University of Edinburgh, Centre for Infectious Diseases, 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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KSHV is closely related to three other herpesviruses with oncogenic potential; herpesvirus saimiri (HVS), murine gammaherpesvirus (MHV-68) and, more distantly, to EpsteinBarr virus (EBV). The complete nucleotide sequence of KSHV DNA has revealed several genes which have probably been captured from the host cell during virus evolution, and whose products could also play a role in cellular transformation and tumour induction (Neipel et al., 1997; Russo et al., 1996
). The three genes encoded by open reading frames (ORFs) K13, 72 and 73 [vFLIP (Fas-associated death domain-like IL-1
-converting enzyme-inhibitory protein) vCyclin and LANA] are transcribed from a common transcription start site in cell lines latently infected with KSHV. The resulting transcript is spliced to yield a 5·32 kb message encoding LANA, vCyclin, vFLIP and a 1·7 kb bicistronic message encoding vCyclin and vFLIP (Dittmer et al., 1998
; Talbot et al., 1999
).
The observation that a bicistronic transcript (Talbot et al., 1999) encodes vCyclin and vFLIP led to the investigation of the mechanism of translation of the vFLIP ORF. We (Bieleski & Talbot, 2001
) and others (Grundhoff & Ganem, 2001
; Low et al., 2001
) were able to identify a novel internal ribosome entry site (IRES) within the latently expressed vCyclin gene that controls the expression of the downstream vFLIP ORF. This IRES is the first such element to be identified in a DNA virus. Recently, an IRES element has been described in the untranslated region of the EpsteinBarr nuclear antigen-1 (EBNA1) gene, which may contribute to the regulation of latent gene expression (Isaksson et al., 2003
).
IRES elements were first identified in the 5' untranslated regions (UTR) of picornaviruses and are essential for the cap-independent translation of the viral polyprotein (Jang et al., 1988; Pelletier & Sonenberg, 1988
). More recently IRES elements have been characterized in several cellular genes which encode growth factors (FGF-2, VEGF) (Stein et al., 1998
; Vagner et al., 1995
), proto-oncogenes (c-myc) (Nanbru et al., 1997
) and an inhibitor of apoptosis (XIAP) (Holcik & Korneluk, 2000
). IRES-dependent translation of these mRNAs may be essential for the survival and proliferation of cells under stressful conditions (Holcik et al., 2000
). IRES elements in two cellular mRNAs [encoding ornithine decarboxlyase (Cornelis et al., 2000
) and PITSLRE protein kinase (Pyronnet et al., 2000
)] have been identified, and are regulated in a cell cycle-dependent manner. These data reveal a novel role for IRES elements in the translational regulation of protein expression during cell cycle progression. The IRES element that we have identified potentially controls the expression of a virus-encoded anti-apoptotic protein, vFLIP (Thome et al., 1997
), which is intimately linked to the expression of a cell growth promoting protein, vCyclin (Cesarman et al., 1996
; Godden-Kent et al., 1997
).
This paper investigates sequence elements within the KSHV IRES essential for efficient translation of the downstream ORF. In addition the cell type-specific activity of the IRES and cell-specific protein factors interacting with the IRES are investigated.
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METHODS |
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Plasmids.
The plasmids pdLUC and pdLUC-SL were constructed as described previously (Bieleski & Talbot, 2001). The IRES sequence from encephalomyocarditis virus (EMCV) or fragments of KSHV vCyclin/vFLIP were cloned into the SmaINcoI or XhoINcoI sites of pdLUC (Fig. 1
a). The following primers were used to PCR amplify specific IRES sequences:
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Primer 2: GCATCTCGAGGCTGGGGGGCTCCCAAC
Primer 3: GCATCCATGGCAACTAAGGCTTTTGTAATCAG
Primer 4: GCATCCATGGAGTCTTTGGGTCAACTAAGGC
Complementary oligonucleotides (TCGAGACGGACGTCACTTCCTTCTTGTTACTTAAATTC and CATGGAATTTAAGTAACAAGAAGGAAGTGACGTCCGTC) were annealed and cloned directly in the XhoINcoI sites of pdLUC and pdLUC-SL to yield the PPT-encoding plasmid.
The KSHV IRES [nucleotides 123206122973, GenBank accession no. U75698 (Russo et al., 1996)] was cloned into the pSP64Poly(A) vector (Promega) for use in the pull-down assay. Primers GTACAAGCTTCCGCGGCAGACTCCTTTTCCC and GTACGAGCTCGCTGATAATAGAGGCGGGCAAT (sense orientation) or GTACGAGCTCCCGCGGCAGACTCCTTTTCCC and GTACAAGCTTGCTGATAATAGAGGCGGGCAAT (antisense orientation) were used to amplify the KSHV IRES by PCR. The DNA was then inserted into the HindIIISacI sites of the vector.
Transfection of cells.
BCP-1 cells (1x105 cells per well), HEK293 (5x104 cells per well) were seeded in 24-well trays and incubated overnight. The cells were infected with vTF7-3 (Fuerst et al., 1986), a recombinant vaccinia virus expressing T7 RNA polymerase, at 5 p.f.u. per cell in 200 µl serum free medium (OptiMEM; Gibco-BRL) for 60 min at 37 °C. The inoculum was removed and the cells washed once with OptiMEM. The cells were then transfected with 0·5 µg of linearized (AflII and NotI) plasmid DNA and 1·5 µl Transfast transfection reagent per well according to the manufacturer's instructions (Promega). After incubation at 37 °C for 60 min, 1 ml of growth medium was added to the wells. The cells were assayed for luciferase activity 24 h later as described below.
Dual luciferase assays.
Transfected cells were washed twice in PBS, and then lysed by addition of 200 µl of passive lysis buffer (PLB, Promega). After incubation for 15 min at room temperature the cell lysates were transferred to Eppendorf tubes and snap-frozen on dry ice. The lysates were then thawed, vortexed for 1 min and the cell debris removed by spinning at 10 000 r.p.m. for 1 min. The activity of Renilla and firefly luciferase was assayed using the dual luciferase system as described by the manufacturer (Promega). Luciferase activities were measured using a Labsystems benchtop luminometer and the ratio of firefly luciferase to Renilla luciferase activity was calculated and used as a measure of IRES function.
Electrophoretic mobility shift assays (EMSAs).
RNA was transcribed in vitro from 0·2 µg of linearized plasmid using T3 RNA polymerase and labelled internally with [-32P]UTP according to manufacturer's instructions (Life Science). Whole-cell lysate was prepared from BCP-1 or HEK293 cells by sonication for 15 min at 4 °C in binding buffer [20 mM HEPES/KOH pH 7·5, 50 mM KCl, 10 mM MgCl2, 0·01 % (v/v) NP40, 5 % (v/v) glycerol], and the cellular debris was removed by centrifugation at 10 000 g for 5 min. The binding reaction was carried out in binding buffer containing 25 000 c.p.m. of labelled RNA, 6 µg protein, 1 unit RNasin, and 0·05 µg poly(dIdT), in a total volume of 20 µl in the presence or absence of 10-fold excess of unlabelled competitor transcripts. After 15 min incubation at 20 °C, the samples were loaded on a 5 % (w/v) non-denaturing polyacrylamide gel containing 0·5x TBE. The gel was run in 0·5x TBE for 1 h at 30 mA before exposure to x-ray film (Hyperfilm) at -80 °C with an intensifying screen.
Preparation of S10 cell extract.
Cells (1x108) were centrifuged and washed three times with isotonic buffer (35 mM HEPES pH 7·4, 146 mM NaCl, 11 mM glucose), resuspended in 2 vols of hypotonic buffer (20 mM HEPES pH 7·4, 10 mM KCl, 1·5 mM magnesium acetate, 1 mM DTT) and incubated on ice for 10 min. The cells were disrupted with 25 strokes of a Dounce homogenizer (on ice), before addition of 0·1 vols of 10x buffer (0·2 M HEPES pH 7·4, 1·2 M potassium acetate, 40 mM magnesium acetate, 50 mM DTT). The nuclei were removed by centrifugation at 2000 r.p.m. for 10 min at 4 °C, followed by addition of CaCl2 (1 mM) and 75 units of S7 nuclease (Roche) per ml of supernatant. After incubation at 20 °C for 15 min the S7 nuclease was inactivated by adding EGTA to 2 mM. The S10 supernatant was centrifuged at 10 000 r.p.m. at 4 °C for 15 min before freezing in aliquots at -80 °C.
RNAprotein pull-down assays.
Plasmids derived from the pSP64Poly(A) vector were linearized with EcoRI, and then used as templates for transcription reactions. RNA transcripts [containing a 3' poly(A) tail of 30 residues] were produced and purified according to the manufacturer's instructions (Ambion; SP6 megascript). The KSHV vCyclin IRES RNA (sense or antisense) was captured onto oligo(dT) Dynabeads (Dynal, 0·5 ml) as described previously (Stassinopoulos & Belsham, 2001). The immobilized RNA transcripts were then incubated with BCP-1 cell S10 extract at 4 °C for 60 min on a rotating wheel. The magnetic beads were captured and the depleted S10 was removed. The beadsRNAprotein complex was washed twice in binding buffer, resuspended in SDS sample buffer, and incubated at 4 °C for 10 min. These samples were analysed by SDS-PAGE and Western blot analysis. The anti-PTB polyclonal antibody was a gift from R. J. Jackson (University of Cambridge, UK) (Hunt & Jackson, 1999
; Mitchell et al., 2001
).
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RESULTS |
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Cell-type specificity of KSHV IRES activity
We noted previously that the IRES activity was high in the PEL cell line BCP-1 (latently infected with KSHV) but that there was little or no activity in the cell lines HEK293, HeLa or KSIMM (Bieleski & Talbot, 2001). This suggests that cell-specific and/or KSHV-specific factors play an important role in the modulation of IRES activity. EMSA is a technique used to study the interaction of proteins with specific nucleic acid targets (DNA or RNA). Radiolabelled RNA encompassing the IRES was produced by in vitro transcription using T7 RNA polymerase mixed with protein (whole-cell lysate) and then electrophoresed through a non-denaturing polyacrylamide gel. Any RNAprotein complexes that are formed run with a slower mobility through the gel in comparison with non-complexed RNA. We have used this system to investigate the possibility of proteins interacting with the KSHV IRES. As shown in Fig. 2
, several specific RNAprotein complexes (indicated by *) are formed when in vitro-transcribed IRES RNA is mixed with crude cell lysate from BCP-1 cells but not with cell lysate from HEK293 cells. The specificity of these RNAprotein interactions was confirmed by the fact that excess unlabelled IRES RNA successfully competed out these complexes. These data confirm the presence of specific protein factors present in the KSHV-positive BCP-1 cell line that may be essential for the activity of the IRES.
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DISCUSSION |
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We have used an EMSA to define IRESprotein complexes within permissive (BCP-1) and non-permissive cells (HEK293). Four distinct IRESprotein complexes were observed with BCP-1 lysate, whereas no distinct species were identified in HEK293 cells. In order to identify potential interacting proteins we used an in vitro binding assay using poly(A)-tailed IRES RNA and oligo(dT) magnetic beads. Proteins interacting with the IRES RNA were enriched from BCP-1 S10 extract and analysed by SDS-PAGE and Western blot. Using this technique we were able to show that the cellular PTB (hnRNP-I) selectively bound to KSHV IRES RNA, but not to the antisense IRES RNA used as a control. Clearly, PTB is not the sole determinant of IRES activity since it is expressed in a wide variety of cell types including HEK293 cells in which the KSHV IRES is non-functional. The potential interaction of PTB with the PPT of the IRES may provide a framework for the binding of further cellular and/or viral factors. We tested whether the expression of PTB in an in vitro transcription/translation system (rabbit reticulocyte lysate) would enhance the activity of the IRES, but found that it had no effect (data not shown). This also supports the idea that multiple protein factors are required for efficient IRES activity.
Some viral IRESs, e.g. the EMCV IRES, do not appear to require proteins other than canonical translation initiation factors for function (Pestova et al., 1996), while others require an additional complex set of factors for activity. Such factors include PTB, which binds specifically to several viral IRESs, although the absolute requirement of viral IRESs for this factor differs. For example, PTB stimulates the initiation of translation by internal ribosome entry from hepatitis C and A virus RNA in vivo (Gosert et al., 2000
) and from the human rhinovirus (HRV) and poliovirus IRESs in vitro (Hunt & Jackson, 1999
) but is not necessary for the activity of wild-type EMCV (Kaminski & Jackson, 1998
). PTB is a cellular protein known to be involved in splicing and branch point selection (Grossman et al., 1998
). It has been shown that the IRES element controlling the expression of the cellular gene Apaf-1, involved in the apoptotic cascade, requires both PTB and, upstream of N-ras (unr), two cellular RNA-binding proteins previously identified to be required for rhinovirus IRES activity (Mitchell et al., 2001
). This study showed that PTB binding to the Apaf-1 IRES occurred only if unr was present.
We have shown that a PPT is essential for the activity of the KSHV IRES and that PTB interacts with KSHV IRES RNA in vitro. Additional IRES RNAprotein complexes were observed using an EMSA that are yet to be identified. Further investigations will be required to determine other binding and regulatory components necessary for the correct functioning of the KSHV IRES.
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ACKNOWLEDGEMENTS |
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Received 20 October 2003;
accepted 5 December 2003.