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BARTs) and cancer
seminars in CANCER BIOLOGY, Vol. 11, 2001: pp. 469–476 doi:10.1006/scbi.2001.0414, available online at http://www.idealibrary.com on
Epstein–Barr virus complementary strand transcripts (CSTs/BARTs) and cancer Paul Smith
associated with specific phases of the viral cycle and also defined a number of potentially oncogenic viral genes. 1 Analysis of these patterns of gene expression suggested that many of these latent genes were not consistently expressed in viral associated tumours however, and identification of the contributions of these genes to tumour development in immunologically competent individuals is still unclear. The lack of a model comparable to that of the B cell lines for EBV infected epithelial cells has meant that analysis of viral genes in this cellular environment has lagged somewhat behind that of B cells. The use of Western blotting showed that NPC tumours expressed a pattern of gene expression consistent with that later defined as latency type II, although only approximately 40% of the tumours expressed LMP1. 4,5 The possibility that EBV encoded genes, which were not associated with B cell latency and which could transform epithelial cells was suggested by the study of Griffin and Karran, 6 who introduced a series of overlapping cosmids covering the entire EBV genome into primary primate cells and demonstrated that one of these cosmids, termed p31, could immortalize these cells. p31 did not, however, encode any of the known viral latent genes. The lack of a tissue culture model for EBV infection of epithelial cells was addressed by Busson and colleagues who successfully isolated a number of EBV positive NPC tumours which could be passaged in immunocompromised mice. 7 Using these materials, Beverly Griffin’s group constructed a cDNA library from mRNA isolated from these xenografts. Viral gene expression across the complete viral genome was analysed and confirmed the presence of a limited number of viral genes consistent with a latency type II phenotype. 8 The surprising result from these studies was the additional identification of a large amount of transcription from a region mapping to the Bam HI A fragment of the viral genome, a region known to contain genes expressed during the late or lytic phase of the viral cycle but not
Epstein–Barr virus (EBV) encodes a family of related transcripts, the complementary strand transcripts (CSTs) or BARTs (Bam A rightward transcripts). These are present in all types of EBV infection but are expressed to particularly high levels in nasopharyngeal carcinomas. Although convincing demonstration of protein expression from these transcripts is still subject to some debate, potential proteins encoded by them have been shown to modify Notch signalling pathways. Key words: Epstein–Barr virus / CSTs / BARTs / cancer / Notch signalling c 2001 Academic Press
Introduction Epstein–Barr virus (EBV) has a well-documented association with a variety of human cancers. 1 Early in the investigations of the biology of the virus, it was shown that EBV was associated with nasopharyngeal carcinoma (NPC), a form of cancer particularly common in specific regions of the world. This association was supported both by physical evidence, the detection of viral DNA in tumour cells, 2 and by serological evidence, the detection of elevated titres of antibodies to viral products. 3 Together, these observations supported the view that EBV is a causative agent in the development of NPC. The availability of B cell lines which could be easily grown in tissue culture systems, enabled the description of patterns of viral gene expression
From the Institute for Cancer Genetics and Pharmacogenomics, Department of Biology, Brunel University, Uxbridge, UB8 3PH, UK. E-mail: [email protected] c
2001Academic Press 1044–579X / 01 / 060469+ 08 / $35.00 / 0
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with a proportion of breast carcinomas (Smith, unpublished observations). In addition, a study which investigated EBV gene expression following tissue culture infection of a wide variety of epithelial cell types showed that CST expression was a consistent feature. 21 The presence of CST expression in a range of epithelial tissues, together with a generally lower level of expression in B cells, suggests that CST functions may be more important in epithelial phases of the viral cycle. However, analysis of peripheral blood samples identified the presence of CSTs in the CD19+ fraction of cells but not in the CD23+ fraction, 22 suggesting that the CSTs are expressed in the normal resting B cell population which is thought to be the reservoir for EBV persistence 23 and, considering the very restricted pattern of viral gene expression, may suggest a role in maintenance of normal B cell latency. Distribution of CSTs is, therefore, widespread throughout a variety of cell types infected with EBV, and studies of cell lines suggest that, generally, expression levels may be increased in epithelial cells. It is clear that CST expression is probably the most consistent viral transcript and should be considered as an alternative to EBER analysis for identification of the presence of EBV infected cells, particularly as CSTs have been shown to be transcribed in hepatocellular carcinomas, where EBER expression could not be demonstrated. 20
thought to be involved in EBV latency. Analysis of a number of cDNA clones from this region further showed that they were transcribed from the strand complementary to these late genes, and were therefore termed complementary strand transcripts or CSTs. [Subsequent groups have termed these transcripts the Bam HI A Rightward Transcripts (BARTs), or BARF0 transcripts, 9 throughout this paper I will use the term CSTs]. Interestingly, this region was within the region coded by the p31 cosmid which had been shown to immortalize monkey cells. 6 Northern blotting of NPC xenografts identified a major band of approximately 4.8 kb with additional products migrating with apparent sizes of 4.2, 5.5 and 6.2 kb. 8,9 A similar pattern of bands was also identified on Northern blots from NPC biopsies, which also confirmed the high levels of expression of these transcripts. 10 Analysis of CSTs has focussed largely in two areas, the distribution of these transcripts in the various types of virally infected cells and examination of the potential products associated with them and analysis of their roles in EBV biology.
Distribution of complementary strand transcripts Following identification of these transcripts in NPC samples a wide range of studies has described CST expression in all EBV associated conditions described to date. Initial studies confirmed the presence of CSTs in B cell lines, but the transcription levels were much lower in all lymphoblastoid cell lines (LCLs) analysed compared to that reported for either NPC biopsies or xenografts. 11 Northern blotting of these cell lines identified a similar pattern of expression to that reported in NPC cells, except in B95-8 cells where the major product was 4.0 kb. This is consistent with the presence of a large deletion in the B95.8 EBV genome. CSTs are present in all types of EBV latency 12 and were also identified in lymphomas developed in Cottontop Tamarins following infection with EBV. 13 A wide variety of EBV associated cancers has been also investigated for CST expression, the majority of these studies utilized PCR amplification with a primer pair across the Exon V/VII boundary. To date, CSTs have been reported in: Burkitt’s lymphomas; 14 gastric carcinomas; 15 salivary gland carcinomas; 16 oral hairy leukoplakia; 17 nasal NKand T cell lymphomas; 18 Hodgkin’s lymphomas; 19 hepatocellular carcinomas; 20 and, more recently,
Structure of the CSTs The isolation of a number of differentially spliced cDNA clones, and the pattern of expression observed on Northern blots, predicts transcription of a number of alternatively spliced members of the CSTs. Identification of CSTs has generally taken two approaches: the isolation of cDNA clones by standard protocols utilizing screening of cDNA libraries, or the use of PCR amplification with primers across specific splice junctions. A number of alternatively spliced species have been isolated using these methods, 24–26 and have led to the description of a transcription pattern outlined in Figure 1 and in Table 1. CST transcription is regulated from a promoter region which, initiating from a TATAAAlike sequence (TAAATAT) at EBV co-ordinate 150640, 24 corresponds to the transcription start site mapped for the BILF2 open reading frame (ORF), albeit from the opposite strand. Subsequent studies 470
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Figure 1. (a) Map showing the positions of complementary strand transcripts. EBV genome co-ordinates are indicated. 46,47 The position of the deletion in B95-8 cells is indicated. The position of identified open reading frames (ORFs) is shown by hatched boxes. Composite clones are taken from Reference 24 and the full-length cDNA clones from Smith et al. 26 (b) Northern blot of C15 NPC xenograft probed for CST expression. Positions of major species are indicated.
have confirmed this, and it has also been suggested that a small proportion of CSTs may initiate from an undetermined alternate start site upstream of this region. 25 Although this was not supported by a subsequent study 26 an alternate start site may, in principle, explain the larger transcripts identified on Northern blots. Little is known of the regulation of CST transcription, DNA methylation analysis has suggested that regions downstream of exon I may be important in controlling CST transcription. 24 The 30 processing of CSTs appears complex (see later), although all transcripts utilize the same polyadenylation site at 160964. CSTs therefore possess many of the characteristics of mRNA, although the presence of a clear large ORF is lacking in most cases. Analysis of protein expression from this family is still a little unclear, however a number of potential protein products have been identified (Figure 2) which are discussed in detail later.
Table 1. Co-ordinates of mapped CST exons. Data from References 25 and 26
BARF0 proteins The original isolation of CST cDNAs, usually from oligo-dT primed libraries, identified the 30 polyadenylation site at EBV genome co-ordinates 160989 following a canonical AATAAA sequence
Exon
Co-ordinates
I IA IB II III IIIA IIIB IV V VA VA0 VB VI VII VIIA VIIB VIIB0 VIID
150641–150769 151736–151841 839–972 6514–6615 9862–10358 9862–9993 10204–10358 10518–10629 155725–157195 155725–155807 155725–156266 156985–157195 157304–157386 159083–160989 159083–159209 160239–160989 160302–160992 159083–160067
at 160964. Sequence analysis of this region showed the existence of a large ORF, termed BARF0, at the 30 end. This ORF largely overlaps the BALF3 ORF on the complementary strand (Figure 1). Although 471
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Two potential BARF0 encoded proteins have been described in detail: BARF0, a 174 amino acid (aa) protein, 28 and RK-BARF0, a splice variant which would extend the BARF0 ORF to encode a potential product of 279 aa. A complete RK-BARF0 clone has not been isolated and the RK-BARF0 cDNA is a chimera of two overlapping cDNAs. 25 The N-terminus of the RK-BARF0 contains hydrophobic sequences with homology to endoplasmic reticulum localization motifs and antibodies generated to a peptide from RK-BARF0 identified a membrane associated protein of approximately 35 kDa, which was expressed in most types of EBV infected cells. 29 Expression of RK-BARF0 peptide was shown to be increased following induction of EBV replication in Akata cells, but was not expressed in oral hairy leukoplakia samples. Screening of human lung cDNA libraries in a yeast hybrid assay has shown an association with the extracellular domains of human Notch3 and Notch4. 30 This interaction caused an alteration in the normal processing of the Notch proteins, by unmasking the Notch nuclear localization signal, RK-BAR0 induced a translocation of a proportion of the Notch protein directly to the nucleus instead of transportation to the cell surface. The Notch/RK-BARF0 interaction also induced expression of the EBV LMP1 protein, although in a large proportion of EBV associated carcinomas which do express CSTs (and presumably, therefore, RK-BARF0) LMP1 is not detected. Expression of BARF0 proteins has also been supported by the observation that cells loaded with BARF0 peptides induced a cytotoxic response from peripheral blood cells isolated from EBV seropositive but not seronegative donors. Matched LCLs expressing RK-BARF0 did not, however, elicit a response. 31 The expression of BARF0 and RK-BARF0 proteins has been complicated by recent publications. Kienzle et al. 32 have shown that RK-BARF0 appears to be largely nuclear in location and that the specific anti-RK-BARF0 antisera cross reacts with a similar sized 35 kDa cellular protein in some EBV negative cell lines. Additionally, following transfection of the RK-BARF0 cDNA into cell lines, alternatively spliced RK-BARF0 products were detected. These alternatively spliced forms remove the specific peptide recognized by immune cells and additionally remove the peptide sequences used for generation of the anti-RK-BARF0 antisera. 33 The existence of alternate splicing patterns following the introduction of RK-BARF0 sequences into cells suggests either
Figure 2. Open reading frames identified in CSTs. ORFs are shown as hatched boxes. Splice junctions are represented by solid lines. Co-ordinates of ORFs are taken from References 24–26 and 33.
BALF3 expression has not been detected in EBV cells, this ORF is conserved in other gammaherpes viruses and, in HSV, has an essential role in capsid localization during replication. 27 The high degree of homology with other herpes viruses suggests that the BALF3 ORF may also be important in EBV biology. Expression of an ORF on the complementary strand may, therefore, be predicted to be constrained by the conserved sequences required of the BALF3 ORF. An additional problem with the translation of the BARF0 ORF is that in the majority of clones isolated no stop codon exists following normal processing of the addition of a polyadenylated tail. 26 This situation, although exceptionally unusual, is not without precedent. 25 However, a detailed analysis of 30 sequences around the site of the addition of the poly A sequence has shown that up to 25% of cDNAs are processed so that a stop codon is present in the BARF0 sequences. 25 472
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that other BARF0 ORF proteins may be expressed or that alternatively the RK-BARF0 splice may be an intermediate step which results in an unstable processed intermediate before additional processing to these additional spliced forms. The production of high quality reagents will be required to clarify the situation.
associated histone deacetylase repressor complex. The interaction between RPMS1 and CIR was confirmed in a number of assays and RPMS1 was shown to partially reverse Notch induced inhibition of muscle cells. This study concluded that RPMS1 can act as a negative regulator of the Notch/EBNA2 signalling via interactions with the CBF1 and CIR proteins within a histone deacetylase associated corepressor complex. One possible implication of these interactions is that RPMS1 may interfere with the EBNA2 up-regulation of the Cp promoter on the EBV genome which controls expression of the six EBNA genes, 1 which may result in the down-regulation of the EBNA proteins transcribed from this promoter. As some of the EBNA proteins induce cytotoxic immune responses, it is possible that the effect of repression by RPMS1 is to restrict recognition of viral infected cells by the immune system. The demonstration that two potential CST proteins, RK-BARF0 and RPMS1, target Notch pathways is intriguing and emphasizes the importance of these signalling pathways to the virus. The contributions of these interactions to cell transformation is at present a matter of conjecture. Notch has been reported to be up-regulated following chromosomal translocations in some T cell cancers 36 and signalling via Notch pathways has been shown to modify a number of cellular responses including cellular differentiation, proliferative and apototic responses. 37,38 It is possible, therefore, that modification of these effects by CST proteins may be a factor in development of EBV associated cancers.
RPMS1 Analysis of EBV transcription has identified situations where a number of genes utilize a common 30 polyadenylation sequence, and usually the most 50 functional ORF within each transcript is expressed. 26 RPMS1 was originally identified from a composite of overlapping cDNA clones 8,24 but the isolation of cDNAs representing complete CSTs enabled identification of RPMS1 as the most 50 ORF in one of these full-length cDNA species. 26 This fulllength cDNA contains a 497 bp untranslated 50 sequence prior to the RPMS1 ATG initiation codon, and the use of this cDNA clone in both in vitro transcription/translation and in vivo transfection assays demonstrated expression of RPMS1. Although direct evidence of RPMS1 expression in EBV infected cells remains elusive, this circumstantial evidence suggests that RPMS1 may be another CST encoded protein and it is likely that RPMS1 will prove to be an important viral protein as the product is translated across the Exon IV/V splice junction which is one of the most abundant spliced forms within the CST family of transcripts. 26 RPMS1 was originally shown to have a low level of homology to the EBV EBNA2 protein, 24 including the WWP motif, a rare sequence shown to be essential for binding of EBNA2 to the cellular transcription factor CBF1. 34 CBF1 is a cellular protein also targeted by EBNA2 and the EBNA3 family of proteins, and is a downstream modulator of Notch signalling pathways. RPMS1 was identified as a nuclear protein which could associate with CBF1 in both in vitro precipitation assays and yeast two-hybrid assays. 24 Interaction of EBNA2 with CBF1 inactivates the CBF1 induced repression of Notch targeted promoters. RPMS1–CBF1 interaction was shown to interfere with the EBNA2 or Notch mediated transactivation of reporter promoters containing CBF1 binding sites and hence repressed transcription from these reporter constructs. 26 Recently, these studies have been extended in an elegant series of experiments 35 which demonstrate that RPMS1 also binds Sin3a and CIR, which are other components of a CBF1
A73 The A73 spliced CST was one of the first identified, 10 and although a full-length cDNA clone encoding A73 has yet to be isolated, RPA assays suggest that this spliced product is one of the more abundant. 26 A potential protein of 126 aa is encoded by this partial cDNA although little evidence demonstrating expression of this protein in vivo is available. However, using epitope-tagged constructs, the A73 product has been shown to be a cytoplasmic protein which in yeast twohybrid assays interacts with the RACK1, 26 a cellular protein reported to modify protein kinase C function. 39 In the same yeast two-hybrid screening, A73 also interacted with integrin beta5 subunits. Intriguingly, RACK1 has also been shown to bind to integrin beta subunits, 40 a provocative observation, one interpretation of which is that A73 may be involved in modulation of the integrin signalling pathways. 473
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CSTs may function as an antisense RNA species controlling late or lytic gene transcription. 11 A recent report showing EBV can transcribe high levels of other RNA species, which may have an effect not dependent upon translation of proteins, suggests that this explanation should be considered. 41
Other CST proteins The diversity of alternatively spliced cDNA clones isolated suggests that a number of other products are likely to be associated with CST transcripts. The most convincing to date is that of RPMS1a, a small (40 amino acid) protein encoded within a full length cDNA clone (Figure 1). RPMS1a initiates from the same ATG initiator codon as RPMS1, and is collinear for the majority of its sequence with RPMS1 but then splices to exon VIIb, which adds a further four amino acids (Figure 2) including two C-terminal cysteine residues, a potential isoprenylation motif. Little else has been described for this protein. The size of this transcript (approximately 2.8 kb) and the failure to identify it by RPA analysis of EBV infected cells suggests that it is likely to be a minor CST species. 26 It is clear that evidence for the production of some proteins from CSTs remains largely circumstantial. The presence of antibodies to the RK-BARF0 protein is still subject to some debate, and conclusive proof of RPMS1/A73 protein expression awaits the availability of good quality reagents to the candidate proteins. It is interesting to note that apart from one study which showed that NPC sera could precipitate the BARF0 protein, no studies have been able to show the presence of antibodies to CST proteins in, for example, NPC patients sera where antibodies to other latent viral proteins are abundant. Analysis of the structure of the CSTs (for example, the use of a promoter already used for transcription of another viral RNA, the presence of a number of spliced forms which do not encode a single clear large ORF, and a common polyadenylation site) allows speculation that transcription of the CSTs may have developed late in the evolution of the virus, possibly associated with infection of epithelial cells. The constraints of conserved ORFs on the other strand presumably preclude translation of a single large protein from CSTs, but evolutionary pressures may have enabled expression of a number of functions which are encoded by a number of small proteins translated from a range of differentially spliced mRNAs. Although the presence of good candidate ORFs encoded by polyadenylated CST mRNA suggests that these transcripts will encode proteins it is possible that an alternative (or additional) role of the CSTs is not protein production but some other mechanism. Considering that a number of important ORFs involved in late or lytic phases are present on the opposing DNA strand, it has been suggested that
Conclusions Complementary strand transcripts have been detected in all types of EBV infection. Indeed, the presence of viral DNA can be determined by expression of CSTs. Their function, therefore, is probably of great importance to the virus. The expression in peripheral blood and the potential role in down-regulation of pathways involved in EBNA expression, suggests a possible role in the maintenance of viral latency. Alternatively, their widespread distribution in epithelial derived cells, and their high levels of transcription may argue for a more epithelial specific role. Their contribution to the development of malignancy is similarly difficult to assess, little evidence exists to support the possibility that CST products can directly induce cellular transformation. Interestingly, a series of experiments focussing on the contribution of EBV genes to the malignant phenotype of Akata cells has shown that while the EBER genes can enhance the tumourigenic potential of EBV negative Akata cells, 42–44 other mechanisms are present which may contribute to malignant phenotype in the parent cell line. As there is a very limited pattern of gene expression in these cells, it is possible that one or more members of the CST family may also be contributing towards these changes. The possibility that CSTs may effect viral transformation, therefore, is still open to debate. The circumstantial evidence of high levels of CST expression in NPC tumours, the ability of some of these putative proteins to induce transformation of fibroblasts in ras co-transfection assays and the immortalization of epithelial cells following transfection of the p31 cosmid containing the CST sequences implies that the transcripts may have a direct role in EBV mediated tumourigenesis. This hypothesis may be balanced somewhat by the observation that they appear to be dispensable for B cell immortalization. 45 The answers to these questions will be aided by a thorough investigation of all the proteins encoded by CSTs and the generation of good quality reagents directed against them. 474
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Griffin BE (2000) Expression of two related viral early genes in Epstein–Barr virus associated tumors. J Virol 74: 2793–2803 Komano J, Sugiura M, Takada K (1998) Epstein–Barr virus contributes to the malignant phenotype and to apoptosis resistance in Burkitt’s lymphoma cell line Akata. J Virol 72:9150–9156 Komano J, Maruo S, Kurozumi K, Takanori O, Takada K (1999) Oncogenic role of Epstein–Barr virusencoded RNAs in Burkitt’s lymphoma cell line Akata. J Virol 73:9827–9831 Ruf IK, Rhyne PW, Yang C, Cleveland JL, Sample JT (2000) Epstein–Barr virus small RNAs potentiate tumorigenicity of Burkitt’s lymphoma cells independently of an effect on apoptosis. J Virol 74:10223–10228 Robertson ES, Tomkinson B, Kieff E (1994) An Epstein– Barr virus with a 58-kilobase deletion that includes BARF0 transforms B lymphocytes. J Virol 68:1449–1458 Farrell PJ (1989) The Epstein–Barr virus genome, in Advances in Viral Oncology (Klein G, ed.) pp. 103–132. Raven Press, New York, NY Parker BD, Bankier A, Satchwell S, Barrell B, Farrell PJ (1990) Sequence and transcription of Raji Epstein–Barr virus DNA spanning the B95-deletion region. Virology 179:339–346
Epstein–Barr virus complementary strand transcripts (CSTs/BARTs) and cancer Paul Smith
associated with specific phases of the viral cycle and also defined a number of potentially oncogenic viral genes. 1 Analysis of these patterns of gene expression suggested that many of these latent genes were not consistently expressed in viral associated tumours however, and identification of the contributions of these genes to tumour development in immunologically competent individuals is still unclear. The lack of a model comparable to that of the B cell lines for EBV infected epithelial cells has meant that analysis of viral genes in this cellular environment has lagged somewhat behind that of B cells. The use of Western blotting showed that NPC tumours expressed a pattern of gene expression consistent with that later defined as latency type II, although only approximately 40% of the tumours expressed LMP1. 4,5 The possibility that EBV encoded genes, which were not associated with B cell latency and which could transform epithelial cells was suggested by the study of Griffin and Karran, 6 who introduced a series of overlapping cosmids covering the entire EBV genome into primary primate cells and demonstrated that one of these cosmids, termed p31, could immortalize these cells. p31 did not, however, encode any of the known viral latent genes. The lack of a tissue culture model for EBV infection of epithelial cells was addressed by Busson and colleagues who successfully isolated a number of EBV positive NPC tumours which could be passaged in immunocompromised mice. 7 Using these materials, Beverly Griffin’s group constructed a cDNA library from mRNA isolated from these xenografts. Viral gene expression across the complete viral genome was analysed and confirmed the presence of a limited number of viral genes consistent with a latency type II phenotype. 8 The surprising result from these studies was the additional identification of a large amount of transcription from a region mapping to the Bam HI A fragment of the viral genome, a region known to contain genes expressed during the late or lytic phase of the viral cycle but not
Epstein–Barr virus (EBV) encodes a family of related transcripts, the complementary strand transcripts (CSTs) or BARTs (Bam A rightward transcripts). These are present in all types of EBV infection but are expressed to particularly high levels in nasopharyngeal carcinomas. Although convincing demonstration of protein expression from these transcripts is still subject to some debate, potential proteins encoded by them have been shown to modify Notch signalling pathways. Key words: Epstein–Barr virus / CSTs / BARTs / cancer / Notch signalling c 2001 Academic Press
Introduction Epstein–Barr virus (EBV) has a well-documented association with a variety of human cancers. 1 Early in the investigations of the biology of the virus, it was shown that EBV was associated with nasopharyngeal carcinoma (NPC), a form of cancer particularly common in specific regions of the world. This association was supported both by physical evidence, the detection of viral DNA in tumour cells, 2 and by serological evidence, the detection of elevated titres of antibodies to viral products. 3 Together, these observations supported the view that EBV is a causative agent in the development of NPC. The availability of B cell lines which could be easily grown in tissue culture systems, enabled the description of patterns of viral gene expression
From the Institute for Cancer Genetics and Pharmacogenomics, Department of Biology, Brunel University, Uxbridge, UB8 3PH, UK. E-mail: [email protected] c
2001Academic Press 1044–579X / 01 / 060469+ 08 / $35.00 / 0
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with a proportion of breast carcinomas (Smith, unpublished observations). In addition, a study which investigated EBV gene expression following tissue culture infection of a wide variety of epithelial cell types showed that CST expression was a consistent feature. 21 The presence of CST expression in a range of epithelial tissues, together with a generally lower level of expression in B cells, suggests that CST functions may be more important in epithelial phases of the viral cycle. However, analysis of peripheral blood samples identified the presence of CSTs in the CD19+ fraction of cells but not in the CD23+ fraction, 22 suggesting that the CSTs are expressed in the normal resting B cell population which is thought to be the reservoir for EBV persistence 23 and, considering the very restricted pattern of viral gene expression, may suggest a role in maintenance of normal B cell latency. Distribution of CSTs is, therefore, widespread throughout a variety of cell types infected with EBV, and studies of cell lines suggest that, generally, expression levels may be increased in epithelial cells. It is clear that CST expression is probably the most consistent viral transcript and should be considered as an alternative to EBER analysis for identification of the presence of EBV infected cells, particularly as CSTs have been shown to be transcribed in hepatocellular carcinomas, where EBER expression could not be demonstrated. 20
thought to be involved in EBV latency. Analysis of a number of cDNA clones from this region further showed that they were transcribed from the strand complementary to these late genes, and were therefore termed complementary strand transcripts or CSTs. [Subsequent groups have termed these transcripts the Bam HI A Rightward Transcripts (BARTs), or BARF0 transcripts, 9 throughout this paper I will use the term CSTs]. Interestingly, this region was within the region coded by the p31 cosmid which had been shown to immortalize monkey cells. 6 Northern blotting of NPC xenografts identified a major band of approximately 4.8 kb with additional products migrating with apparent sizes of 4.2, 5.5 and 6.2 kb. 8,9 A similar pattern of bands was also identified on Northern blots from NPC biopsies, which also confirmed the high levels of expression of these transcripts. 10 Analysis of CSTs has focussed largely in two areas, the distribution of these transcripts in the various types of virally infected cells and examination of the potential products associated with them and analysis of their roles in EBV biology.
Distribution of complementary strand transcripts Following identification of these transcripts in NPC samples a wide range of studies has described CST expression in all EBV associated conditions described to date. Initial studies confirmed the presence of CSTs in B cell lines, but the transcription levels were much lower in all lymphoblastoid cell lines (LCLs) analysed compared to that reported for either NPC biopsies or xenografts. 11 Northern blotting of these cell lines identified a similar pattern of expression to that reported in NPC cells, except in B95-8 cells where the major product was 4.0 kb. This is consistent with the presence of a large deletion in the B95.8 EBV genome. CSTs are present in all types of EBV latency 12 and were also identified in lymphomas developed in Cottontop Tamarins following infection with EBV. 13 A wide variety of EBV associated cancers has been also investigated for CST expression, the majority of these studies utilized PCR amplification with a primer pair across the Exon V/VII boundary. To date, CSTs have been reported in: Burkitt’s lymphomas; 14 gastric carcinomas; 15 salivary gland carcinomas; 16 oral hairy leukoplakia; 17 nasal NKand T cell lymphomas; 18 Hodgkin’s lymphomas; 19 hepatocellular carcinomas; 20 and, more recently,
Structure of the CSTs The isolation of a number of differentially spliced cDNA clones, and the pattern of expression observed on Northern blots, predicts transcription of a number of alternatively spliced members of the CSTs. Identification of CSTs has generally taken two approaches: the isolation of cDNA clones by standard protocols utilizing screening of cDNA libraries, or the use of PCR amplification with primers across specific splice junctions. A number of alternatively spliced species have been isolated using these methods, 24–26 and have led to the description of a transcription pattern outlined in Figure 1 and in Table 1. CST transcription is regulated from a promoter region which, initiating from a TATAAAlike sequence (TAAATAT) at EBV co-ordinate 150640, 24 corresponds to the transcription start site mapped for the BILF2 open reading frame (ORF), albeit from the opposite strand. Subsequent studies 470
EBV encoded CST/BARTs and cancer
Figure 1. (a) Map showing the positions of complementary strand transcripts. EBV genome co-ordinates are indicated. 46,47 The position of the deletion in B95-8 cells is indicated. The position of identified open reading frames (ORFs) is shown by hatched boxes. Composite clones are taken from Reference 24 and the full-length cDNA clones from Smith et al. 26 (b) Northern blot of C15 NPC xenograft probed for CST expression. Positions of major species are indicated.
have confirmed this, and it has also been suggested that a small proportion of CSTs may initiate from an undetermined alternate start site upstream of this region. 25 Although this was not supported by a subsequent study 26 an alternate start site may, in principle, explain the larger transcripts identified on Northern blots. Little is known of the regulation of CST transcription, DNA methylation analysis has suggested that regions downstream of exon I may be important in controlling CST transcription. 24 The 30 processing of CSTs appears complex (see later), although all transcripts utilize the same polyadenylation site at 160964. CSTs therefore possess many of the characteristics of mRNA, although the presence of a clear large ORF is lacking in most cases. Analysis of protein expression from this family is still a little unclear, however a number of potential protein products have been identified (Figure 2) which are discussed in detail later.
Table 1. Co-ordinates of mapped CST exons. Data from References 25 and 26
BARF0 proteins The original isolation of CST cDNAs, usually from oligo-dT primed libraries, identified the 30 polyadenylation site at EBV genome co-ordinates 160989 following a canonical AATAAA sequence
Exon
Co-ordinates
I IA IB II III IIIA IIIB IV V VA VA0 VB VI VII VIIA VIIB VIIB0 VIID
150641–150769 151736–151841 839–972 6514–6615 9862–10358 9862–9993 10204–10358 10518–10629 155725–157195 155725–155807 155725–156266 156985–157195 157304–157386 159083–160989 159083–159209 160239–160989 160302–160992 159083–160067
at 160964. Sequence analysis of this region showed the existence of a large ORF, termed BARF0, at the 30 end. This ORF largely overlaps the BALF3 ORF on the complementary strand (Figure 1). Although 471
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Two potential BARF0 encoded proteins have been described in detail: BARF0, a 174 amino acid (aa) protein, 28 and RK-BARF0, a splice variant which would extend the BARF0 ORF to encode a potential product of 279 aa. A complete RK-BARF0 clone has not been isolated and the RK-BARF0 cDNA is a chimera of two overlapping cDNAs. 25 The N-terminus of the RK-BARF0 contains hydrophobic sequences with homology to endoplasmic reticulum localization motifs and antibodies generated to a peptide from RK-BARF0 identified a membrane associated protein of approximately 35 kDa, which was expressed in most types of EBV infected cells. 29 Expression of RK-BARF0 peptide was shown to be increased following induction of EBV replication in Akata cells, but was not expressed in oral hairy leukoplakia samples. Screening of human lung cDNA libraries in a yeast hybrid assay has shown an association with the extracellular domains of human Notch3 and Notch4. 30 This interaction caused an alteration in the normal processing of the Notch proteins, by unmasking the Notch nuclear localization signal, RK-BAR0 induced a translocation of a proportion of the Notch protein directly to the nucleus instead of transportation to the cell surface. The Notch/RK-BARF0 interaction also induced expression of the EBV LMP1 protein, although in a large proportion of EBV associated carcinomas which do express CSTs (and presumably, therefore, RK-BARF0) LMP1 is not detected. Expression of BARF0 proteins has also been supported by the observation that cells loaded with BARF0 peptides induced a cytotoxic response from peripheral blood cells isolated from EBV seropositive but not seronegative donors. Matched LCLs expressing RK-BARF0 did not, however, elicit a response. 31 The expression of BARF0 and RK-BARF0 proteins has been complicated by recent publications. Kienzle et al. 32 have shown that RK-BARF0 appears to be largely nuclear in location and that the specific anti-RK-BARF0 antisera cross reacts with a similar sized 35 kDa cellular protein in some EBV negative cell lines. Additionally, following transfection of the RK-BARF0 cDNA into cell lines, alternatively spliced RK-BARF0 products were detected. These alternatively spliced forms remove the specific peptide recognized by immune cells and additionally remove the peptide sequences used for generation of the anti-RK-BARF0 antisera. 33 The existence of alternate splicing patterns following the introduction of RK-BARF0 sequences into cells suggests either
Figure 2. Open reading frames identified in CSTs. ORFs are shown as hatched boxes. Splice junctions are represented by solid lines. Co-ordinates of ORFs are taken from References 24–26 and 33.
BALF3 expression has not been detected in EBV cells, this ORF is conserved in other gammaherpes viruses and, in HSV, has an essential role in capsid localization during replication. 27 The high degree of homology with other herpes viruses suggests that the BALF3 ORF may also be important in EBV biology. Expression of an ORF on the complementary strand may, therefore, be predicted to be constrained by the conserved sequences required of the BALF3 ORF. An additional problem with the translation of the BARF0 ORF is that in the majority of clones isolated no stop codon exists following normal processing of the addition of a polyadenylated tail. 26 This situation, although exceptionally unusual, is not without precedent. 25 However, a detailed analysis of 30 sequences around the site of the addition of the poly A sequence has shown that up to 25% of cDNAs are processed so that a stop codon is present in the BARF0 sequences. 25 472
EBV encoded CST/BARTs and cancer
that other BARF0 ORF proteins may be expressed or that alternatively the RK-BARF0 splice may be an intermediate step which results in an unstable processed intermediate before additional processing to these additional spliced forms. The production of high quality reagents will be required to clarify the situation.
associated histone deacetylase repressor complex. The interaction between RPMS1 and CIR was confirmed in a number of assays and RPMS1 was shown to partially reverse Notch induced inhibition of muscle cells. This study concluded that RPMS1 can act as a negative regulator of the Notch/EBNA2 signalling via interactions with the CBF1 and CIR proteins within a histone deacetylase associated corepressor complex. One possible implication of these interactions is that RPMS1 may interfere with the EBNA2 up-regulation of the Cp promoter on the EBV genome which controls expression of the six EBNA genes, 1 which may result in the down-regulation of the EBNA proteins transcribed from this promoter. As some of the EBNA proteins induce cytotoxic immune responses, it is possible that the effect of repression by RPMS1 is to restrict recognition of viral infected cells by the immune system. The demonstration that two potential CST proteins, RK-BARF0 and RPMS1, target Notch pathways is intriguing and emphasizes the importance of these signalling pathways to the virus. The contributions of these interactions to cell transformation is at present a matter of conjecture. Notch has been reported to be up-regulated following chromosomal translocations in some T cell cancers 36 and signalling via Notch pathways has been shown to modify a number of cellular responses including cellular differentiation, proliferative and apototic responses. 37,38 It is possible, therefore, that modification of these effects by CST proteins may be a factor in development of EBV associated cancers.
RPMS1 Analysis of EBV transcription has identified situations where a number of genes utilize a common 30 polyadenylation sequence, and usually the most 50 functional ORF within each transcript is expressed. 26 RPMS1 was originally identified from a composite of overlapping cDNA clones 8,24 but the isolation of cDNAs representing complete CSTs enabled identification of RPMS1 as the most 50 ORF in one of these full-length cDNA species. 26 This fulllength cDNA contains a 497 bp untranslated 50 sequence prior to the RPMS1 ATG initiation codon, and the use of this cDNA clone in both in vitro transcription/translation and in vivo transfection assays demonstrated expression of RPMS1. Although direct evidence of RPMS1 expression in EBV infected cells remains elusive, this circumstantial evidence suggests that RPMS1 may be another CST encoded protein and it is likely that RPMS1 will prove to be an important viral protein as the product is translated across the Exon IV/V splice junction which is one of the most abundant spliced forms within the CST family of transcripts. 26 RPMS1 was originally shown to have a low level of homology to the EBV EBNA2 protein, 24 including the WWP motif, a rare sequence shown to be essential for binding of EBNA2 to the cellular transcription factor CBF1. 34 CBF1 is a cellular protein also targeted by EBNA2 and the EBNA3 family of proteins, and is a downstream modulator of Notch signalling pathways. RPMS1 was identified as a nuclear protein which could associate with CBF1 in both in vitro precipitation assays and yeast two-hybrid assays. 24 Interaction of EBNA2 with CBF1 inactivates the CBF1 induced repression of Notch targeted promoters. RPMS1–CBF1 interaction was shown to interfere with the EBNA2 or Notch mediated transactivation of reporter promoters containing CBF1 binding sites and hence repressed transcription from these reporter constructs. 26 Recently, these studies have been extended in an elegant series of experiments 35 which demonstrate that RPMS1 also binds Sin3a and CIR, which are other components of a CBF1
A73 The A73 spliced CST was one of the first identified, 10 and although a full-length cDNA clone encoding A73 has yet to be isolated, RPA assays suggest that this spliced product is one of the more abundant. 26 A potential protein of 126 aa is encoded by this partial cDNA although little evidence demonstrating expression of this protein in vivo is available. However, using epitope-tagged constructs, the A73 product has been shown to be a cytoplasmic protein which in yeast twohybrid assays interacts with the RACK1, 26 a cellular protein reported to modify protein kinase C function. 39 In the same yeast two-hybrid screening, A73 also interacted with integrin beta5 subunits. Intriguingly, RACK1 has also been shown to bind to integrin beta subunits, 40 a provocative observation, one interpretation of which is that A73 may be involved in modulation of the integrin signalling pathways. 473
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CSTs may function as an antisense RNA species controlling late or lytic gene transcription. 11 A recent report showing EBV can transcribe high levels of other RNA species, which may have an effect not dependent upon translation of proteins, suggests that this explanation should be considered. 41
Other CST proteins The diversity of alternatively spliced cDNA clones isolated suggests that a number of other products are likely to be associated with CST transcripts. The most convincing to date is that of RPMS1a, a small (40 amino acid) protein encoded within a full length cDNA clone (Figure 1). RPMS1a initiates from the same ATG initiator codon as RPMS1, and is collinear for the majority of its sequence with RPMS1 but then splices to exon VIIb, which adds a further four amino acids (Figure 2) including two C-terminal cysteine residues, a potential isoprenylation motif. Little else has been described for this protein. The size of this transcript (approximately 2.8 kb) and the failure to identify it by RPA analysis of EBV infected cells suggests that it is likely to be a minor CST species. 26 It is clear that evidence for the production of some proteins from CSTs remains largely circumstantial. The presence of antibodies to the RK-BARF0 protein is still subject to some debate, and conclusive proof of RPMS1/A73 protein expression awaits the availability of good quality reagents to the candidate proteins. It is interesting to note that apart from one study which showed that NPC sera could precipitate the BARF0 protein, no studies have been able to show the presence of antibodies to CST proteins in, for example, NPC patients sera where antibodies to other latent viral proteins are abundant. Analysis of the structure of the CSTs (for example, the use of a promoter already used for transcription of another viral RNA, the presence of a number of spliced forms which do not encode a single clear large ORF, and a common polyadenylation site) allows speculation that transcription of the CSTs may have developed late in the evolution of the virus, possibly associated with infection of epithelial cells. The constraints of conserved ORFs on the other strand presumably preclude translation of a single large protein from CSTs, but evolutionary pressures may have enabled expression of a number of functions which are encoded by a number of small proteins translated from a range of differentially spliced mRNAs. Although the presence of good candidate ORFs encoded by polyadenylated CST mRNA suggests that these transcripts will encode proteins it is possible that an alternative (or additional) role of the CSTs is not protein production but some other mechanism. Considering that a number of important ORFs involved in late or lytic phases are present on the opposing DNA strand, it has been suggested that
Conclusions Complementary strand transcripts have been detected in all types of EBV infection. Indeed, the presence of viral DNA can be determined by expression of CSTs. Their function, therefore, is probably of great importance to the virus. The expression in peripheral blood and the potential role in down-regulation of pathways involved in EBNA expression, suggests a possible role in the maintenance of viral latency. Alternatively, their widespread distribution in epithelial derived cells, and their high levels of transcription may argue for a more epithelial specific role. Their contribution to the development of malignancy is similarly difficult to assess, little evidence exists to support the possibility that CST products can directly induce cellular transformation. Interestingly, a series of experiments focussing on the contribution of EBV genes to the malignant phenotype of Akata cells has shown that while the EBER genes can enhance the tumourigenic potential of EBV negative Akata cells, 42–44 other mechanisms are present which may contribute to malignant phenotype in the parent cell line. As there is a very limited pattern of gene expression in these cells, it is possible that one or more members of the CST family may also be contributing towards these changes. The possibility that CSTs may effect viral transformation, therefore, is still open to debate. The circumstantial evidence of high levels of CST expression in NPC tumours, the ability of some of these putative proteins to induce transformation of fibroblasts in ras co-transfection assays and the immortalization of epithelial cells following transfection of the p31 cosmid containing the CST sequences implies that the transcripts may have a direct role in EBV mediated tumourigenesis. This hypothesis may be balanced somewhat by the observation that they appear to be dispensable for B cell immortalization. 45 The answers to these questions will be aided by a thorough investigation of all the proteins encoded by CSTs and the generation of good quality reagents directed against them. 474
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References
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