Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
Correspondence
Philip G. Stevenson
pgs27{at}mole.bio.cam.ac.uk
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ABSTRACT |
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INTRODUCTION |
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How episome maintenance functions in vivo is germane to both combating gammaherpesvirus disease and developing episomal vectors for gene delivery. However, such questions are difficult to address with human gammaherpesviruses due to their strict species tropisms. Consequently, the murine gammaherpesvirus 68 (MHV-68) has become an important experimental tool for understanding in vivo gammaherpesvirus gene functions. Like EBV, MHV-68 persists in memory B cells (Flano et al., 2002; Marques et al., 2003
; Willer & Speck, 2003
). The episome maintenance proteins of MHV-68 and the Kaposi's sarcoma-associated herpesvirus (KSHV) are encoded by ORF73 (Ballestas et al., 1999
; Rainbow et al., 1997
). The MHV-68 ORF73 is transcribed in latency both in vitro and in vivo (Marques et al., 2003
; Martinez-Guzman et al., 2003
; Virgin et al., 1999
), and ORF73-deficient MHV-68 shows a profound latency deficit in vivo (Fowler et al., 2003
; Moorman et al., 2003
). The task now with MHV-68 is to define the molecular details that underlie in vivo ORF73 function. For this we must identify how ORF73 production is controlled and how this control relates to its function.
Latent KSHV in B cell tumour lines transcribes ORF73, ORF72 and ORF71 from a common promoter just upstream of ORF73 (Dittmer et al., 1998; Rainbow et al., 1997
; Sarid et al., 1999
; Talbot et al., 1999
). In contrast, MHV-68 has no ORF71, its ORF16 homologue is sited between ORF72 and ORF73 (Virgin et al., 1997
), and there are consensus polyadenylation signals just 3' of ORF73. In vivo analysis indicates that ORF73 transcription is largely distinct from that of ORF72 (Virgin et al., 1999
). Rightwards of ORF73, MHV-68 lacks a K14 homologue, may not have a K15 homologue, and has three copies of ORF75 rather than one. Otherwise, MHV-68 appears fairly similar to KSHV.
Surprisingly, analyses of MHV-68 latent gene transcription have identified loci rightwards of ORF73 that are not transcribed in KSHV latency, including ORF74 in latently infected peritoneal exudate cells (Virgin et al., 1999) and ORF74, ORF75a and ORF75c in latently infected S11E cells (Martinez-Guzman et al., 2003
). The S11E cell line contains at least one integrated viral genome (Husain et al., 1999
) and may therefore show abnormal patterns of transcription, but ORF74, ORF75a, ORF75b and ORF75c transcripts in 3T3 cells are resistant to inhibition by cycloheximide (Martinez-Guzman et al., 2003
), consistent with latency-associated promoter activity. These results have raised the possibility that MHV-68 latency gene expression is fundamentally different to that of KSHV. However, the splicing patterns of MHV-68 latency transcripts remain largely undefined, and without knowing the structure of each gene it is difficult to interpret definitively the detection of small mRNA segments. In order to understand more about MHV-68 latency, we have mapped the 5' ends of the major ORF73 transcripts and identified the functional cores of the associated promoters.
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METHODS |
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Promoter constructs and CAT ELISA.
The MHV-68 terminal repeats were cloned from the MHV-68 BAC with BglII (117840) and PvuII, which cuts at the left end of the BAC cassette, into the BglII and EcoRV sites of pSV40-ZEO2 (Invitrogen). This plasmid was then digested with PstI and ligated back to itself, leaving one intact repeat unit. This was subcloned as a PflFI/XhoI fragment into the XbaI/SalI sites of pCAT-enhancer (Promega), inserting the CAT gene in ORF73 exon 2 with a single upstream terminal repeat unit (118210119450). We generated a 5' truncation of this construct by digestion with PstI (118700) and HinDIII, which cuts in pCAT-enhancer, blunting with T4 DNA polymerase (New England Biolabs) and self-religation of the plasmid.
CAT was joined to ORF73 exon 1 by first PCR amplifying genomic DNA, co-ordinates 118616118719, including XbaI and HinDIII sites in the respective downstream and upstream primers, and ligating this into the XbaI/HinDIII sites of pCAT-enhancer. A 1·2 kb PstI genomic clone (Efstathiou et al., 1990), comprising the 5' 750 bp of one terminal repeat unit and the 3' 500 bp of the next, was then cloned into this plasmid at the 118700 PstI site, upstream of CAT. This construct was truncated at the 5' end by digestion with HinDIII and either PstI (118700), SacII (119078), Bsu36I (119087), NotI (119105), AscI (119151), SfiI (119348), XcmI (119359) or BbvCI (118616, cuts in the upstream terminal repeat copy). We used MspA1I (118720) to truncate at the 3' end of the SacII clone and PstI (118700) plus XbaI to truncate at the 3' end of the BbvCI clone. We also used BAL-31 exonuclease to digest from the 5' end of the promoter construct after linearization with HinDIII. The cut ends were blunted with T4 DNA polymerase and ligated together. These constructs are summarized in Fig. 6(a)
.
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Rapid amplification of cDNA ends (RACE).
RNA was extracted from MHV-68-infected 3T3 cells (5 p.f.u. per cell, 18 h) by using RNAzol B (Tel-Test). For 5' RACE of ORF73 (Roche Diagnostics), total RNA was reverse transcribed from a primer corresponding to genomic co-ordinates 104638104659. A 5' poly(A) tail was added with terminal transferase, and the product was amplified by PCR, using a 5' poly(A)-specific primer and a 3' ORF73 primer (genomic co-ordinates 104699104717). The products were gel-purified (QIAquick gel extraction kit; Qiagen), sequenced directly, and T/A-cloned into pGEM-T-easy. For 3' RACE of ORF73, we used a poly(A)-specific RACE kit primer for reverse transcription, which incorporated a 3' tail for subsequent PCR amplification. RACE products were amplified by using a tail-specific primer and sequence-specific primers, matching genomic co-ordinates 104503104483, 104383104363, 104263104243 or 104050104030. For 5' RACE of ORF75a (genomic co-ordinates 117904114032), the reverse transcription primer matched 117720117740 and the PCR amplification primer matched 117741117760. PCR products were cloned into pSP73 by using the ClaI site in the 5' poly(A) tail-specific primer and a PstI site at 117755.
For RT-PCR based confirmation of the RACE-mapped ORF73 transcript, we paired a 104699104717 ORF73 primer with upstream primers corresponding to genomic co-ordinates 117904117920, 118100118117, 118182118199 or 118616118632. A positive control for these primers was a BglII/PstI genomic fragment (117840118700) cloned into the NruI/PstI sites of pcDNA3-ORF73, i.e. an 860 bp genomic fragment covering the right-hand end of the viral unique sequence, 32 bp upstream of ORF73.
Northern blotting.
RNA was purified from uninfected or MHV-68-infected (5 p.f.u. per cell, 18 h) 3T3 cells using RNAzol-B, electrophoresed (20 µg per lane) on a 0·8 % formaldehyde agarose gel and blotted overnight onto uncharged nylon membranes (Roche Diagnostics). Probe templates for -actin and ORF73 (a BsaI/Bsu36I genomic fragment, co-ordinates 103126104829) were random-prime labelled (Qbiogene) with 32P-dCTP (AP Biotech). Blots were hybridized with probe overnight at 45 °C in 50 % formamide, 5x Denhardt's solution, 6x SSC, 0·1 % SDS, 100 µg sonicated salmon sperm DNA ml1, followed by washing (0·1x SSC, 0·1 % SDS, 65 °C) and exposure to X-ray film (Coleman et al., 2003
).
RNase protection assay.
We generated four probes. A StyI genomic fragment (104714105433) was cloned into the EcoRV site of pSP72 (Promega), linearized with XmnI (105088), and transcribed with T7 RNA polymerase (Ambion). A BglII/PstI genomic fragment (117840118700) was cloned into the BglII/PstI sites of pSP72, linearized with BseYI (118292) and transcribed with T7 RNA polymerase. An ORF73 RACE clone (genomic co-ordinates 104699104871, 118055118160, 118605118658) in pGEM-T-easy was linearized with SalI and transcribed with T7 RNA polymerase to give a 514 nt probe (60 nt of plasmid sequence, 121 nt of poly(A) tail added during RACE, and 333 nt of MHV-68 exons). An SfoI/PstI genomic fragment (118552118700) cloned into pSP72 was linearized with XhoI, 28 bp downstream of the PstI cloning site, and transcribed with T7 RNA polymerase. Each anti-sense riboprobe was labelled with 32P-UTP (Amersham Biosciences) and hybridized to mRNA from MHV-68-infected 3T3 cells (5 p.f.u. per cell, 6 h) by using the Direct Protect lysate RPA kit (Ambion). Following RNase A/RNase T1 treatment, protected fragments were purified, separated on 8 M urea/6 % polyacrylamide gels, and exposed to X-ray film.
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RESULTS |
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We mapped the predominant 3' end of the ORF73 transcript by 3' RACE (Fig. 1d). There is a consensus polyadenylation signal at genomic co-ordinate 103789, 138 bp 3' of the ORF73 termination codon. The 5' primers sited in ORF73 amplified a 3' RACE product consistent with polyadenylation very close to this site. DNA sequence analysis of all these PCR products identified a poly(A) tail at genomic co-ordinate 103781103786. The predominant 3' end of the ORF73 mRNA therefore matched the site predicted by DNA sequence analysis.
RT-PCR based confirmation of the RACE-mapped ORF73 mRNA
We confirmed the presence of the spliced ORF73 mRNA in different virus-infected cell populations by RT-PCR. A 3' primer in the ORF73 coding sequence amplified a product from MHV-68-infected 3T3 cells with a 5' primer sited in ORF73 exon 1 or exon 2, but not with a 5' primer in either intron (Fig. 2a). Cycloheximide treatment (Fig. 2b
) reduced the abundance of a thymidine kinase mRNA but not that of the spliced ORF73 mRNA, indicating that the latter had immediate-early kinetics. The spliced mRNA was also detectable in cDNA derived from lungs, spleens and purified germinal centre B cells of MHV-68-infected mice (Fig. 2c
). Germinal centre B cells appear to support almost entirely latent infection (Stevensons et al., 2002
). Thus, in vivo latent ORF73 transcripts also initiated in the viral terminal repeats.
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A probe derived from the longest ORF73 RACE clone (333 nt of MHV-68 sequence: 104699104871, 118055118160, 118605118658) was fully protected (Fig. 3d), confirming the existence of the RACE-mapped mRNA in infected cells. There was also a 170 nt band, consistent with 3' probe protection by an unspliced ORF73 mRNA (Fig. 3b
). A protected 5' end would have required an ORF75a rather than ORF73 mRNA and would have protected a 321 nt fragment of the BglII/BseYI probe, of which we saw no sign (Fig. 3c
).
We used an SfoI/PstI genomic clone (118552118700) to look for evidence of mRNAs initiating upstream of genomic co-ordinate 118640 (Fig. 3e). Protected fragments were seen below 50 nt even with unhybridized probe, presumably because this G/C-rich RNA has considerable secondary structure (see also Fig. 8c
). It was therefore not possible to identify or to rule out a 4050 nt band corresponding to exon 1 of the RACE clones. The 150 nt fragment corresponded to the full-length probe (148 nt of MHV-68 sequence). This probably reflected protection by G/C-rich, terminal repeat DNA, since cycloheximide treatment of the infected cells, which would prevent viral DNA replication, reduced its abundance. In contrast, a 9095 nt protected fragment was more abundant in cycloheximide-treated cells, indicating protection by a latency transcript. This was presumably a 5' probe fragment, from the splice donor site at genomic co-ordinate 118605 to the PstI site at genomic co-ordinate 118700. 3'-Probe protection would have required an mRNA that missed the 118605 splice donor site, and was therefore inconsistent with the RACE product sequencing and with direct RT-PCR (primer C, Fig. 2
).
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With both a splice acceptor and a splice donor in each terminal repeat, there was the potential for the 5' untranslated region of an ORF73 mRNA to accumulate multiple copies of the 118605118695 exon. RACE would not have detected these, since internal PCR priming would have preferentially amplified from the most 3' terminal repeat copy. Northern blotting (Fig. 4) showed a predominant ORF73 mRNA at least 4000 nt in size, consistent with published data (Virgin et al., 1999
). There was also a 2000 nt mRNA, consistent with an unspliced ORF73 mRNA initiating around genomic co-ordinate 105100 (see below). The 4000 nt mRNA was too large for this, and so was likely to be the RACE-mapped transcript.
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Overlapping ORF73 and ORF75a transcripts
We first cloned a single repeat unit and tested its capacity to drive CAT expression in transfected 3T3 cells. Initially, we retained ORF73 exon 1 and intron 1 and fused CAT to ORF73 exon 2 at genomic co-ordinate 118117 (Fig. 5a, 1xTR). However, even a negative control plasmid, extending only 40 bp upstream of the RACE-mapped ORF73 transcription start site, retained some promoter activity (Fig. 5b
, exon 2/PstI clone).
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We confirmed that genomic co-ordinates 118117118598, essentially ORF73 intron 1, contained a constitutively active promoter (Fig. 5d). Crucially, transcription from this promoter initiated in the terminal repeats (ORF75a, RACE start site 2). The copy in the proximal repeat unit transcribed ORF75a: all ORF73 RACE clones were spliced to genomic co-ordinate 118605; PCR with a primer at the 3' end of intron 1 gave no ORF73-specific product (Fig. 2b
); and RNase protection assays indicated an ORF75a transcript, but not an ORF73 transcript, starting close to genomic co-ordinate 118210 (Fig. 3c
). However, transcripts from distal copies of this ORF75a promoter would run into the next terminal repeat rather than the unique right-hand end of the genome, and would therefore join the 118605118695 exon to become ORF73 mRNAs. Interestingly, a cDNA clone derived from S11E cells has a terminal repeat exon matching genomic co-ordinates 119373119195, followed by the 118605118695 exon (Husain et al., 1999
). This fitted nicely with transcription from the promoter in the left-hand portion of an upstream terminal repeat copy.
A major ORF73 promoter in the MHV-68 terminal repeats
Although transcription starting from the left-hand end of an upstream repeat unit could have generated the ORF73 RACE product, the greater activity of the 1xTR clone than the exon 2/PstI clone in CAT assays (Fig. 5b) suggested additional promoter elements in the terminal repeats. To look at this further, we generated MHV-68 genomic clones with a 3' end at 118616, thereby fusing CAT to ORF73 exon 1, upstream of the ORF75a promoter (Fig. 6
a). Extending this region to genomic co-ordinate 118700 gave no CAT transcription. Further 5' extensions up to the end of the terminal repeat unit gave increasing CAT transcription; adding portions of the next terminal repeat unit gave little additional activity (Fig. 6b
). Thus the promoter driving transcription from ORF75a RACE start site 2 had, in this setting, less impact on ORF73 transcription than a promoter in the right-hand portion of the terminal repeat, upstream of the 118700 PstI site. Deleting genomic co-ordinates 118700119105 abolished promoter activity (Fig. 6b
, clone BbvCI-PstI/NotI), as did reversing the orientation of the region upstream of 118700 (Fig. 6c
, clone PstI-TR-rev). The lack of transcription from the upstream ORF75a promoter in the BbvCI-PstI/NotI construct probably reflected a loss of the 118695 splice acceptor site.
The activity of the SacII genomic clone (5' limit 119078) (Fig. 6b, d) argued against transcription initiation much upstream of 118800. We used 3' deletions to try to identify a more precise transcription start site. A 3' deletion up to 118720 abolished promoter activity (Fig. 6d
, compare clones SacII-MspA1I and SacII). A deletion up to 118705 reduced promoter activity, but did not abrogate it entirely (Fig. 6d
, clone BbvCI-PstI/XbaI). Both 3' deletions also destroyed the 118695 acceptor site. Thus transcription initiation probably occurred just 5' of the 118700 PstI site, suggesting a TATAA box function for the striking, A/T-rich region at genomic co-ordinates 118756118778 in the otherwise G/C-rich repeat unit (Fig. 6a
, see also Fig. 1b
).
Unbiased transcription initiation in the terminal repeats would vary the 5' untranslated region by 2500 nt, with about 30 different, discrete lengths. There was some evidence for a variable ORF73 mRNA length (Fig. 4), but the predominance of a 4000 nt band implied that initiation was biased, with distal repeat units transcribing ORF73 more actively than proximal ones. A possible explanation was that the unique left end of the viral genome promoted transcription from its adjacent repeat unit, that furthest away from ORF73. Consistent with this idea, a 1·8 kb PstI genomic clone, which contained genomic co-ordinates 118700119450 of the right-most terminal repeat copy plus 1225 bp of the left end of the genome, gave very high transcriptional activity (Fig. 6e
). A 3' deletion of this genomic clone up to its SacII site at genomic co-ordinate 118720 abolished its promoter activity (data not shown), indicating that transcription remained dependent on the promoter in the right-hand portion of the terminal repeat. Thus, the predominant 4000 nt ORF73 mRNA appeared to reflect that the unique left end of the viral genome enhanced ORF73 transcription from the A/T-rich region of the adjacent terminal repeat copy. Overall, CAT assays provided strong supporting evidence for ORF73 transcription from the viral terminal repeats.
A third ORF73 promoter proximal to the ORF
Despite all RACE clones starting in the terminal repeats, RNase protection assays (Fig. 3b, d) had suggested that the MHV-68 ORF73 was also transcribed from a promoter overlapping the 5' end of ORF74 (Fig. 7
a). RT-PCR (Fig. 7b
) confirmed the existence of an unspliced ORF73 transcript, corresponding to the protection of the StyI probe. An unspliced transcript across the 5' end of ORF73 was also present in cycloheximide-treated, MHV-68-infected cells (Fig. 7c
), consistent with the RNase protection data. CAT assays (Fig. 7d
) established that the 3' end of the RACE-mapped ORF73 intron 2 (104872105430) contained a constitutive promoter, albeit a weaker one than those in the terminal repeats. Thus, at least three separate promoters could transcribe ORF73 with immediate-early kinetics. The 104871105430 region in reverse orientation also gave CAT transcription, possibly reflecting an ORF74 promoter.
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DISCUSSION |
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In conjunction with previously published cDNA clones (Husain et al., 1999), our data explain some of the structure of the MHV-68 terminal repeats (Fig. 8
). The downstream transcription initiation site lay within 27 bp that are duplicated at the ends of each repeat unit (see the annotation of GenBank accession number af105037). Nineteen basepairs of this 27 bp are duplicated at the extreme right end of the viral unique sequence, thereby mimicking the junction between repeat units. This arrangement may be important for promoter function, although the significance of generating an immediate-early ORF75a promoter in the proximal repeat unit is unclear. Both the terminal repeat promoters lay mainly within introns, separated by a 91 nt 5' untranslated ORF73 exon. The considerable secondary structure predicted for this exon (Fig. 8c
) and supported by the self-protection of the SfoI RNase protection probe (Fig. 3e
) may, particularly in tandem copies, contribute to the control of mRNA splicing, nuclear export or translation. Thus an involvement in splicing would explain why the downstream promoter in the proximal repeat unit gave an unspliced ORF75a mRNA, whereas the upstream promoter gave a spliced ORF73 mRNA; there is precedent from the 5' untranslated regions of other latency transcripts for internal ribosome entry site function (Bieleski & Talbot, 2001
; Coleman et al., 2003
; Grundhoff & Ganem, 2001
; Isaksson et al., 2003
; Low et al., 2001
); and translational autoregulation is an important immune evasion mechanism of EBNA-1 (Yin et al., 2003
).
Transcription from the MHV-68 terminal repeats was evident in infected lungs, spleens and germinal centre B cells, as well as in fibroblasts. In fibroblasts, ORF73 transcription appeared to initiate close to the left end of the viral genome, perhaps explaining the latency deficit seen with some left-end viral mutants (Adler et al., 2001; Clambey et al., 2000
). One function of viral tRNA transcription (Bowden et al., 1997
) might therefore be to keep the left end of the genome open for ORF73 transcription. The KSHV ORF73 gene product binds to the viral terminal repeats (Ballestas & Kaye, 2001
; Garber et al., 2002
). In MHV-68, this would provide an opportunity for transcriptional autoregulation. We saw little sign of this in our promoter constructs, but binding to multiple repeat units may be required for a large effect.
The large ORF73 intron 2 highlighted a general problem with tracking MHV-68 latency transcripts by RT-PCR. This is necessary to study low-abundance viral transcripts, but the data can be difficult to interpret without transcript mapping. For example, ORF74, which is spanned by ORF73 intron 2 but encoded on the opposite strand, has been identified as an immediate-early transcript (Martinez-Guzman et al., 2003) and as a latency transcript (Virgin et al., 1999
) by RT-PCR, but as a late-lytic transcript by strand-specific RNase protection assays (Rochford et al., 2001
). Unspliced, nuclear ORF73 mRNAs are a potential source of RT-PCR signal for ORF74, as well as for ORF75a, ORF75b, ORF75c. Also, ORF75a could be transcribed from the proximal repeat unit with immediate-early kinetics, but in the absence of ORF57 (Malik et al., 2004
) the unspliced mRNA may not be exported from the nucleus. One way to tackle such ambiguity would be to restrict RT-PCR to cytoplasmic extracts rather than whole-cell lysates.
The promoter just upstream of ORF73 was not a major one in infected fibroblasts, but may be more active in other cell types. It may also play a role in the lytic/latent switch of viral gene expression, since it was strongly downregulated by ORF50; there is clearly the potential for reciprocal inhibition between ORF73 and ORF50 (Lan et al., 2004). Defining the roles of the three different ORF73 promoter in host colonization will probably require in vivo analysis, since normal gene regulation is unlikely to be reproduced in immortalized tumour cells. The present data provide a basis for such a molecular description, and ultimately for understanding how gammaherpesvirus episome maintenance works in vivo.
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ACKNOWLEDGEMENTS |
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Received 31 August 2004;
accepted 16 November 2004.