Microbiology and Tumor Biology Center, Karolinska Institute, S171 77, Stockholm, Sweden1
Department of Microbiology, University Medical School of Debrecen, H-4012 Debrecen, Hungary2
Author for correspondence: Laszlo Szekely.Fax +46 8 330498. e-mail lassze{at}ki.se
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Abstract |
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Introduction |
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HHV-8 is uniformly present in all forms of Kaposi's sarcoma (KS) including classic, endemic, AIDS-associated and post-transplant KS and is thought to contribute to the malignant transformation (Knowles et al., 1997 ; Kennedy et al., 1998
). HHV-8 can immortalize primary endothelial cells (Flore et al., 1998
). It is also present in CD19+ peripheral B-cells of KS patients.
Although HHV-8 was not found to transform B-cells in vitro, double infection together with EBV generated immortalized lymphoblastoid cell lines (LCLs) that carried both viruses (Kliche et al., 1998 ). HHV-8 is also present in body cavity (BC) lymphomas and in multicentric Castleman's disease, suggesting that the virus may play an aetiological role in these diseases.
HHV-8 contains at least 14 genes that are very similar to cellular genes (Nicholas et al., 1998 ; Whitby et al. , 1998
). Some of them, such as the orf72-encoded v- cyclin, are mainly expressed during the latent phase of the infection and are important candidates for the putative transforming function of HHV-8 (for reviews see Moore et al., 1998
; Weiss et al., 1998
).
The major latency-associated nuclear antigen (LNA-1) has no cellular homologue. It shows no sequence similarity to any of the EBV-encoded nuclear antigens either. It is encoded by orf73 and transcribed as part of a polycistronic message. It migrates on SDSpolyacrylamide gels with an apparent mobility of 226234 kDa. KS patients regularly have very high antibody titres against LNA-1. The antibodies are directed against the immunodominant epitopes located at the C terminus (Kedes et al., 1997 ; Rainbow et al. , 1997
; Boshoff et al., 1998
; Dittmer et al., 1998
; Sarid et al., 1998
).
LNA-1 accumulates in nuclear bodies in interphase nuclei, producing a characteristic stippled pattern. We have shown that LNA-1 bodies remain associated with the mitotic chromosomes. They did not co- localize with EBNA-1 in the doubly infected BC lymphoma line BC-1 (Szekely et al., 1998 ).
In the present study we have investigated the high resolution intranuclear distribution of LNA-1 in relation to the chromatin and known nuclear structures in BC cells, cell hybrids and freshly infected cells.
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Methods |
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HHV-8 was produced by treating BCBL-1 cells with 20 ng/ml TPA for 7 days. The cell-free supernatant was filtered through a 0·45 µm filter and used in a dilution of 1:5 to infect the target cells for 48 h.
Visualization of heterochromatin by endonuclease treatment and chromatin spreading.
Mechanical spreading of unfixed nuclei was done as previously described (Szekely et al., 1996 ). To visualize heterochromatin by endonuclease digestion, methanolacetone fixed cells were digested with Sau3A restriction enzyme in 20 mM TrisHCl pH 7·5, 7 mM MgCl2 , 100 mM KCl, 2 mM ß-mercaptoethanol for 2 h at 37 °C using a fivefold dilution series of the enzyme. Slides showing a high contrast between loose and compact chromatin on Hoechst 33258 staining were used for detailed analysis.
DNase and RNase treatment of isolated nuclei.
BCBL-1 cells were treated with ice-cold nucleus isolation buffer containing 50 % glycerol, 10 mM TrisHCl, pH 7·5, 10 mM NaF, 1 mM EDTA and 0·1% NP40 for 10 min. For hypotonic lysis the glycerol concentration was decreased to 10%. The single cell suspension was passed through 25 g twice, pelleted for 5 min at 1000 g and the nuclei were resuspended in isolation buffer supplemented with 10 mM Mg2-acetate. The isolated nuclei were digested at 37 °C for 30 min with 0·5 mg/ml DNase I or 0·5 mg/ml RNase or with both. The digested nuclei were centrifuged on glass slides in Cytospin, fixed with methanolacetone and stained for LNA-1.
Production of cell hybrids.
HHV-8-carrying BC-1 and BCBL-1 cells were fused with the mouse plasmacytoma cell line Sp2-0 or with EBV-carrying IB4 cells using a modified hybridoma protocol. Briefly the cells were washed with serum- free Iscove's medium, mixed with each other. After centrifugation for 5 min at 1200 g the pellet was exposed to 50% PEG 1500, 5% DMSO, 45% Iscove's medium and preheated to 37 °C for 60 s with constant stirring. The fusion mixture was gradually diluted to 10 ml with Iscove's medium during the following 2 min, centrifuged for 5 min at 1200 g and resuspended in Iscove's medium containing 10% foetal calf serum. To select stable mousehuman hybrids of the HGPRT- Sp2-0 and the ouabain-sensitive BCBL-1 cells, the medium was supplemented with 5x10-4 M ouabain (Sigma) and 1x hypoxanthineaminopterinthymidine (HAT) selection mixture.
Immunofluorescence staining and image analysis.
Sera with high anti-LNA-1 titres were collected from patients suffering from the classical form of KS at the Dermatology Unit of Debrecen Medical School, Hungary, and stored at -20 °C.
The cells were regularly fixed with methanolacetone (1:1) at room temperature for at least 10 min and rehydrated in PBS. Sera were diluted in blocking buffer (2% BSA, 0·2% Tween-20, 10% glycerol, 0·05% NaN3 in PBS). The first antibody was incubated with the cells for 30 min at room temperature followed by three 2 min washes in PBS. The secondary, fluorochrome- conjugated antibody was diluted in blocking buffer (1:20) and incubated with the cells for an additional 30 min. Following four PBS washes the slides were mounted with 70% glycerol containing 2·5% DABCO (Sigma) anti-fading agent.
Double immunofluorescence staining for PML, Sp100, EBNA-1, EBNA-2, EBNA-5, EBNA-6, LMP-1 and LNA-1 was carried out as described (Szekely et al., 1995 , 1996
, 1998
; Pokrovskaja et al., 1997
).
The images were produced using a Leitz DM RB microscope, equipped with Leica PL Fluotar 100x, 40x and PL APO Ph 63x oil immersion objectives. Leica L4, Tx and A composite filter cubes were used for the FITC, Texas red and Hoechst 33258 fluorescence respectively. The pictures were captured with a Hamamatsu dual mode cooled CCD camera (C4880) and recorded and analysed on a Pentium PC computer equipped with an AFG VISIONplus-AT frame grabber board using Hipic3.2.0 (Hamamatsu), Image-Pro Plus (Media Cybernetics) and Adobe Photoshop image capturing and processing software.
Three-dimensional microscopy and image quantification.
Images were acquired by a Zeiss Axiophot microscope, equipped with 16x oil Plan-Neofluar NA 0·5, 63x oil Plan- Apochromat NA 1·4 and 100x oil Plan-Neofluar NA 0·71·3 objectives, illuminated with Osram HBO 100 W mercury short arc lamp. The following excitation filters, mounted in the computer-controlled filter wheel were used: single band UV exciter for Hoechst (84360), single band blue exciter for FITC (84490), single band green exciter for TRITC (84555). The emission filter was a multiple band pass filter (84000) mounted on a static stage. All filters were from Chroma Technology. The filter wheel, dual shutter and Z axis motor were controlled through a LEP MAC2000 Communication Interface 73000400 using RS-232 serial connection (from Ludl Electric Products, Hawthorne, NY, USA). Images were captured with a PXL cooled CCD camera (Photometrix) operating at -25 °C, using 12 bit (4096 grey scale level) capture mode. The hardware control and image processing was provided by an Indy Silicon Graphics computer equipped with a MIPS R4600 CPU and a MIPS R4610 floating point processor. The operation system was IRIX 5.3. The imaging programs were written and operated using the Isee 3.4 graphical programming system (Inovision) as described (Holmvall et al., 1999 ).
GFPLNA-1-C' fusion construct.
A 418 nucleotide long fragment covering the coding region aa 9391121 was PCR-amplified from BCBL-1 cells using the primers: 5' GAGGATCCGAATACCGCTATGTACTCAG 3' and 5' CTGAATTCCTAGGCGGGCCATTTGTACT 3'. The fragment was subcloned into the GST-2TK vector (Pharmacia), sequenced and further subcloned into the pEGFP vector (Clontech). The GFPLNA-1-C' plasmid was transfected into CV-1 cells as described.
Detection of EBV and HHV-8 by PCR.
PCR amplification of genomic fragments of EBV and HHV-8 was done in glass capillaries using Rapid Cycler (Idaho Technologies). The reactions were carried out in 10 µl vol. using 2 mM MgCl 2, 1 µM primer and 200 µM dNTP mixture and 0·1 U Platinum Taq polymerase. The initial 15 s denaturation at 94 °C was followed by 35 cycles of denaturation at 94 °C for 0 s, annealing at 50 °C for 0 s and elongation at 72 °C for 45 s. The primer pair for EBV was 5' CTAGGCCACCTTCTCAGT 3' and 5' CTTCTCTGCTCGTTACCA 3', which amplifies a 1087 bp long fragment from the W-repeats. The LNA-1 C terminus primers were used to detect HHV-8.
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Results |
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LNA-1 in DNase-, RNase- and detergent-treated nuclei
In order to study the association of LNA-1 with DNA, RNA or nucleoskeleton, BCBL-1 nuclei were isolated by hypotonic or isotonic lysis in the presence of a non-ionic detergent. Neither the nucleus isolation nor the subsequent treatment with RNase had any effect on LNA- 1 staining. In contrast, digestion of isolated nuclei with DNase I almost completely eliminated the LNA-1 staining while neither DNase nor RNase treatment affected the staining of nucleoskeleton-associated PML bodies (Fig. 4AF
).
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Immunostaining of LNA-1 272 h after fusion showed that the LNA-1 bodies were confined to the human nucleus in the heterokaryons during the whole period (Fig. 5A). The same phenomenon was observed in BCBL-1Sp2-0 and BCBL-1NIH3T3 heterokaryons (data not shown). Following the completion of a full cell cycle and fusion of the two nuclei, LNA-1 bodies were associated with the human chromatin and avoided the mouse chromatin (Fig. 5B
).
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Interestingly LNA-1 bodies were still present in the late hybrids after the loss of human chromosomes. They were associated with the murine pericentromeric heterochromatin (Fig. 5C).
Spreading the chromatin with the help of a rapidly expanding fluid drop generated extended nuclear sheets with sharp borders between euchromatin and heterochromatin. Immunostaining confirmed the close association between the LNA-1 bodies and mouse heterochromatin (Fig. 5D ).
Association of LNA-1 with metaphase chromosomes
In dividing cells LNA-1 containing nuclear bodies remains attached to the chromosomes (Szekely et al., 1998 ). The number of nuclear bodies varies greatly among individual metaphases (Fig. 6A
). LNA-1 bodies appear often as symmetric doublets in both interphase and metaphase cells. The presence of these structures suggested that they might associate with both copies of a given sequence that could indicate sequence-specific DNA interaction. Analysis of metaphase cells showed, however, that the LNA- 1 doublets were frequently located on only one side of one of the sister chromatids (Fig. 6B
). This was also observed in metaphases where intact chromosomes were visualized by three- dimensional reconstitution from a series of optical sections.
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LNA-1 in apoptotic cells
Small numbers of apoptotic cells are present in almost all cell cultures. Apoptotic cells are easily recognized by Hoechst 33258 staining by the loss of nuclear structures followed by the condensation and eventual fragmentation of the nuclear remnants, leading to the formation of apoptotic bodies. In both BC-1 and BCBL-1 cells LNA-1 foci persisted even in advanced apoptotic cells but were not included in the apoptotic bodies (Fig. 6D).
Nuclear distribution of LNA-1 and EBNAs in BCBL-1LCL hybrids
Since LNA-1 is the major latency-associated nuclear protein of HHV- 8, it might share functions with some of the six EBNAs encoded by EBV. We have previously shown that BC-1 cells that harbour both viruses show strict type I EBV latency (express only EBNA-1 and LMP2A). They were found to accumulate EBNA-1 and LNA-1 in distinct nuclear foci with no significant overlap (Szekely et al., 1998 ).
In the present study we have extended these experiments to study the possible relationship between EBNA-2, -5, -6 and LNA-1 in cell hybrids between the EBV-negative BCBL-1 cells and the EBV-immortalized LCL IB- 4, which expresses all six EBNAs. Double fluorescence staining of the heterokaryons for LNA-1 and the EBNAs at different time-points after the fusion showed that while EBNA-1, -2, -5, and -6 equilibrated between the nuclei within 40 h, LNA-1 remained associated with the original BCBL-1 nucleus during the entire observation time (>>72 h). There was no co-localization between any of the EBNAs and LNA-1 in the double positive nuclei. Interestingly, the different EBNAs equilibrated between the nuclei of the heterokaryons with different kinetics. EBNA-5 was the most mobile, showing full equilibration by 4 h, whereas EBNA-6 needed more than 40 h (Table 1 and Fig. 7A
I
). We also stained the EBV-encoded latent membrane protein LMP-1 together with LNA-1 and showed that it was distributed on the entire surface of the hybrids as early as 5 h after fusion (not shown). The staining intensity of EBNA-6 and LMP-1 but not EBNA-2 and EBNA-5 was reduced in the older (66 h) hybrids.
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GFPLNA-1-C' in transfected cells
The C-terminal part of LNA-1 harbours a putative nuclear translocation signal. We tested the surrounding 182 amino acid long region for subcellular localization by fusing the LNA-1 fragment to green fluorescent protein (GFP). Upon transfection into CV-1 cells the fusion protein accumulated in the nucleus. In most cells the protein showed a nearly homogeneous nuclear distribution, but occasionally accumulated in distinct foci. However, these foci primarily localized to the euchromatin (Fig. 6E).
Relationship between LNA-1 and ND10 bodies
The ND10 bodies (Daniel et al., 1993 ) are nuclear structures that are frequently targeted by viral regulatory proteins. Their components, PML and Sp100 proteins, are induced by interferon alfa and gamma. Double fluorescence staining of LNA-1 and either PML or Sp100 showed that the LNA-1 bodies and ND10 occupy different nuclear domains. (Fig. 7L
and J
respectively).
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Discussion |
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The degree of chromatin condensation affects gene expression. Nuclear areas with highly condensed DNA are transcriptionally inactive, as a rule (Wallrath, 1998 ). Chromatin condensation is regulated in several ways, such as methylation of CpG sites, acetylation of core histones or association of protein complexes such as the Polycomb group proteins (Jenuwein et al., 1998
; Saurin et al., 1998
; Turner, 1998
). The latter form distinct bodies that associate with heterochromatin during the interphase and remain associated with the chromosomes during cell division.
We have found that LNA-1-containing nuclear foci associate with the border of highly condensed chromatin. These foci are primarily bound to DNA but not to the nuclear matrix. No RNA is needed to maintain their structural integrity. Their number, size and morphology varies from cell to cell in established cell lines. This heterogeneity is also present in metaphase cells, indicating that it is not cell-cycle- dependent.
The association with the heterochromatin border is not restricted to BC cells but is also detectable in freshly infected erythroleukaemia and endothelial cells.
Binding of LNA-1 bodies to murine pericentromeric heterochromatin blobs in the nuclei of the BC-1Sp2-0 hybrids but not to the pericentromeric regions of the murine metaphase chromosomes suggests that LNA-1 has a specific affinity to the interphase heterochromatin but not to specific sequences of the pericentromeric region. This is consistent with the random association of LNA-1 with human metaphase chromosomes. The persistent association of LNA-1 with the human chromatin in the mousehuman hybrids and its failure to equilibrate with the partner nucleus in both humanhuman and humanmouse heterokaryons suggests that LNA-1 has a high affinity to certain low turnover structures on the heterochromatin of the donor nuclei.
The recent demonstration of co-localization between LNA-1 foci and the HHV-8 DNA suggests that these low turnover structures are the viral genomes themselves (Ballestas et al.,1999 ).
LNA-1 has a very unusual primary structure. It contains a highly hydrophilic central domain of multiple repeats of glutamate, glutamine and asparagine, flanked by proline-rich N-terminal and basic C-terminal domains. The latter also harbours a putative nuclear translocation signal that we found capable of accumulating in the nucleus in the absence of other domains.
The peculiar distribution of LNA-1 might suggest that it participates in the control of euchromatin/heterochromatin transition. Upon docking to the border of the heterochromatin, the strongly acidic middle domain might facilitate the opening of closed heterochromatic areas in a similar way as the histone acetylation-induced charge shift opens up condensed chromatin.
The LNA-1 foci are clearly different from the ND10 bodies that are frequent targets of viral regulatory proteins (Maul, 1998 ). LNA-1 does not co-localize with the ND10 components PML and Sp100. Unlike ND10, the LNA-1 bodies do not resist mechanical spreading and stay on the mitotic chromosomes. High resolution three-dimensional reconstitution of LNA-1 clusters also suggest that unlike the ND10s the LNA-1 bodies do not have rigid borders but tend to fill up spaces excluded from other structures. These spaces often communicate with each other.
Since LNA-1 is the major latency-associated nuclear antigen of HHV- 8, it has been suggested that it may have a similar function to some of the EBV-encoded EBNAs. No co-localization was detectable between LNA-1 and EBNA-1, -2,-5 and -6 in the BCLCL heterokaryons.
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Acknowledgments |
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References |
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Received 21 May 1999;
accepted 9 July 1999.