32P-labelled double-stranded TR probe in 1x reaction buffer [20 mM Tris pH 7·5, 10 % (v/v) glycerol, 50 mM KCl, 0·1 mM dithiothreitol, 10 mM MgCl2, 1 mM EDTA, 20 µg poly(dIdC) ml1] as described previously (Ballestas & Kaye, 2001
). Reactions were resolved by electrophoresis through 5 % (w/v) non-denaturing polyacrylamide gels in 1x TBE. The gels were fixed (acetic acid/methanol) and dried before exposure to X-ray film (X-OMAT-AR) at 80 °C with an intensifying screen.
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RESULTS |
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DISCUSSION |
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The C terminus of LANA has been shown to interact with a number of cellular proteins (p53, Rb, RING3) (Friborg et al., 1999; Platt et al., 1999
; Radkov et al., 2000
) as well as being involved in the transcriptional regulation of cellular and viral genes mediated through transcription factors such as CREB, CBP and Sp1 (An et al., 2002
; Krithivas et al., 2000
; Lim et al., 2000
; Radkov et al., 2000
). The C terminus of LANA also mediates dimerization (Schwam et al., 2000
) and is responsible for binding to two short sequence motifs within the terminal repeats (TR) of the KSHV genome (Garber et al., 2002
). Although localized in the nucleus, the LANA-
76 protein does not co-localize with LANA in KSHV-infected cells, suggesting that it has lost the domain responsible for dimerization and/or the domain required for TR binding. Using an EMSA, LANA-
76 failed to bind to the KSHV TR motif (Ballestas & Kaye, 2001
). Viejo-Borbolla et al. (2003)
have identified a short domain in the C terminus of LANA (aa 11291143) that plays a role in heterochromatin binding, probably by modulating the heterochromatin-binding domain identified at the N terminus (aa 522) (Piolot et al., 2001
). These data are consistent with the nuclear localization of LANA-
76 (lacks aa 10861162), which also fails to associate with either heterochromatin or full-length LANA.
Although it does not bind to the KSHV TR or associate with full-length LANA, LANA-76 still possesses the domains responsible for interacting with p53, Rb and RING3. The interaction of RING3 has been shown to result in the phosphorylation of serine and threonine residues in the C terminus of LANA (Platt et al., 1999
). There are three predicted serine and one predicted threonine phosphorylation sites in the terminal 76 aa of LANA (Fig. 1a
), but these fall outside the domain phosphorylated through interaction with RING3 (aa 9511107) (Platt et al., 1999
). The role of LANA-
76 and whether it interacts with these proteins in KSHV-infected cells still remains to be determined.
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ACKNOWLEDGEMENTS |
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Received 12 November 2003;
accepted 18 February 2004.
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