Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea
Correspondence
Joonho Choe
jchoe{at}kaist.ac.kr
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ABSTRACT |
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INTRODUCTION |
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Latency-associated nuclear antigen 1 (LANA1), encoded by open reading frame 73 of KSHV genome, is one of the virus genes expressed during latent infection (Russo et al., 1996; Rainbow et al., 1997
). Based on the primary amino acid sequence, it was suggested that this 222234 kDa nuclear protein was a transcription factor. LANA1 interacts with several cellular transcription factors such as p53, pRb, ATF4/CREB2 and CREB-binding protein, and modulates their transcriptional activities (Friborg et al., 1999
; Lim et al., 2000
, 2001
; Radkov et al., 2000
). When tethered to promoters via the heterologous DNA-binding domain, both the N terminus and the C terminus of LANA1 exhibit cell-type and promoter-specific transcriptional repressor activity, possibly through interactions with the components of the mSin3 corepressor complex and heterochromatin protein 1, respectively (Krithivas et al., 2000
; Lim et al., 2003
; Schwam et al., 2000
). In addition, LANA1 has also been shown to participate in the transcriptional regulation of several cellular and virus promoters (An et al., 2002
; Friborg et al., 1999
; Groves et al., 2001
; Hyun et al., 2001
; Knight et al., 2001
; Krithivas et al., 2000
; Radkov et al., 2000
; Renne et al., 2001
).
Maintenance of the herpesvirus genome during latent infection is accomplished by two distinct events. These include replication of the virus genome, which is synchronized with the DNA synthesis of host cells, and the equal segregation of replicated virus genomes to daughter cells after mitosis. A long-term replication assay, in which these two activities can be collectively analysed by selecting drug-resistant cells containing origin of replication (oriP)-plasmids, revealed that KSHV LANA1 and TR sequences located at both ends of the virus genome act as virus trans- and cis-acting elements, respectively, in the latent replication of KSHV (Ballestas et al., 1999; Ballestas & Kaye, 2001
). LANA1 binds to sequences within TR via the C-terminal DNA-binding domain (Ballestas & Kaye, 2001
; Cotter et al., 2001
; Garber et al., 2001
, 2002
; Lim et al., 2002
) and associates with the chromosomes of host cells via the N-terminal chromosome-binding sequence (CBS) (Ballestas et al., 1999
; Cotter & Robertson, 1999
; Piolot et al., 2001
), suggesting a model in which LANA1 tethers virus genome to host chromosome for its persistence during latent infection. Recently, we and others showed that LANA1 actively participates in replication of the virus genome using a short-term replication assay whereby a methylation-sensitive restriction enzyme removes unreplicated oriP-containing plasmid from transiently transfected cells (Garber et al., 2002
; Hu et al., 2002
; Lim et al., 2002
). It was also shown that LANA1 might recruit the cellular replication machinery to KSHV TR, possibly via interaction with origin recognition complexes (Lim et al., 2002
) as in the case of EBNA-1 (EpsteinBarr virus nuclear antigen-1), a functional analogue of KSHV LANA1 (Chaudhuri et al., 2001
; Dhar et al., 2001
; Schepers et al., 2001
).
In this report, we verified the transient replication of KSHV TR-containing plasmid in cell lines derived from different species, and determined the minimum domain of LANA1 required for replication of the TR-containing plasmid, using a transient replication assay. We also identified dominant-negative mutants of LANA1 for the replication activity and speculated on their replication-inhibitory mechanisms using other cell lines and virus replication systems.
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METHODS |
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Cell culture, transfection and reporter assay.
Human embryonic kidney cell lines, 293 and 293T, and human cervical carcinoma cell line, C33A, were maintained and transfected as described previously (Lim et al., 2000, 2003
). Simian virus 40 (SV40)-transformed African green monkey kidney cell line, COS-1, and mouse embryo cell line, NIH/3T3, were maintained in Dulbecco's modified Eagle's medium supplemented with 10 % fetal bovine serum, and were transfected using LIPOFECTAMINE plus reagent according to the manufacturer's instructions (Invitrogen). The quantity of total DNA used in the transfections was kept constant by including an appropriate blank vector. The transient reporter assay was performed as described previously (Lim et al., 2000
).
Western blotting.
293T cells in 60 mm dishes were transfected with the indicated mammalian expression vectors. At 36 h after transfection, cells were harvested and cell pellets were resuspended in 100 µl of PBS. After addition of 100 µl of 2xSDS buffer, lysates were briefly sonicated, and then boiled for 5 min. Total proteins were separated by SDS-PAGE, transferred to Hybond-C membrane (Amersham Biosciences), immunoblotted with an anti-FLAG M2 monoclonal antibody (Sigma) and rabbit polyclonal anti-LANA1 serum (a generous gift from Dr Jung, Harvard Medical School), and detected by ECLTM (Amersham Biosciences).
Immunofluorescence assay.
293T cells grown on coverslips were transfected with the indicated expression vectors. At 24 h after transfection, cells were fixed in 3·7 % formaldehyde at room temperature for 30 min, and permeabilized in PBS containing 0·2 % Triton X-100 at 4 °C for 25 min. FLAG-tagged proteins were observed using an anti-FLAG M2 monoclonal antibody (Sigma) and a rhodamine-conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch Laboratories). Coverslips were mounted with Vectashield (Vector Laboratories) and examined by confocal laser scanning microscopy (Pascal, Carl Zeiss, Jena, Germany).
Transient replication assay.
A transient replication assay was performed as described previously (Lim et al., 2002), with minor modifications. For the KSHV replication assay, 293, 293T, C33A, COS-1 and NIH/3T3 cells grown in 100 mm dishes were cotransfected with 2 µg of p4TR-luc containing KSHV TR, 2 µg of pGL2-basic as an internal control, and the indicated amount of trans-element expression plasmid. For the EBV replication assay, 293, 293T and C33A cells grown in 100 mm dishes were cotransfected with 2 µg of pOLP containing EBV oriP, 2 µg of pGL2-basic as an internal control, and the indicated amount of trans-element expression plasmid. For the SV40 replication assay, 293T cells grown in 100 mm dishes were cotransfected with 2 µg of pcDNA3 containing SV40 oriP and 8 µg of the indicated trans-element expression plasmid. For the HPV replication assay, 293 and C33A cells grown in 100 mm dishes were cotransfected with 2 µg of p105core-luc containing HPV oriP, and the indicated amount of trans-element expression plasmid. Cells were split at
2436 h after transfection and harvested at 96 h after transfection, unless otherwise indicated. Low molecular mass DNA was extracted from transfected cells by the Hirt lysis method (Hirt, 1967
), followed by phenol/chloroform/isoamyl alcohol and chloroform extraction. Ethanol-precipitated DNA was dissolved in RNase-containing distilled water. Plasmids p4TR-luc, pOLP, pcDNA3 and p105core-luc were linearized by digestion with Alw44I, SacI, EcoRI and BamHI, respectively. Unreplicated DNA was removed by digestion with DpnI, and the complete digestion of unreplicated oriP-containing plasmid was monitored by comparison with that of unreplicating internal control, pGL2-basic in the cases of KSHV and EBV replication assay. Digested DNA was separated by electrophoresis on a 0·8 % agarose gel and analysed by Southern blot hybridization. Probes specific for the luciferase gene of pGL2-basic and neomycin open reading frame of pcDNA3 were synthesized with a NEBlot Phototope kit and detected with a Phototope-Star Detection kit, according to the manufacturer's instructions (New England Biolabs). All transient replication assays were performed more than twice and the representative result was shown.
[3H]Thymidine incorporation assay.
293T cells in six-well plates or in 60 mm dishes were transfected with the indicated mammalian expression vectors. Transfected cells in six-well plates were incubated with 0·6 µCi ml-1 (22·2 kBq ml-1) of [methyl-3H]thymidine (90 Ci mmol-1, Amersham Biosciences) for the final 12 h before the assay that was performed at 36 h post-transfection. Transfected cells in 60 mm dishes were split into six-well plates 36 h after transfection, and incubated with radioactive thymidine as above for the final 24 h before the assay that was performed at 96 h post-transfection. At the indicated time, culture medium was removed and transfected cells were washed twice with PBS and extracted three times with 10 % trichloracetic acid. Precipitated materials were solubilized in 1 M NaOH, 1 % SDS at room temperature for 1 h and counted by liquid scintillation (LS6500 Liquid Scintillation Counters, Beckman Coulter). More than two independent experiments were performed in duplicate.
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RESULTS |
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As described previously (Hung et al., 2001), EBV oriP-containing plasmid, pOLP, replicated only in the presence of EBNA-1, but not LANA1 (Fig. 4
A, left panel). Overexpression of the wild-type and the N terminus of LANA1 dramatically reduced the replication of pOLP by EBNA-1, while the C-terminal LANA1 did not show any effect on it. Western blot of total lysates from transfected cells indicated that this inhibitory effect of the wild-type and the N terminus of LANA1 on the transient replication of EBV oriP-containing plasmid is not due to the change in the expression level of EBNA-1 (Fig. 4A
, right panel). Reciprocal transient replication assay was also performed to investigate the effect of EBNA-1 overexpression on the replication of KSHV TR-containing plasmid, p4TR-luc, which only replicates in the presence of LANA1. As shown in Fig. 4(B)
, overexpression of EBNA-1 neither inhibited the replication of KSHV TR-containing plasmid nor affected LANA1 expression. Since the N-terminal LANA1 seemed to possess the general inhibitory activity on the herpesvirus replication systems under the condition of our transient replication assay in 293T cells, we questioned whether this domain of LANA1 could also suppress other virus replication other than herpesviruses. When these herpesvirus replication proteins were overexpressed in the transient replication assay of pcDNA3 containing SV40 oriP in 293T cells, only the wild-type and the N terminus of LANA1 showed significant inhibitory effect on the SV40 replication (Fig. 4C
). To determine which region of the N-terminal LANA1 is responsible for the inhibitory activity of the virus replication, a series of LANA1-deletion mutants depicted in Fig. 2(A)
was included in the transient replication assay of EBV. As shown in Fig. 4(D)
, LANA1-deletion mutants supporting the replication of KSHV TR-containing plasmid were different from those restraining the replication of EBV oriP-containing plasmid, and only the LANA1
91950 showed the inhibitory activity comparable to N-terminal LANA1.
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Cell-type specificity of dominant-negative effect of LANA1 and its derivatives on the virus replication systems
Cell lines such as 293T and COS-1 endogenously express SV40 large T antigen, thereby permitting the replication of SV40 oriP-containing plasmid. The transient replication assay of virus replication systems in those cell lines is obligatorily coupled to SV40 replication system if any SV40 oriP-containing plasmid is used. Since all mammalian expression vectors used in our study contain SV40 oriP, it is possible that SV40 replication system affects the outcome of transient replication assay in 293T cells and the general replication-inhibitory activity of the N-terminal LANA1 results from the reduced replication of SV40 oriP-containing plasmid by the N-terminal LANA1. To exclude this possibility, we performed similar transient replication assays in other cell lines that do not express SV40 large T antigen. These include human embryonic kidney cell line 293 (Fig. 5A) and human cervical cancer cell line C33A (Fig. 5B
). In transiently transfected 293 cells, LANA1 and its derivatives showed similar effects on the transient replication of KSHV TR- and EBV oriP-containing plasmid as in 293T cells. The N-terminal LANA1 inhibited both virus replication systems, but the replication-inhibitory activity of C-terminal LANA1 was KSHV-specific (Fig. 5A
, top and middle). In contrast, only the C-terminal LANA1 inhibited the replication of KSHV TR-containing plasmid, albeit weakly but reproducibly, and we could not observe any comparable replication-inhibitory activity of wild-type and the N-terminal LANA1 in transiently transfected C33A cells (Fig. 5B
, top and middle).
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DISCUSSION |
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From the facts that the C-terminal LANA1 alone can bind sequences within TR and repress the transcription from TR (Garber et al., 2001, 2002
; Lim et al., 2002
), but not facilitate the replication of TR-containing plasmid in the transient replication assay under our experimental condition (Lim et al., 2002
), we reasoned that it can compete with the wild-type LANA1 for binding sites within TR, and act as a dominant-negative inhibitor of the replication of oriP-containing plasmid, although Hu et al. (2002)
reported the partial replication activity of C-terminal LANA1. As expected, the C terminus of LANA1 inhibited the replication of TR-containing plasmid by LANA1, but not other virus replication systems including EBV, SV40 and HPV in other cell lines. However, unexpectedly, the N terminus of LANA1 also showed the inhibitory activity of KSHV, EBV and SV40 replication systems in transiently transfected 293 and 293T cells, but not in C33A cells. The mode of replication-inhibitory activities of these two LANA1-deletion mutants may be distinct. In the case of the C-terminal LANA1, competitive binding with the wild-type LANA1 for specific sequences within KSHV TR could be the explanation for the observed KSHV-specific inhibition of replication. It seems unlikely that cellular replication factors including origin recognition complexes, which are commonly targeted by both the C terminus of LANA1 and EBNA-1 (Chaudhuri et al., 2001
; Dhar et al., 2001
; Lim et al., 2002
; Schepers et al., 2001
), were limiting for virus replication because the overexpression of C-terminal LANA1 did not influence the replication of EBV oriP-containing plasmid by EBNA-1 which recruits origin recognition complexes to the virus origin of replication. In contrast, the N terminus of LANA1, which is required for the association with host chromosome and the replication activity, may squelch cellular replication factors and/or replication sites on host chromosomes where cellular replication factors are assembled for virus replication, resulting in the inhibition of virus replication systems in human embryonic kidney cell lines. Since most of mammalian expression vectors used in our experiments contain SV40 oriP, the transient replication assay of other virus replication systems in 293T cells is obligatorily coupled to SV40 replication system. Therefore, it is also possible that the observed suppression of virus replication by the N-terminal LANA1 involves the effect of SV40 replication by large T antigen and arises from the reduced replication of expression vectors for trans-acting element of virus replication, theoretically resulting in the reduced expression of them. However, we could not observe any significant effect of the N-terminal LANA1 on the expression of wild-type LANA1 and EBNA-1 from transiently transfected expression vectors. In addition, the population of multifunctional LANA1, which actually takes part in the replication activity of virus genome may be small. This is inferred from two observations: (i) that the titration experiment in the transient replication assay showed that even a small amount of LANA1 expression vector achieved the maximum efficiency in TR-containing plasmid replication (Hu et al., 2002
; Lim et al., 2002
); (ii) a relatively low copy number of virus genome is maintained in latently infected cells, suggesting that the expression level of virus trans-acting element may not significantly affect the replication efficiency of herpesvirus replication systems under our experimental condition. Finally, we could observe the replication-inhibitory activity of N-terminal LANA1 in transiently transfected 293 cells, which does not express SV40 large T antigen and support the replication of SV40 oriP-containing plasmid, excluding that possibility. Interestingly, we could not detect any transcriptional repression activity of N-terminal LANA1 using transient reporter assay in C33A cells (Lim et al., 2003
), where the N-terminal LANA1 did not also show the replication-inhibitory activity, suggesting the possible link between them.
While performing transient replication assays, we observed that the replication efficiency of virus oriP-containing plasmid showed a certain degree of variation in different experiments. There could be several reasons for the fluctuations in the replication efficiency. First, the different batch of serum and passage numbers of cells used in the transient replication assay may affect the cellular context such as the rate of cell cycle and the signal pathways up- or down-regulating the replication of virus oriP-containing plasmid. It is noteworthy that the overexpression of the catalytic subunit of protein kinase A or the treatment of phorbol 12-myristate 13-acetate during the transient replication assay negatively regulated the replication of KSHV TR-containing plasmid, supporting this possibility (data not shown). Secondly, the transiently transfected plasmid could be differentially chromatinized in separate transfections, which may lead to variations in the accessibility and process of cellular replication machinery on the virus oriP. Finally, since the copy number of herpesvirus genomes maintained by infected cells is limited by some unknown mechanism, it is possible that the initial amount of virus oriP-containing plasmid transfected into cells may determine its replication efficiency in the transient replication assay.
Because of its functional importance, we were interested in cellular proteins targeted by the CBS of LANA1. We found several candidate proteins that interact with CBS of LANA1 by proteomics approach. Currently, their functional roles on the chromosome association and replication activity of LANA1 are under investigation.
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ACKNOWLEDGEMENTS |
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Received 16 July 2003;
accepted 8 December 2003.