1 Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Church Street, Glasgow G11 5JR, UK
2 Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD, USA
Correspondence
J. Barklie Clements
b.clements{at}vir.gla.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Currently there is no efficient cell culture system for KSHV; the only viable experimental system uses naturally infected B cell lines, such as BCBL-1 (Renne et al., 1996), generated by culture of PELs that contain the genome of KSHV. The majority of PEL cells are latently infected with KSHV, with a low level of spontaneous virus reactivation. Addition of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) to BCBL-1 cells efficiently induces the lytic cycle, producing virions (Renne et al., 1996
). ORF50 (also known as RTA), a replication and transcription activator, and ORF57 (also known as MTA), immediate-early proteins, the earliest KSHV regulatory genes to be induced and expressed (Lukac et al., 1999
; Paulose-Murphy et al., 2001
), are both essential for lytic virus replication (Sun et al., 1999
; Zhu et al., 1999
).
ORF57 protein exhibits nucleocytoplasmic shuttling (Bello et al., 1999) and, like its EBV MTA (BMLF-1) and HVS ORF57 homologues (Lieberman et al., 1986
; Nicholas et al., 1988
; Kenney et al., 1989
; Whitehouse et al., 1998
), has a post-transcriptional mode of action (Gupta et al., 2000
; Kirshner et al., 2000
). An ORF57 counterpart is present in every herpesvirus sequenced, reflecting the importance of this signature viral protein. ORF50 protein is analogous to gammaherpesvirus transactivators HVS ORF50 and EBV RTA (BRLF-1) (Hardwick et al., 1988
; Whitehouse et al., 1997
; Lukac et al., 1999
). Expression of ORF50 alone is necessary and sufficient for the switch from latent to lytic KSHV replication (Lukac et al., 1998
; Sun et al., 1998
; Gradoville et al., 2000
) in cultured cells (Liang & Ganem, 2003
) and may determine viral host range and spread in vivo (Bechtel et al., 2003
). ORF50 protein exhibits sequence-specific DNA binding (Lukac et al., 2001
; Song et al., 2001
) and positively regulates its own promoter (Seaman et al., 1999
; Deng et al., 2000
) via an octamer element and Oct-1 protein (Sakakibara et al., 2001
). Overexpression of ORF57 and ORF50 proteins together, driven by a human cytomegalovirus (HCMV) promoter, synergistically enhanced expression from several viral lytic gene promoters; however, at that time no physical association was demonstrated between the two proteins (Kirshner et al., 2000
).
We have shown that simultaneous expression of ORF57 and ORF50 proteins stimulates reporter gene expression from the immediate-early ORF50 gene promoter itself by a further 13-fold above the level obtained with ORF50 protein alone. We have also shown that ORF57 and ORF50 proteins are present together in the same complex in extracts of KSHV-infected BCBL-1 cells, and a physical interaction was also demonstrated using a pull-down assay with a glutathione S-transferase (GST)ORF57 fusion protein. A chromatin immunoprecipitation (ChIP) assay showed that ORF50 promoter sequences were preferentially associated with immunoprecipitated chromatin using both anti-ORF50 and anti-ORF57 antibodies (Abs). Thus, the co-ordinated activities of these two KSHV regulatory proteins will promote the cascade of lytic viral gene expression, overcoming the inefficiency of spontaneous lytic virus reactivation.
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METHODS |
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Chemical induction of the KSHV lytic cycle and preparation of cell extracts.
BCBL-1 cells (0·2x106 cells ml1) were treated with TPA (20 ng ml1) (Renne et al., 1996) for 72 h or left untreated. Cell extracts prepared as described by Wadd et al. (1999)
in 800 µl lysis buffer (50 mM Tris/HCl, pH 7·5, 100 mM NaCl, 1 mM EDTA, 0·1 % NP-40, 0·5 mM PMSF) containing protease inhibitor cocktail (Roche) were passed five times through a 26-gauge needle. RNase treatment was with 10 U (ONE; Promega) at 37 °C for 20 min.
Recombinant protein expression, in vitro GST pull-down assays and Western immunoblotting.
GSTORF50 (FL) fusion protein was prepared as described previously (Wang et al., 2003a). GSTORF57 fusion protein was prepared as described for GSThnRNP K (Wadd et al., 1999
) with modifications: Escherichia coli BL21 cells (500 ml) were treated with 1·0 mM IPTG for 16 h at 28 °C and resuspended in 5 ml modified NETN lysis buffer (20 mM Tris/HCl, pH 8·0, 150 mM NaCl, 1 mM EDTA, 0·1 % NP-40, 10 % w/v glycerol, 5 mM
-mercaptoethanol) with 100 µg lysozyme ml1 and sonicated on ice. Triton X-100 was added to 2 % (v/v), extracts were kept at 4 °C for 30 min and the GSTORF57 fusion protein was purified on glutathioneSepharose 4B beads. GST pull-down assays performed as described previously (Wadd et al., 1999
; Koffa et al., 2001
) used untreated or TPA-treated BCBL-1 extracts (200 µg protein) or 10 µl each [35S]methionine-labelled ORF57 (FL), ORF57 deletion mutants and luciferase (Promega) proteins, synthesized in vitro using the TNT T7 Quick Coupled transcription/translation system (Promega) according to the manufacturer's instructions. Bound proteins were resolved by SDS-PAGE and visualized either by Western blotting (Wadd et al., 1999
) using anti-ORF57 (GH) Ab diluted 1 : 2500, with the ECL detection system (Amersham Pharmacia Biotech) or by autoradiography.
In vitro co-immunoprecipitation assays.
For immunoprecipitations with [35S]methionine-labelled ORF57 and luciferase proteins synthesized in vitro, 10 µl [35S]methionine-labelled in vitro-translated protein was incubated with 6 µl rabbit anti-ORF57 (GH) Ab in 200 µl in vitro immunoprecipitation buffer (50 mM Tris/HCl, pH 8·0, 100 mM NaCl, 1 mM EDTA, 2 mM EGTA, 0·1 % NP-40, 1 % BSA, 0·5 mM PMSF) and protease inhibitor cocktail (Roche) for 1 h at 4 °C. Then, 75 µl of a 50 % slurry in immunoprecipitation buffer of protein Aprotein G (50 : 50 ratio) Sepharose beads (Amersham Pharmacia) was added to the mixture and incubated for 1 h at 4 °C. The beads were washed four times with cold immunoprecipitation buffer at 15 min intervals, resuspended and boiled in 2x SDS gel loading buffer, and bound proteins were resolved by SDS-PAGE and visualized by autoradiography.
In vivo co-immunoprecipitation assays.
BCBL-1 cell extracts (200 µg protein) were pre-cleared with 5 µl rabbit pre-immune serum and 100 µl of a 50 % slurry of protein Aprotein G (50 : 50) Sepharose beads for 1 h at 4 °C. After pre-clearing, 3 µl each of 718, GH and 721 ORF57 Abs, 5 µl anti-ORF50 Ab or pre-immune rabbit serum was added to pre-cleared cell extracts in 300 µl immunoprecipitation buffer (10 mM Tris/HCl, pH 8·0, 100 mM NaCl, 5 % glycerol, 1 mM EDTA, 2 mM EGTA, 0·1 % NP-40, 1 % BSA, 0·5 mM PMSF) with protease inhibitor cocktail (Roche), and immunoprecipitations were performed as described previously (Koffa et al., 2001). Immunoprecipitated proteins were resolved by SDS-PAGE and visualized by Western blotting (Wadd et al., 1999
) using anti-ORF50 Ab diluted 1 : 3000, with the ECL detection system (Amersham Pharmacia Biotech).
ORF50 promoter-driven luciferase assay.
Human 293 cells (6x105), a cell line useful for KSHV propagation from KS biopsy specimens (Foreman et al., 1997) and infectious virus production, following induction by overexpression of ORF50 from KSHV-infected cells (Bechtel et al., 2003
), were transfected using Polyfect (Qiagen) following the manufacturer's protocol. Transfections used 250 ng pORF50/500-luc promoter DNA, 10 ng pRL-SV40 DNA (expressing Renilla luciferase), specified amounts of GFP-gORF57 and pcDNA4-gORF57 (or pcDNA-ORF57 deletion mutants) DNAs with or without pcDNA3-gORF50 DNA. The final amount of DNA in each transfection was kept constant with empty pcDNA3/pcDNA4 or pEGFP-C1 vector DNAs. Control experiments were performed with the heterologous promoter containing the interferon stimulatory response element driving a luciferase gene (pISRE-luc, 250 ng DNA), and recombinant IFN-
was added at 200 U ml1 at 5 h post-transfection. Transfected cells were harvested 48 h after transfection and frozen at 70 °C for 2 h. Luciferase activity of transfected cell extracts was measured using the Dual Luciferase Reporter assay kit (Promega). All firefly luciferase data were normalized with respect to Renilla luciferase activity expressed from a separate, constitutively active plasmid that was co-transfected with the firefly luciferase reporter plasmid. This normalization compensated for differences in transfection efficiencies between replicate cultures.
Chromatin immunoprecipitation assay.
BCBL-1 cells were treated with TPA for 48 h to induce the KSHV lytic cycle before harvesting. Chromatin extracts, cross-linking, sonication, immunoprecipitation, agarose bead elution and protein removal were carried out with a ChIP assay kit (Upstate Biotechnology) based on the manufacturer's protocol with slight modifications as described previously (Wang et al., 2004). DNA recovered from immunoprecipitates with anti-ORF50 or anti-ORF57 (GH) polyclonal Abs or a no-Ab control was used as a template for PCR amplifications. Primers LGH4354 (5'-GAACTACTCGAGCTGTGCCCTCCAGCTCTCAC-3') and LGH4355 (5'-GGACGTAAGCTTACAGTATTCTCACAACAGAC-3'), specific for a 261 bp region in the KSHV ORF50 promoter from 241 to +20, were used to detect the ORF50 promoter region. Primers LGH5297 (5'-CGTGTTCAACGACCACGGACTTCAGGTGCG-3') and LGH5298 (5'-CTGGGTTTCCTCGCGCGATGCTTTTCTCTG-3'), specific for KSHV ORF64 coding region (aa 13291425), were used as a negative control to detect a non-promoter region. The PCR products were analysed by electrophoresis on a 2 % agarose gel. Quantification of the PCR products was conducted with a MultiImage light cabinet (Alpha-Innotech Corp.) and the accompanying FluorChem (version 1.02) software.
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RESULTS |
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To identify the regions of ORF57 protein involved in stimulation of luciferase activity from the ORF50 promoter, pcDNA4-ORF57 DNA expressing full-length ORF57 or various deletion mutant proteins (Fig. 1b) were co-transfected with the pORF50/500-luc reporter construct (Fig. 1c
). Compared with the level obtained with full-length ORF57 protein, the ORF57 N-terminal (aa 1215) and middle region (aa 181328) mutants showed low levels of luciferase activity (Fig. 1c
). By contrast, a luciferase value of 108 % was obtained with a mutant expressing ORF57 aa 329455 (Fig. 1c
). The activation was reduced to 65 % with ORF57 aa 387455 (Fig. 1c
). Thus, residues at the ORF57 protein C terminus are important for the activation of luciferase expression.
Simultaneous expression of ORF57 and ORF50 proteins co-operatively stimulates ORF50-mediated promoter activity
During lytic KHSV replication, ORF57 is not expressed in isolation; rather it is produced with other viral regulatory proteins and is always accompanied by the KSHV transactivator protein ORF50. We therefore examined the effect of co-expressing ORF57 and ORF50 proteins on the ORF50 promoter. Human 293 cells were transfected with the pORF50/500-luc reporter plasmid and vectors expressing the following proteins: ORF57 alone (GFP-gORF57; 600 ng) or ORF50 alone (pcDNA3-gORF50; 500 ng) or ORF50 and ORF57 together. The functionality of the 5'-UTR of pORF50/500-luc was verified in co-transfection studies when, in several independent experiments, ORF50 protein expressed alone (pcDNA3-gORF50; 500 ng) activated basal promoter activity of pORF50/500-luc by a maximum of 4-fold (Fig. 2a). These data were consistent with levels seen by others for a comparable ORF50 5'-UTR reporter plasmid in KSHV-uninfected cells (Seaman et al., 1999
; Sakakibara et al., 2001
; Wang et al., 2003b
). Expression of ORF57 alone activated luciferase expression by a maximum of 8-fold (Fig. 2a
; also shown in Fig. 1a
). Simultaneously, when ORF50 DNA (pcDNA3-gORF50; 500 ng), which resulted in maximum activity from the pORF50/500-luc reporter vector, was co-transfected with increasing amounts of ORF57 DNA (GFP-gORF57; 0900 ng), normalized luciferase activity rose in a dose-dependent manner (Fig. 2b
). ORF50 promoter activity was upregulated further to a maximum of 13-fold with the addition of 600 ng ORF57 DNA (Fig. 2b
), above the 4-fold level obtained with ORF50 DNA alone (as shown in Fig. 2a
). Similar results were obtained using a pORF50/3000-luc reporter plasmid containing 3000 bp of DNA upstream of the ORF50 translation initiation codon (data not shown). These data demonstrated functional co-operation between ORF57 and ORF50 proteins on gene expression from the ORF50 promoter.
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Kinetics of ORF57 and ORF50 expression in TPA-treated BCBL-1 cells
Given the functional co-operation between the ORF57 and ORF50 proteins on expression from the pORF50/500-luc reporter plasmid, we tested for simultaneous expression of the ORF57 and ORF50 proteins following TPA treatment of BCBL-1 cells. Western blotting with anti-ORF57 (GH) Ab on BCBL-1 cell extracts revealed that an increase in the amount of ORF57 protein was detected between 2 and 4 h post-TPA treatment (Fig. 3a, compare lanes 3 and 4). Up to 17 h after TPA treatment, anti-ORF57 Ab stained one major band of 5052 kDa, and, between 17 and 24 h after TPA treatment, a faster-migrating protein band (4548 kDa) was detected (Fig. 3a
, compare lanes 6 and 7), suggestive of ORF57 protein processing. The ORF57 Ab showed some non-specific staining of a faint protein band present above ORF57 in both untreated and TPA-treated BCBL-1 cell extracts (Fig. 3a
, lanes 18). The amount of this protein band did not increase with TPA treatment.
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ORF57 and ORF50 gene products co-immunoprecipitate from TPA-treated BCBL-1 cell extracts
To search for a possible ORF57ORF50 interaction, anti-ORF50 Ab was used in immunoprecipitations with untreated and TPA-treated BCBL-1 cell extracts, and ORF50 protein was detected by Western blotting. ORF50 protein was readily immunoprecipitated from TPA-treated cell extracts (Fig. 4a, lane 2) and some was also detected in untreated cell extracts (Fig. 4a
, lane 1). Control pre-immune rabbit serum did not bring down ORF50 (Fig. 4a
, lanes 3 and 4). ORF50 protein was present in input TPA-treated cell extracts and a small quantity was also in untreated extracts (Fig. 4a
, lanes 5 and 6) due to spontaneous viral reactivation, as discussed above. The presence of ORF57 in immunoprecipitates obtained with anti-ORF50 Ab could not be shown by Western blotting with anti-ORF57 (GH) Ab as the IgG heavy chain of the available anti-ORF50 and ORF57 rabbit Abs is of similar size to ORF57 protein and gave a strong masking signal on Western blots (data not shown). The rabbit heavy chain IgG that masks the ORF57 protein band can be seen in the Western blot obtained with anti-ORF50 Ab (Fig. 4a
, lanes 14).
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To confirm the specificity of the anti-ORF57 Ab, an immunoprecipitation assay was performed using 35S-labelled ORF57 protein synthesized in vitro. Autoradiography showed that 35S-labelled ORF57 protein (Fig. 4c, input, lane 3) was immunoprecipitated with anti-ORF57 (GH) Ab (Fig. 4c
, lane 1) but not with control pre-immune rabbit serum (Fig. 4c
, lane 2). A control unrelated protein, 35S-labelled luciferase, which is of similar size to ORF57, was not immunoprecipitated with anti-ORF57 (GH) Ab (Fig. 4c
, lane 4).
ORF57 and ORF50 proteins interact in GST pull-down assays
To verify the physical interaction between ORF57 and ORF50 proteins, glutathione beads carrying either recombinant GSTORF57 or GST alone, purified from E. coli (strain BL21 cells), were assayed for binding to ORF50 protein present in extracts of KSHV-infected BCBL-1 cells. Coomassie blue staining of the GSTORF57 fusion protein and GST protein showed a detectable amount of full-length GSTORF57 fusion protein plus smaller truncation products and a single GST band (Fig. 5a, lanes 2 and 3). Bound proteins were separated by SDS-PAGE and analysed by Western blotting with anti-ORF50 Ab. This experiment showed that GSTORF57 pulled down ORF50 from TPA-treated, but not from untreated, BCBL-1 extracts (Fig. 5b
, compare lanes 3 and 4). Negative control GST alone showed no interaction with ORF50 protein from the cell extracts (Fig. 5b
, lanes 1 and 2). The existence of multiple forms of ORF50 protein in BCBL-1 cells (Fig. 5b
, lanes 5 and 6) has been reported (Wang et al., 2003a
) and is due in part to extensive post-translational modification (Lukac et al., 1999
). Full-length ORF57 protein interacted with ORF50 protein in GST pull-down assays. To map the interacting ORF57 domain(s), ORF57 deletion mutants (see Fig. 1b
) synthesized by in vitro translation and labelled with [35S]methionine were tested for their abilities to interact with GSTORF50 (FL, 1691): ORF57 (FL) synthesized in vitro from pcDNA4-ORF57 gave two truncation products. Bound proteins were separated by SDS-PAGE and analysed by autoradiography. A region of ORF57 between aa 17 and 215 was involved in the binding to ORF50 protein (Fig. 5c
). ORF57 (FL) (aa 1455) and ORF57 deletion mutants containing aa 17455 and 1215 interacted with GSTORF50 (Fig. 5d
, lanes 57). However, ORF57 aa 181328, aa 329455 and aa 387455 failed to interact with ORF50 protein (Fig. 5d
lanes 8, 3 and 4). A control unrelated protein, 35S-labelled luciferase, was not pulled down with GSTORF50 (Fig. 5d
, lane 2), and GST alone showed no interaction with 35S-labelled ORF57 (FL) protein (Fig. 5c
, lane 1).
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DISCUSSION |
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In the present study, the contribution of the ORF57 protein to the transcriptional activity from the ORF50 promoter in the absence of the downstream ORF50 coding region was assessed independently of its possible effects on ORF50 RNA sequences. In 293 cells, ORF57 protein alone activated expression from the ORF50 promoter independently of B cell-specific factors and other virus-specific factors. The ORF57 C-terminal region, comprising aa 329455, stimulated expression from the ORF50 promoter as efficiently as full-length protein, whereas ORF57 aa 387455 showed much reduced reporter gene activity. However, the N terminus of ORF57 and not the C terminus was shown to interact with ORF50, indicating that activation by the ORF57 C terminus may be via a cellular factor. Interestingly, KSHV ORF57, unlike its homologues in other herpesviruses, contains a putative leucine zipper motif located between aa 343 and 364 (our unpublished observations; Gupta et al., 2000) with a possible role in protein dimerization, and KSHV ORF57 is capable of interacting with itself via the C terminus (data not shown). Repressing and enhancing functions have both been shown to map to the C-terminal regions of HVS ORF57 and the herpes simplex virus 1 (HSV-1) ICP27 proteins (McMahan & Schaffer, 1990
; Goodwin et al., 2000
). These regions are required to modulate viral gene expression very early in HSV-1 infection (McMahan & Schaffer, 1990
).
When ORF57 and ORF50 proteins were co-expressed, activity from the ORF50 promoter was further upregulated by 13-fold compared with the 4-fold level obtained with ORF50 alone. Thus, ORF57 augments the ORF50-inducible effect on its own promoter and these two proteins act co-operatively to promote expression from the ORF50 promoter. The synergistic effects of ORF57 and ORF50 protein co-expression, previously observed with viral early promoters, used ORF50 expressed under the control of the HCMV promoter (Kirshner et al., 2000), which is unaffected by ORF57 (Gupta et al., 2000
; Kirshner et al., 2000
). The synergy was attributed to a post-translational enhancement by ORF57 of ORF50 transcriptional activity (Kirshner et al., 2000
).
We have shown by co-immunoprecipitation and pull-down assays that ORF57 and ORF50 proteins are present in the same complex and therefore interact physically. To ensure the biological relevance of this interaction, studies were performed with naturally infected BCBL-1 cells, in which these two proteins are expressed during the course of KSHV reactivation following TPA treatment. Kirshner et al. (2000) were unable to detect an interaction between the products of the ORF57 and ORF50 genes (Kirshner et al., 2000
) but no experimental details were provided. A possible explanation could reflect the fact that our binding assays used recently generated Abs specific to viral ORF57 and ORF50 proteins, and that studies were performed in KSHV-infected TPA-treated BCBL-1 cells, where other viral proteins are also expressed. A ChIP assay using ORF50 promoter region primers revealed that ORF50 promoter sequences were indeed preferentially associated with immunoprecipitated chromatin using both anti-ORF50 and anti-ORF57 Abs. This result is fully consistent with both an in vivo physical association between ORF57 and ORF50 and a potential role for ORF57 at the transcriptional level, although a post-transcriptional action could also explain the presence of ORF57 in close association with active promoter-associated complexes. In the case of the ORF50 promoter, ORF50 itself may not bind directly to the promoter DNA sequences, but rather be present in a complex with enhanced levels of both the cellular C/EBP
and cJUN/cFOS proteins (Wang et al., 2003a
, b
, 2004
), and ORF57 might also be a part of either these or other similar large complexes.
This is the first demonstration of an interaction between these two immediate-early regulatory proteins in the gammaherpesviruses. However, in alphaherpesviruses, the HSV-1 counterpart of ORF57, ICP27, complexes with the ICP4 protein, the HSV-1 counterpart of ORF50 (Panagiotidis et al., 1997). ICP27 affects intracellular localization (Zhu & Schaffer, 1995
) and electrophoretic mobility of ICP4 (Rice & Knipe, 1988
; Su & Knipe, 1989
). ICP27 acts at transcriptional (Jean et al., 2001
) and post-transcriptional levels, influencing pre-mRNA processing and promoting export of viral RNAs (Sandri-Goldin & Mendoza, 1992
; Koffa et al., 2001
). Recently, ICP27 protein has been demonstrated to associate with RNA polymerase II (Zhou & Knipe, 2002
). Similarly, varicella-zoster virus proteins IE62 and IE4, counterparts of KSHV ORF50 and ORF57, interact with each other (Spengler et al., 2000
). A functional and physical interaction between these two protein counterparts, which have transcriptional and post-transcriptional actions, could be a general characteristic of herpesviruses and it will be interesting to look for it in the betaherpesviruses. The association of herpesviral regulatory proteins that have transcriptional and post-transcriptional actions reflects the close interconnection of transcription and pre-mRNA processing steps with each other (reviewed by Proudfoot et al., 2002
; Reed & Hurt, 2002
) and facilitates cross-regulation of protein activities.
Studies of cultured cells have indicated that inefficient spontaneous lytic reactivation is a likely explanation for the narrow host range of KSHV and, as ORF50 protein is sufficient to overcome this block, control of its expression may be the key determinant of KSHV spread in vivo (Bechtel et al., 2003). Activation of ORF50 promoter activity could be one mechanism by which KSHV lytic gene expression is regulated by the ORF50 and ORF57 proteins together, since basal transcription from the ORF50 promoter is upregulated by these two proteins in reporter gene assays. In the present study, new insights into the co-ordinated action of these two key regulatory proteins have been demonstrated in which augmentation of ORF50 activity by ORF57 protein, and vice versa, would facilitate the cascade of KSHV lytic viral gene expression, overcoming the inefficiency of spontaneous lytic viral reactivation and breaking latency. The co-operative effect between ORF57 and ORF50 proteins observed here could be due to one or more transcriptional, post-transcriptional, translational or post-translational effects.
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ACKNOWLEDGEMENTS |
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Received 5 November 2003;
accepted 6 May 2004.