1 INSERM EPI 03-34, IUH, Hôpital Saint-Louis, Paris, France
2 Hématologie Biologique, Hôpital Avicenne, EA 1625, UFR SMBH, Université Paris 13 Bobigny, France
Correspondence
Irène Joab
i.joab{at}chu-stlouis.fr
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ABSTRACT |
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INTRODUCTION |
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B lymphocytes are immortalized by EBV infection in vitro, generating permanent lymphoblastoid cell lines (LCLs), in which an array of virus-encoded proteins, including six EBV nuclear antigens (EBNA-1, -2, -3A, -3B, -3C and leader protein) and three latent membrane proteins (LMP-1, -2A and -2B), are expressed. The small EBV-encoded non-polyadenylated nuclear RNAs (EBER-1 and -2) are also expressed. In LCLs, all six EBNAs are generated by differential splicing of a primary transcript that originates from one of the two promoters located in the BamHI C or W fragments (Cp or Wp). This form of latency is termed latency III (Lat III). In human tissue, EBV expression is often more restricted. EBNA-1, the LMPs and the EBERs are expressed in latency II (Lat II), whereas only EBNA-1 and the EBERs are expressed in latency I (Lat I). In Lat I and Lat II, EBNA-1 transcription originates from a different promoter, located in the BamHI Q fragment of the EBV genome (Qp). During the lytic cycle, EBNA-1 mRNA is transcribed from the F promoter (Fp), which lies upstream of Qp (reviewed by Kieff & Rickinson, 2001).
Recently, Kelly et al. (2002) identified a subset of BL tumours in which the Lat III-associated EBNA promoter Wp drove expression of the EBNA-3 genes. EBNA-2 production was abrogated by a gene deletion.
Here, we analysed extensively EBV gene expression in NHL arising in HIV-infected patients using immunohistochemistry (IHC) and/or RT-PCR to monitor the expression of EBNA-1, -2, -3A, -3B, -3C and LMP-1 and -2, as well as BZLF1 (the EBV immediate-early antigen), in 14 biopsies of NHL of HIV-infected patients. Moreover, our results show that expression of EBNA-3 genes can be directed from Fp in BL from HIV-infected patients as well as in Akata and Mutu I BL cell lines.
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METHODS |
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Preparation of RNA and RT-PCR.
Frozen specimens were pulverized and RNA extracted using RNasol B (Bioprobe Systems). RNA was then treated with RQ1 DNase (Promega). RNA (1 µg) was reverse-transcribed with MoMLV reverse transcriptase (Gibco) after priming with oligo(dT). PCR was carried out with the cDNA samples obtained from 33 ng of total RNA. PCR was performed as described (Martel-Renoir et al., 1995). Second-round PCR was carried out with 1/50 of the first-round PCR mixture. Primers used are listed in Table 2
.
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ISH.
EBER ISH (Barletta et al., 1993) was carried out on routinely processed paraffin sections with FITC-labelled EBER-1- and -2-specific oligonucleotides, according to the manufacturer's instructions (Dako).
Western blots.
Western blots were performed using anti-EBNA-3B (Exalpha Biologicals) and the A10 anti-EBNA-3C (Radkov et al., 1997), as described previously (Fahmi et al., 2000
).
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RESULTS |
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PCR was then performed with primers specific for type A and type B EBV (Rowe et al., 1989) on each of the tumour samples, with the exceptions of tumours #1, #7 and #14 (due to a lack of material). Most of the tumour biopsies analysed contained the type A variant, while two samples (#3 and #10) contained both type A and type B strains (Table 1
).
EBV gene expression
RT-PCR and IHC were used to determine the specific pattern of EBV gene expression in each tumour cell. RT-PCR was performed for EBNA-2, -3A, -3B and -3C and LMP-1, -2A and -2B and ZEBRA. IHC was performed on EBNA-2, LMP-1 and ZEBRA.
Of the 14 tumours studied, four followed a typical pattern of gene expression: tumour #14 (BLL) showed the classical Lat I pattern, tumour #1 (BLL) showed the classical Lat II pattern and tumours #3 (BL) and #17 (DLCL) showed the Lat III pattern.
Non-canonical patterns that did not follow one of these expressions were also observed. Indeed, the remaining tumours showed various levels of heterogeneity in their patterns of viral gene expression. The majority of these tumours express at least one of the EBNA-3 genes in the absence of EBNA-2, either with or without LMP-1.
Markedly, in one DLCL (#13), we observed expression of EBNA-2 and LMP-1 and -2 genes without the detection of any transcripts of the EBNA-3 gene family. Moreover, in one IBP (#7), three DLCL (#5, #6 and #16), two BL (#10 and #11) and three BLL (#4, #8 and #9), expression of transcripts of the EBNA-3 gene family was observed without detection of the EBNA-2 gene product. An illustration is detailed in Fig. 1: in tumour #11, in the absence of EBNA-2 expression (Fig. 1b
, ii), amplification of EBNA-3A, -3B and -3C cDNAs was observed (Fig. 1b
, iiiv). Furthermore, in tumour #4, only expression of two members of the EBNA-3 gene family (EBNA-3B and -3C) was detected (Fig. 1b
, iv and v), again without EBNA-2. The same results were obtained in three independent experiments.
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Characterization of promoter usage
In three lymphomas, #3, #4 and #11, RT-PCR was performed to determine promoter activity. Cp and Wp activities were assessed as described previously (Tierney et al., 1994). F/Qp activity was assayed using a sense primer in the F/Qp region and an anti-sense primer within the BKRF1 open reading frame (ORF) encoding EBNA-1 (Fig. 2
). The activity of all promoters, Cp, Wp and F/Qp, was detected in tumour #3, while only the F/Qp promoter seems to be active in tumours #4 and #11, even though EBNA-3 expression was detected in these samples.
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Fp-driven EBNA-3 gene expression
In Akata and Mutu I cells, the entire EBNA-3 gene family can be expressed through Fp. This expression occurs predominantly after induction of the EBV lytic cycle. EBNA-3A, -3B and -3C expression through Fp was assayed by RT-PCR of RNA from Akata cells. In each case, two rounds of PCR were necessary to visualize a band of the expected size (data not shown). However, if RNA was prepared from Akata cells, in which the EBV lytic cycle had been induced by treatment with 1 % anti-human IgG (Dako), only one round of PCR was needed to amplify EBNA-3A (Fig. 4a), -3B (Fig. 4b
) and -3C (Fig. 4c
) cDNAs. The three amplified bands hybridized with the 32P-labelled oligonucleotide U172AS, suggesting that the cDNAs harbour the U172 exon. This result shows that in Akata cells, EBNA-3 expression from Fp occurs predominantly after the induction of the EBV lytic cycle. Similar results were obtained for Mutu I cells. Fig. 4(df
) shows expression of EBNA-3A, -3B and -3C, respectively, in Mutu I cells in which the lytic cycle has been induced by treatment with TGF-
1 (transforming growth factor-
1). As with Akata cells, no expression of mRNA for any of the EBNA-3 genes was detectable in the absence of lytic cycle induction, though, as expected, EBNA-3B and -3C proteins were detected by Western blot in Mutu III cells (Fig. 5
). Fig. 4
(df) shows that no expression of EBNA-3A, -3B or -3C occurs through Fp in Mutu III cells, irrespective of whether the virus is induced to lytic cycle or not (virus induction was verified by Western blot of the ZEBRA protein, data not shown).
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EBNA-3B and -3C proteins were detected in Akata and Mutu I BL cell lines
To verify that the EBNA-3B and -3C transcripts detected in our RT-PCR experiments could in fact give rise to their corresponding proteins, Western blots were performed using anti-EBNA-3B and the A10 anti-EBNA-3C mAbs (Fig. 5). EBNA-3B protein was detected in Akata and Mutu I cells, as well as in Mutu III and B95-8 cells, which were used as controls. In both Akata and Mutu I cells, the levels of detected protein were similar, irrespective of whether or not the lytic cycle was induced (Fig. 5
). However, in Akata cells, the signal for EBNA-3B expression was only seen after overexposure of the blot. Hence, the EBNA-3B protein is expressed in certain group I BL cell lines and it is possible that at least some of the protein detected in our Western blots was the product of the mRNA transcripts observed by RT-PCR. Results obtained for EBNA-3C were slightly different. Unlike EBNA-3B, EBNA-3C protein was not detected in Akata cells, whether induced or not. However, EBNA-3C was detected at similar levels in both induced and uninduced Mutu I cells.
Lytic gene expression is detected in NHL of HIV-infected patients
In 7 of 14 lymphomas, EBV expression was not wholly latent, since the immediate-early protein ZEBRA was detected. Fig. 6 shows IHC detection of ZEBRA in frozen sections of tumour #4. The large nuclei of numerous tumour cells stain positive, demonstrating that virus reactivation had occurred in this tumour. This correlates with results described in Fig. 3
showing that the lytic promoter Fp is active in tumour #4.
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DISCUSSION |
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The BL cell lines Oku and Sal have been shown to produce Wp/Cp-driven EBNA-3 transcripts from a virus in which the EBNA-2 gene has been deleted (Kelly et al., 2002). In Akata and Mutu I cell lines, the EBNA-2 gene is not deleted, since we could obtain amplification of the corresponding ORF using primers E2S and E2AS (data not shown). In this case, the EBNA-2 gene is silent, as the promoter that drives EBNA-3 gene expression is located downstream of the EBNA-2 ORF. Our results show that EBV can use a mechanism other than EBNA-2 deletion to produce EBNA-3 without EBNA-2.
The Q promoter is used in Lat I and II to generate EBNA-1 transcripts. During the lytic cycle, transcription from Fp generates EBNA-1 mRNA (Lear et al., 1992; Sample et al., 1991
). We show that expression of the EBNA-3 gene family occurred through Fp. This promoter is used for EBNA-1 and -3 expression during the lytic cycle and we did not observe any pre-mRNA containing both EBNA-3B- and -1-encoding sequences. The U172 exon is present in EBNA-1 transcripts and in the latent mRNA harbouring the EBNA-3C gene (Speck & Strominger, 1985
; Bodescot & Perricaudet, 1986
). Here, results show that the U172 exon is also present in the Fp-driven EBNA-3 transcripts and sequence analysis shows that U172 is spliced to the BERF2a exon. We then confirmed by sequencing the splicing acceptor site of BERF2a, which has been predicted from early RNase mapping analyses (Kerdiles et al., 1990
) but has never been identified definitively.
It has been reported that EBNA-1 (Lear et al., 1992; Nonkwelo et al., 1996
) as well as the truncated form of the LMP-1 protein (Hudson et al., 1985
) are expressed during the lytic cycle. Results presented here show that the EBNA-3 gene family can also be expressed during the lytic cycle. This represents a new type of transcription pattern observed in some type I BL cell lines as well as in lymphomas of immunocompromised patients. Expression of EBNA-3 proteins in lymphomas of immunodeficient patients is important as these proteins are immunodominant targets for CD8+ cells (Rickinson & Moss, 1997
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 July 2002;
accepted 27 November 2002.