1 MRC Virology Unit, Institute of Biological and Life Sciences, University of Glasgow, Church Street, Glasgow G11 5JR, UK
2 Division of Virology, Institute of Biological and Life Sciences, University of Glasgow, Church Street, Glasgow G11 5JR, UK
Correspondence
Andrew Davison
a.davison{at}vir.gla.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Cellular IRFs operate in interferon signal transduction, acting as transcriptional activators or repressors that are regulated by class I interferon receptor signalling. They consist of a conserved N-terminal DNA-binding domain and a C-terminal regulatory domain. The DNA-binding domain is also partially conserved in vIRF-1, although the protein appears to inhibit interferon-induced transcription in reporter transfection studies by mechanisms other than binding directly to DNA (Gao et al., 1997; Li et al., 1998
; Zimring et al., 1998
; Flowers et al., 1998
; Burysek et al., 1999a
). These mechanisms involve binding to some of the cellular IRFs and to coadaptors that link transcription factors to the RNA polymerase holoenzyme (Jayachandra et al., 1999
; Burysek et al., 1999a
; Seo et al., 2000
; Li et al., 2000
; Lin et al., 2001
). The multifunctional properties of vIRF-1 are illustrated further by the observation that, although the protein is a transcription inhibitor, in some settings it can act as an activator (Roan et al., 1999
). In addition, vIRF-1 is able to transform cells in culture which are then able to form tumours in nude mice (Gao et al., 1997
). It exerts anti-apoptotic properties through transcriptional repression (Kirchhoff et al., 2002
) and by binding to the p53 tumour suppressor (Nakamura et al., 2001
; Seo et al., 2001
) and a cell death regulator, GRIM19 (Seo et al., 2002
).
Burysek et al. (1999b) carried out initial studies on an ORF of 163 codons that was subsequently identified as the first exon of vIRF-2. In order to distinguish this ORF from the spliced vIRF-2 gene, we term it vIRF-2x1. The vIRF-2x1 protein is related to the DNA-binding domain of cellular IRFs. Burysek et al. (1999b)
showed from Northern blotting that even the smallest transcript detected (2·2 kb) is considerably larger than the vIRF-2x1 ORF, and from RT-PCR that transcription was only marginally inducible. A recombinant version of the vIRF-2x1 protein could form homodimers, and bound the consensus NF-
B-binding site. In reporter transfection assays, the vIRF-2x1 protein bound to several cellular IRFs and a transcriptional coadaptor, and inhibited interferon-induced transcription. Burysek & Pitha (2001)
concluded that vIRF-2x1 is transcribed constitutively and characterized the protein as a 20 kDa species that is localized to the nucleus. The vIRF-2x1 protein was shown to inhibit the antiviral effect of interferon by binding to and inhibiting double-stranded RNA-activated protein kinase. Kirchhoff et al. (2002)
showed that vIRF-2x1 is able to inhibit apoptosis by specific transcriptional repression.
Despite these studies, the expression pattern of vIRF-2 has remained controversial. Jenner et al. (2001) detected the spliced transcript by RT-PCR, whereas Burysek & Pitha (2001)
excluded splicing by similar experiments. Moreover, in contrast to Burysek et al. (1999b)
and Burysek & Pitha (2001)
, Jenner et al. (2001)
and Fakhari & Dittmer (2002)
catalogued vIRF-2 as an inducible gene from microarray and quantitative PCR studies, respectively. In another microarray study, Paulose-Murphy et al. (2001)
noted that K11, which forms the second exon of vIRF-2, is inducible. Similarly, Sarid et al. (1998)
listed K11 as inducible from Northern blot experiments. In addition to these conflicting conclusions regarding splicing and inducibility of vIRF-2, the locations of the mRNA ends were not determined.
vIRF-3 specifies a spliced mRNA of 2·2 or 1·8 kb (Lubyova & Pitha, 2000; Rivas et al., 2001
; Jenner et al., 2001
). Lubyova & Pitha (2000)
and Jenner et al. (2001)
characterized vIRF-3 as inducible. In contrast, Rivas et al. (2001)
and Fakhari & Dittmer (2002)
showed that the gene is expressed constitutively. Lubyova & Pitha (2000)
noted a potential TATA box and polyadenylation signal in the DNA sequence, but the 5'-end of a single cDNA clone analysed by Rivas et al. (2001)
does not correspond with use of this TATA box. The vIRF-3 protein (LANA-2) is expressed in the nuclei of PEL cell lines but not in KS tissue, and appears to be involved in inhibiting p53-mediated apoptosis (Rivas et al., 2001
).
The splicing pattern of vIRF-4 has been demonstrated (Jenner et al., 2001), and the transcript characterized as inducible (Sarid et al., 1998
; Paulose-Murphy et al., 2001
; Jenner et al., 2001
; Fakhari & Dittmer, 2002
). The size of the mRNA and the locations of its ends have not been determined.
In summary, the deduced transcriptional patterns of the vIRFs are incompletely understood and in some respects contentious. In this paper, we have evaluated transcription of all four genes in PEL cell lines by Northern blot hybridization to determine the sizes and inducibility of mRNAs, RT-PCR to examine splicing patterns, and RACE to detect mRNA ends. We have also confirmed the splicing pattern of vIRF-2 in HHV-8-infected endothelial cells.
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METHODS |
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Transformed primary human dermal microvascular endothelial cells (tDMVECs) were cultured as described by Moses et al. (1999) in EGM-2 MV medium (BioWhittaker) and infected with HHV-8 prepared from JSC-1 cells treated with 1 mM sodium butyrate, essentially as described by Cannon et al. (2000)
. Briefly, JSC-1 cells were diluted to 2x105 viable cells ml-1 and cultured for 3 days, resuspended at 8x105 viable cells ml-1 and treated with sodium butyrate for 18 h. The cells were then resuspended in the original volume of culture medium for 4 days in the absence of sodium butyrate. The medium was filtered (0·45 µm), and virus was harvested from the filtrate by centrifugation at 16 900 g for 2 h at 4 °C and resuspended in EGM-2 MV medium. The virus suspension was used to infect tDMVECs that had been cultured for 48 h in the absence of heparin and G418 and pre-treated with 100 µg polybrene (hexadimethrine bromide; Sigma) ml-1 for 1 h. The infected cells were subcultured after incubation for 48 h, and induced with 20 ng PMA ml-1.
Purification of polyadenylated RNA.
Total cellular RNA was extracted with Trizol (Invitrogen) at 107 cells ml-1 according to the manufacturer's instructions. Except for experiments involving tDMVECs, polyadenylated [poly(A)] RNA was isolated by resuspending the total cellular RNA from approximately 1·5x108 cells in 2·7 ml TE/5 (2 mM Tris/HCl pH 7·5, 0·2 mM EDTA), heated at 55 °C for 10 min, mixed with 300 µl 5 M NaCl, 15 µl 2 M Tris/HCl pH 7·5 and 120 mg oligo(dT)cellulose (Sigma) and incubated at room temperature for 30 min. Poly(A) RNA bound to oligo(dT)cellulose was pelleted by centrifuging for 10 min at 2000 g and washed twice by resuspending in 2 ml binding buffer (10 mM Tris/HCl pH 7·5, 0·5 M NaCl) and centrifuging. The pellet was resuspended in 2 ml binding buffer, distributed between six Spin-X tubes (Costar) and centrifuged for 2 min at 6000 g. The pellets were then washed twice by resuspending in 400 µl washing buffer (10 mM Tris/HCl pH 7·5, 0·25 M NaCl) and centrifuging. The Spin-X columns were transferred to fresh tubes and poly(A) RNA was eluted by resuspending twice in 200 µl elution buffer (10 mM Tris-HCl pH 7·5) and centrifuging. The two fractions from each column were combined, mixed with 2 µl Pellet Paint (Novagen), 40 µl 3 M sodium acetate pH 5·2 and 800 µl ethanol, incubated at room temperature for 5 min and centrifuged at 12 000 g for 5 min. The RNA pellets were washed by resuspending and centrifuging in 200 µl 70 % (v/v) ethanol and then in 100 % ethanol. Each pellet was air-dried and resuspended in 10 µl TE/5. The aliquots were combined and the RNA concentration was estimated by spectrophotometry.
RT-PCR.
Except for experiments involving RNA from tDMVECs, RT-PCR was carried out in 50 µl reaction volumes using the Titan kit (Boehringer) with poly(A) RNA and primers shown in Fig. 2. The conditions consisted of heating at 50 °C for 30 min, ten cycles at 94 °C for 30 s, 55 °C for 3 s and 68 °C for 60 s, 25 cycles under the same conditions except that the final step was prolonged by increments of 5 s at each cycle, and a final cycle with an extension step of 7 min. RT-PCR analyses of infected tDMVECs were performed using total cellular RNA essentially as described by Blackbourn et al. (1992)
. An aliquot (5 µl) of each reaction was subjected to agarose gel electrophoresis and ethidium bromide staining, and gels photographed under shortwave UV irradiation. Product sizes were estimated by comparison with marker DNA ladders (New England Biolabs). The remainder of each reaction was subjected to gel electrophoresis in low melting point agarose and ethidium bromide staining, and fragments were excised under longwave UV irradiation. DNA fragments were recovered by treatment with
-agarase (New England Biolabs) and cloned using a pGEM-T Vector System 1 kit (Promega). Plasmid DNA was prepared by standard protocols and the sizes of inserts were estimated by agarose gel electrophoresis of DNA digested with SalI and SphI (which each cut the vector once, on opposite sides of the insert). Sequences were obtained for several clones of each fragment with universal and custom primers, using an ABI PRISM 377 instrument.
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The conditions for PCR and nested PCR consisted of five cycles at 94 °C for 30 s and 72 °C for 180 s, five cycles at 94 °C for 30 s, 70 °C for 30 s and 72 °C for 180 s, and 2027 cycles at 94 °C for 30 s, 68 °C for 30 s and 72 °C for 180 s. RACE products were isolated by agarose gel electrophoresis, cloned into pGEM-T and characterized and sequenced as described above.
Northern blotting.
Aliquots of 4 µg poly(A) RNA were electrophoresed in formaldehydeagarose gels alongside synthetic poly(A) RNA markers (Life Technologies). The markers were visualized by photographing the ethidium bromide-stained gel under shortwave UV irradiation alongside a ruler. The RNA was transferred to Nytran Supercharge membranes (Schleicher & Schuell) by standard methods, and the positions of markers marked. A 32P-labelled double-stranded DNA probe was made using a random DNA nonaprimer kit (Appligene) from inserts purified from plasmids. A control probe for cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (approx. 1·4 kb) was also generated. Hybridization was carried out by standard procedures and the results were visualized using a phosphorimager (Bio-Rad Personal Molecular Biology Imager FX). Induction ratios for vIRF mRNAs, standardized to GAPDH mRNA levels established by reprobing the blot with the control probe, were obtained using the phosphorimager software.
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RESULTS |
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An experiment was undertaken to determine whether vIRF-2 is spliced in other cell lines. Fig. 4 shows that HHV-8 DNA yielded an unspliced product of 402 bp (B) and that a major spliced fragment of 281 bp (A) was produced from the JSC-1 and BCBL-1 PEL cell lines and also from tDMVECs infected with HHV-8 from JSC-1 cells. Very small amounts of unspliced (B) and heteroduplex (C) products were detected. Marginally more spliced product was generated from induced JSC-1 cells and tDMVECs than from uninduced cells.
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Sizes and inducibility of vIRF mRNAs assessed by Northern blot hybridization
Representative results of Northern blot hybridization of RNA from uninduced and induced BCP-1 and HBL-6 cells using probes encompassing the entire protein-coding regions of the vIRF genes are shown in Fig. 5. The sizes of the major RNAs were 2·4 kb for vIRF-2, 1·9 kb for vIRF-3, 2·9 kb for vIRF-4 and 1·5 kb for vIRF-1. This implies that each gene possesses its own promoter and polyadenylation site. Although quantitative comparisons were not carried out, levels of transcription of the spliced genes appeared low even in induced cells. Several minor inducible transcripts were detected for the spliced genes that may represent read-through RNAs or spurious hybridization. The relative levels of induction given at the foot of Fig. 5
indicate that vIRF-2, vIRF-4 and vIRF-1 were induced in BCP-1 and HBL-6 cells. The accuracy of these values is particularly sensitive to the low levels of transcripts in uninduced cells. vIRF-3 was not induced in HBL-6 cells, and was induced weakly in BCP-1 cells.
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Chen et al. (2000) reported a minor vIRF-1 transcript in uninduced RNA commencing 84 nucleotides upstream from the major 5'-end (TACCCA near the 5'-end of the sequence in Table 1
). In nested PCR experiments using the Smart RACE kit with uninduced RNA (data not shown), a minor RACE product (denoted L in Table 1
) was generated that was 6070 bp larger than the product corresponding to the major 5'-end (denoted S). As shown in Table 1
, the corresponding 5'-ends were distributed over a region upstream from the major end, but none corresponded to the minor end of Chen et al. (2000)
. No distinct product indicating 5'-ends upstream of the major end were identified using the GeneRacer kit followed by nested PCR, and cloned products from the relevant region of the gel exhibited 5'-ends no different from those of the major product. However, 5'-ends close downstream from the major end were identified in the S product made using the GeneRacer kit. These are located 2526 nucleotides downstream from a TATA box (TATATC). Corresponding 5'-ends were not detected using the Smart RACE kit. Therefore, the existence and relevance of minor vIRF-1 transcripts in uninduced cells should be considered inconclusive. The results for vIRF-1 indicated that the locations of 5'-ends derived using the GeneRacer kit are the most reliable, and can be supported by data obtained using the Smart RACE kit.
Table 1 shows that the 5'-end of vIRF-2 mRNA in induced cells is located 23 nucleotides downstream from a TATA box (TATATT). vIRF-4 mRNA has two inducible promoters, with the 5'-ends located 25 and 2226 nucleotides downstream, respectively, from TATA boxes (TATAAA and TATAAG). Neither vIRF-2 nor vIRF-4 was expressed at sufficient levels in uninduced cells to permit identification of 5'-ends. In contrast, the 5'-ends of vIRF-3 transcripts from uninduced and induced cells were spread over a region, and the major end in both is not located downstream from a TATA box.
As expected, in experiments where the gene-specific RACE primer was positioned downstream from the intron in the three spliced genes, the spliced transcript was detected. Alternative splicing was also evident in induced vIRF-4mRNA, occurring between a donor site upstream and an acceptor site downstream of the initiation codon, as well as in the usual intron (Fig. 2). Jenner et al. (2001)
reported this splicing pattern for uninduced vIRF-4 mRNA and noted that the translation product would lack the N-terminal portion of the vIRF-4 protein. In addition, we found that other sequences were also spliced to the vIRF-4 region via the alternative acceptor site, some from other regions of the HHV-8 genome and some from cellular sequences.
The Marathon kit was used to map the 3'-ends of vIRF genes (Table 1). The data are considered reliable as, in contrast to 5'-end mapping, the method does not depend on the generation of full-length cDNAs. Each 3'-end is located close downstream from a polyadenylation signal (AATAAA or ATTAAA). These data confirm that each vIRF gene has its own promoter (or promoters) and polyadenylation site. No evidence was found for splicing between different vIRF genes.
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DISCUSSION |
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Comparisons of three RACE kits for mapping the 5'-end of vIRF-1 revealed that the Smart RACE and GeneRacer kits were reliable, the latter probably performing with greater accuracy. The situation regarding the use of alternative promoters to generate minor vIRF-1 transcripts in uninduced cells remains unresolved. We were unable to confirm the minor end reported by Chen et al. (2000), and instead detected a different minor end, but with only one kit. The pattern of vIRF-1 expression in uninduced cells would thus bear greater scrutiny. Nonetheless, the success of the Smart RACE and GeneRacer kits with the major vIRF-1 mRNA was taken as strong support for the validity of the 5'-ends mapped for other vIRF genes.
There is disagreement in the literature regarding vIRF-2 structure and transcription. Our data confirm that the vIRF-2 mRNA is inducible and spliced. It is possible that the gene expresses two products, the full-length protein from spliced mRNA and a C-terminally truncated form (the vIRF-2x1 protein) from unspliced mRNA. However, Jenner et al. (2001) did not detect the unspliced RNA in induced cells, and the RT-PCR results in Fig. 4
show that little or no unspliced RNA is present in uninduced or induced cells. Taken together, these data indicate that the vIRF-2x1 protein is probably produced in very small amounts, if at all. Nonetheless, it must be registered that Burysek & Pitha (2001)
detected by Western blotting a protein in several PEL cell lines that had a size consistent with it being the product of vIRF-2x1, and apparently did not detect the larger protein anticipated from the spliced mRNA.
Of the four genes investigated, 5'-ends were the most difficult to detect for vIRF-4. Transcription of vIRF-4 was also unusual in that alternatively spliced transcripts were detected in induced mRNA. It should be noted that these RNAs were detected as contaminants of 5'-RACE products originating from the conventionally spliced transcripts. They were expressed by splicing of a variety of exons to vIRF-4 sequences via a range of donor sites, and consequently possessed a variety of 5'-sequences. They were probably fragmented products of longer RNAs that are unlikely to specify vIRF-4-related proteins. In any case, these RNAs would at best specify only the C-terminal portion of the vIRF-4 protein. Therefore, it is our opinion that these transcripts are likely to be biologically irrelevant, and were detected because of the relatively low level of vIRF-4 transcription even in induced cells and the fortuitous presence of an alternative acceptor site near the 5'-end of the gene.
The vIRF-3 (LANA-2) promoter is unusual in that it contains no obvious TATA box and directs transcriptional initiation over a relatively wide region, although a single major 5'-end was evident. The putative TATA box identified by Lubyova & Pitha (2000) is located 500 bp further upstream and appears not to direct initiation. The 5'-ends of another latent mRNA, encoding LANA-1, have been mapped to a region that is an unusually large distance (3455 nucleotides) from a proposed TATA box (Dittmer et al., 1998
; Talbot et al., 1999
; Sarid et al., 1999
). Sarid et al. (1999)
mapped another 5'-end about 50 bp upstream that lacks a TATA box. Various potential transcriptional regulatory sites have been identified in the LANA-1 promoter region, one of which is an Oct-1/TAATGARAT element (AAGGTAATGAAAT) identified by Talbot et al. (1999)
about 250 bp upstream from the initiation site. A closely related sequence (AAGGTAATGAGGT) is located a similar distance upstream from the major vIRF-3 initiation site (Fig. 2
). We note that the Oct-1/TAATGARAT element has been characterized through its involvement in expression of herpes simplex virus immediate early genes (O'Hare, 1993
).
Fig. 6 shows an alignment of the conserved domain in HHV-8 and RRV vIRFs in relation to the corresponding DNA-binding region of human IRFs, updated and expanded from alignments presented by Moore et al. (1996)
and Jenner et al. (2001)
. Some of the residues involved in interactions between IRF1 and its binding site (Escalante et al., 1998
) are conserved in vIRFs, but much conservation concerns other residues. Although it is clear that the DNA-binding domain of a cellular IRF has been captured by an ancestor of HHV-8 and RRV, it remains to be determined whether the viral proteins have retained functions that are dependent on binding DNA.
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ACKNOWLEDGEMENTS |
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Received 27 November 2002;
accepted 26 January 2003.