Laboratory for Clinical and Molecular Virology, The University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK1
Author for correspondence: James Stewart. Fax +44 131 650 6511. e-mail james.stewart{at}ed.ac.uk
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Abstract |
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Introduction |
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The G protein-coupled receptor (GPCR) super-family of seven transmembrane spanning receptors comprises over 250 members. They all share characteristic structural features that most likely reflect their common mechanism of action (Strader et al., 1995 ). Mutations in GPCRs resulting in constitutive signalling activity have been associated with human disease (Parma et al., 1993
; Schipani et al., 1995
). In addition, a number of cellular GPCRs including the muscarinic acetylcholine receptor (Gutkind et al., 1991
) and the serotonin 1c receptor have been shown to be oncogenic when expressed ectopically (Julius et al., 1989
).
Several gamma-2 herpesviruses including herpesvirus saimiri(HVS) and KSHV contain an open reading frame (ORF74) encoding a functional GPCR that has significant sequence identity (32 and 30% respectively) with CXCR2, the high-affinity interleukin 8 receptor (Ahuja & Murphy, 1993 ; Arvanitakis et al., 1997
). In the case of KSHV, the GPCR binds a wide range of chemokines and has constitutive (agonist-independent) activity. It also exhibits transforming activity in 3T3 cells and is tumorigenic in nude mice (Arvanitakis et al., 1997
; Bais et al., 1998
). Thus, it has been proposed that the KSHV GPCR is a viral oncogene that can exploit cell-signalling pathways to induce cell proliferation that may be important in viral pathogenesis and oncogenesis.
MHV-68 encodes a number of cellular homologues that are postulated to be involved in immune evasion and virus persistence (Virgin et al., 1997 ). A cluster of such genes is encoded at the right-hand end of the genome. The products of two of these ORFs [homologues of cyclin D (ORF72), Bcl-2 (M11)] have been shown to be functional (Roy et al., 2000
; van Dyk et al., 1999
; Wang et al., 1999
). A third gene in this cluster is ORF74. Like the analogous genes in HVS and KSHV, MHV-68 ORF74 shares significant (28%) sequence similarity with CXCR2.
The aim of this study was to characterize the expression and activity of the MHV-68 ORF74 gene product. We describe the transcription pattern of the gene and the expression pattern of the protein in cells. We also show that the ORF74 gene product is a viral oncogene with transforming activity in vitro.
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Methods |
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Mice.
Female BALB/c mice were obtained from Bantin and Kingman, UK. Mice were infected at 4 weeks of age intranasally with 4x105 p.f.u. MHV-68 under halothane anaesthesia.
RNA preparation from MHV-68 infection in vitro.
BHK cells (8x107) were infected at 5 p.f.u. per cell. RNA specific for the immediate early stage of infection was harvested at 8 h p.i. from cells that were incubated with 100 µg/ml cycloheximide (CHX) for 30 min prior to adsorption of the virus and for the rest of the infection. Early RNA was isolated at 24 h p.i. from cells that were pre-treated for 2 h with 100 µg/ml of phosphonoacetic acid (PAA) prior to and during infection. Late RNA was derived from infected cells maintained without treatment for 24 h. At appropriate time-points, monolayers were washed and harvested by scraping. Cells were pelleted and total RNA purified as described previously (Stewart et al., 1994 ). Poly(A)+ RNA was prepared from total RNA using an mRNA Purification kit (Amersham Pharmacia).
Northern analysis.
Poly(A)+ RNA (10 µg/lane) was electrophoresed through formaldehyde gels, transferred to nylon membranes and probed with [-32P]dCTP-labelled DNA probes as described previously (Stewart et al., 1994
). Molecular mass determinations were made using an RNA ladder (Life Technologies).
Preparation of RNA from MHV-68-infected mouse tissues.
BALB/c mice were infected intranasally with 4x105 p.f.u. of MHV-68 as previously described (Sunil-Chandra et al., 1992a ). Three mice were sacrificed at 2 weeks and 10 months p.i. and lung and spleens were removed and snap-frozen at -70 °C. A portion of tissue from each organ was removed and total RNA prepared using an RNeasy kit (Qiagen).
RTPCR analysis.
Total RNA (2 µg) was treated with 10 U of RNase-free DNase I (Amersham Pharmacia) at 37 °C for 30 min. cDNA was synthesized at 42 °C with 200 U of reverse transcriptase (Superscript II, Life Technologies) in the presence of 100 pM random primers (Roche) and 100 nM dNTPs (Amersham Pharmacia). A portion (1·5 µl) of cDNA was subject to PCR with primer sets specific for the MHV-68 ORFs vBcl-2 (M11), ORF74, ORF50 and ORF57. Separate PCR reactions for murine -actin were done for each cDNA sample. The sequences of primer sets used and the predicted sizes of PCR products are given in Table 1
. Primers for ORF50 and ORF57 were as described by Virgin et al. (1999)
. PCR reactions were carried out with 1 U per reaction of Taq DNA polymerase (Life Technologies) in the presence of 100 nM dNTP, 2·0 mM MgCl2 and 50 pmol of each primer in a total reaction volume of 50 µl. Cycling parameters were the same for each primer set used and were: 96 °C, 45 s; 45 °C, 60 s; 72 °C, 120 s for 35 cycles with a final extension at 72 °C for 7 min. Electrophoresed PCR products were transferred to Hybond N+ (Amersham Pharmacia) in 0·4 M NaOH. Blots were hybridized using standard techniques with DNA probes random prime-labelled with [
-32P] dCTP. Viral gene-specific DNA probes for labelling were obtained from cloned viral DNA fragments that were cleaved from plasmid vectors and purified prior to labelling. Murine
-actin blots were hybridized with a full-length chick
-actin probe (Cleveland et al., 1980
). A series of PCR controls was incorporated. To control for the carry over of viral DNA, PCR was performed on cDNA synthesized in the absence of reverse transcriptase. Controls for false positive signals included PCR analysis of cDNA from spleen and lung tissue from uninfected mice. Contamination was also controlled for with PCR performed in the absence of cDNA. These control reactions were negative in all cases.
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DNA electroporation.
Cells (2x106) were mixed with 20 µg of linearized plasmid DNA and electroporated using a double-pulse setting (HV=600 V, 25 µF, 99 ; LV=260 V, 1500 µF, 329
; 0·1 s inter-pulse delay) on an Easyject electroporator (EquiBio). The efficiency of transfection was estimated using a control plasmid expressing GFP. Transiently transfected cells were generally harvested 24 h p.i.
Immunofluorescence.
Indirect immunofluorescence was carried out as described by Stewart et al. (1994) . The primary antibody was rat anti-haemagglutinin (clone 3F10, Roche) used at a dilution of 1:1000. The secondary antibody was FITC-labelled goat anti-rat (Pharmingen) used at a dilution of 1:250 After staining the cells were incubated with PBS containing 1 µg/ml propidium iodide and 10 µg/ml RNaseA for 30 min at 37 °C in darkness. Cells were then washed twice in PBS and mounted in Citifluor (UKC). Slides were examined using a Leica TCSNT confocal microscope.
Focus formation assay.
Stably transfected cell lines were grown to confluence in six-well plates. The medium (DMEM+10% foetal calf serum) was replaced on alternate days for 34 weeks until cellular foci were observed under the light microscope.
Growth in soft agar.
Soft agar assays were performed as described by Baichwal & Sugden (1989) . Cellular foci (diameter >50 µm) were counted with the aid of a calibrated graticule and a light microscope.
Prediction of protein structure.
Structural motifs were predicted from protein sequence using the following programs (which can be found at the listed web sites). The position of transmembrane helices and the orientation of the molecule in the membrane was predicted using TMpred (Hofmann & Stoffel, 1993 ), which is found at http://www.ch.embnet.org/software/TMPREDform.htm. The presence of common motifs was determined using Scan Prosite, which is found at http://expasy.cbr.nrc.ca/tools/scnpsit1.html. The alignment of multiple proteins was performed using the Blosum scoring matrix with the ClustalW program (Thompson et al., 1994
), which is found at http://www.ch.embnet.org/software/Clustalw.htm. The prediction of phosphorylation site was made using the program NetPhos (Blom et al., 1999
), which is found at http://www.cbs.dtu.dk/services/NetPhos/.
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Results |
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The size of all transcripts exceeded that of the 1·0 kb GPCR coding region. It was therefore possible that they represented polycistronic messages. The closest ORF on the same strand of the MHV-68 genome is M11, which encodes the viral Bcl-2 homologue and lies upstream (see Fig. 3C). A Northern blot containing mRNA from cells 24 h p.i. was therefore screened using a probe specific for vBcl-2. As shown in Fig. 2
, messages of 8·7, 6·6, 4·4, 3·4, 2·4 and 1·4 kb were revealed. The blot had to be exposed to film for a similar length of time as that probed with GPCR and therefore the vBcl-2-specific transcripts were also low abundance. Thus, a subset comprising the larger four of these mRNAs were of a similar size to the GPCR-specific mRNAs. It was possible, therefore, that these mRNAs were bicistronic and contained vBcl-2 and GPCR.
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Thus, as shown in Fig. 3(C), at least one of the forms of GPCR mRNA is unspliced, bicistronic, and also contains the vBcl-2 gene.
Expression of GPCR in vivo
To confirm previous data on MHV GPCR transcription (Virgin et al., 1999 ) and to study the expression during persistent infection of lungs, we performed RTPCR on lung and spleen tissue from mice intranasally infected with MHV-68. We analysed expression at 2 weeks p.i. where acutely productive infection is resolving in the lung, persistence is establishing and latently infected cells are present (Sunil-Chandra et al., 1992a
, b
). Analysis was also performed at 10 months p.i. where a steady state of persistence and latency exists. We used primers specific for ORF50, ORF57, GPCR, vBcl-2 and
-actin. ORFs 50 and 57 are only transcribed during the productive cycle (Liu et al., 2000
; Mackett et al., 1997
; Wu et al., 2000
). The sensitivity of the PCRs was checked by limiting dilution of cloned templates and was one copy in all cases. Control amplifications performed without reverse transcriptase (no RT) were negative. As shown in Fig. 4
,
-actin transcription was detected in all samples. Expression of ORF50 and ORF57 was detected in both the spleen and the lung 2 weeks p.i. in all three mice, but by 10 months expression was negative. This indicated that productive viral gene expression was still occurring at 2 weeks p.i. but by 10 months p.i. was undetectable. GPCR and vBcl-2 were expressed in the spleen and lung of all mice at 2 weeks p.i. By 10 months p.i., GPCR and vBcl-2 were expressed in the spleen of one mouse but were found in all lungs tested.
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Expression pattern of the GPCR protein in cells
No antibodies to the MHV GPCR are available. To study GPCR protein we utilized a transient transfection system with a gene tagged at the 3' end with a sequence encoding three copies of an influenza virus haemagglutinin (HA) epitope. The tagged GPCR gene was inserted into the expression vector, pVR1255 [pVR1255/GPCR-(HA)3]. Human 293 cells were transfected with expression constructs and protein expression was detected with rat monoclonal anti-HA; the cell nuclei were counterstained with propidium iodide (PI) and fluorescence then visualized by confocal microscopy. Cells transfected with empty vector alone showed no positive staining with anti-HA and cells transfected with a construct known to express an HA-tagged version of J exhibited characteristic positive nuclear staining (not shown). As shown in Fig. 5
, cells transfected with pVR1255/GPCR-(HA)3 showed positive staining in patches with anti-HA. This staining was quite separate from the intra-nuclear PI staining and, when the two images were overlaid, it could be seen that the pattern of anti-HA staining was consistent with expression at the plasma membrane. Thus, the pattern of MHV GPCR expression is in keeping with the fact that it is homologous to growth factor receptors that are found at the cell surface.
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Discussion |
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In contrast to a previous report (Virgin et al., 1999 ), we were able to detect GPCR and vBcl-2 mRNAs by Northern analysis. However, the level of expression was low, bordering on the level of detection (Fig. 2
). This indicates a high degree of transcriptional control. There were four GPCR-specific messages (3·4, 4·4, 6·6 and 8·7 kb). Similar sized messages were detected by a vBcl-2 probe suggesting co-transcription of the two ORFs. This interpretation is strengthened by the fact that RTPCR using primers specific for the 5' end of vBcl-2 and the 3' end of GPCR demonstrated an unspliced bicistronic transcript. 3'-RACE data showed that GPCR-containing messages terminate at 106637. The combined length of the overlapping RTPCR and 3'-RACE products is approximately 3·2 kb. Thus, it is probable that the 3·4 kb transcript corresponds to this with additional 5' sequences and a poly(A) tail (Fig. 3C
). Analysis of potential promoter elements using the program Promoter 2.0 (Knudsen, 1999
) produced strong predictions upstream of the vBcl-2 gene at around positions 102000 and 103000. No such elements were predicted directly upstream of the GPCR. Put together, these data are consistent with the hypothesis that the 4·4 and 3·4 kb mRNAs are bicistronic, originate upstream of vBcl-2 at around nucleotides 102000 and 103000 respectively and terminate downstream of the GPCR.
The vBcl-2 probe hybridized to two additional unshared transcripts (1·4 and 2·4 kb). There is a non-canonical polyadenylation signal (AGTAAA) at position 104216, downstream of the vBcl-2 but upstream of the GPCR gene. It has been shown experimentally that an identical signal sequence is used by the MHV-68 latency-associated M2 gene as a polyadenylation signal (Husain et al., 1999 ). It seems likely, therefore, that the two smaller vBcl-2 specific mRNAs terminate just downstream of this. This signal is clearly ignored in the production of the longer bicistronic transcripts. The size of the vBcl-2-specific transcripts and the distance between the two poly(A) signal sequences (2·4 kb) makes it possible for them to have the same starting points as the 3·4 and 4·4 kb transcripts. To date, we have been unable to locate the precise 5' end of the mRNAs by using 5'-RACE. Due to the complex nature of the transcription across this region, precise definition of the composition of these mRNA demands the cloning and sequencing of their cDNAs. However, as yet no cDNAs corresponding to the GPCR or vBcl-2 have been found in a library from which both lytic and latent genes have been isolated (J. Sample, personal communication).
The observation that the vBcl-2 ORF is contained on the GPCR mRNAs is interesting. This is therefore similar to KSHV where the homologous GPCR gene is expressed as the 3' ORF in a bicistronic transcript, although in this case the upstream gene is K14 (v-Ox-2) (Kirshner et al., 1999 ; Talbot et al., 1999
). The co-expression of MHV-68 vBcl-2 and GPCR backs up the finding of transcription of both ORFs during persistence in the mouse. This confirms the results of previous work by Virgin et al. (1999)
, who described expression of both in the lymphoid compartment of B cell-deficient mice. Our results therefore extend this analysis to intact mice and show that co-expression of these genes occurs most consistently in the lung, an organ that we have previously shown to be important for MHV-68 persistence (Stewart et al., 1998
). The fact that both mRNAs are found during persistence in the absence of lytic cycle genes (ORF50 and ORF57) strongly implies that they are expressed and function in latently infected cells. Definitive confirmation of this and the definition of the cell type involved (epithelial or lymphoid) will rely upon the direct visualization of both transcripts in the same cells. However, to date we have been unable to detect either GCPR or vBcl-2 by in situ hybridization in vitro or in vivo due to the low abundance of the transcripts. The fact that MHV-68 vBcl-2 and GPCR are both expressed during lytic infection and during latency suggests either that the promoter elements are active during both phases of the life-cycle or that there are separate promoters. There is a precedent for the former in that both EBV EBNA 1 and LMP transcripts are produced during latency but are also induced during lytic replication (Torii et al., 1998
; Zetterberg et al., 1999
).
Previous work has shown that the KSHV GPCR is oncogenic in the sense that it is able to induce transformation of mouse 3T3 cells in vitro (Arvanitakis et al., 1997 ; Bais et al., 1998
). Our data therefore show that the MHV GPCR is not only similar in sequence but also in transforming activity to the KSHV homologue. The transforming ability of KSHV GPCR is thought to be a function of constitutive signalling in the absence of ligand (Arvanitakis et al., 1997
). Light has recently been shed on the molecular basis of this by the finding that mutation of the aspartate (D) in the highly conserved DRY motif in the second intracellular loop of CXCR2 and histamine H(2) receptor (mimicking KSHV GPCR, see Fig. 1B
) confers constitutive signalling and transforming ability (Alewijnse et al., 2000
; Burger et al., 1999
). The equivalent motif in the MHV-68 GPCR is HRC (Fig. 1B
). Although the DRY motif is highly conserved, C replacing the Y is seen in certain functional GPCRs such as the fMLPR receptor (Boulay et al., 1990
). Thus MHV-68, like KSHV has a mutated form of this motif that may underlie the ability of the protein to confer growth-inducing properties on cells.
The fact that vBcl-2 and GPCR are encoded on the same mRNA suggests that these ORFs may be co-translated and may have coordinated functions in the virus life-cycle. We have shown here that the GPCR can enhance the growth properties of cells and it has been shown that vBcl-2 can protect cells from apoptotic death (Bellows et al., 2000 ; Wang et al., 1999
). Thus it may be envisaged the GPCR functions to activate virus infected cells and that the vBcl-2 acts at the same time to prolong the life of the same cells not only during acute infection but also during persistence in the lung. Considerable sub-pleural lymphoid accumulations are seen after MHV-68 infection (Sunil-Chandra et al., 1992a
). Thus, it is possible that the vBcl-2 and GPCR expression seen in latency by RTPCR is derived from either lymphoid or epithelial cells. Both genes might therefore play a role in the development of the lymphoproliferative disease seen in long-term infected mice (Sunil-Chandra et al., 1994
). The exact function of the GPCR in the virus life-cycle, however, awaits the characterization of a virus with this ORF mutated. Further, the functional similarities between the MHV-68 GPCR and the KSHV homologue make this and future studies particularly pertinent with respect to the modelling the pathogenesis of KSHV-associated disease.
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Acknowledgments |
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Received 8 December 2000;
accepted 29 January 2001.