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Journal of Virology, December 2007, p. 13544-13551, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.01521-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

RNA Editing of the Human Herpesvirus 8 Kaposin Transcript Eliminates Its Transforming Activity and Is Induced during Lytic Replication{triangledown}

Sharon Z. Gandy, Sarah D. Linnstaedt, Sumitra Muralidhar, Kathleen A. Cashman, Leonard J. Rosenthal, and John L. Casey*

Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20007

Received 11 July 2007/ Accepted 20 September 2007


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ABSTRACT
 
Human herpesvirus 8 is the etiologic agent associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The K12 RNA, which produces as many as three variants of the kaposin protein, as well as a microRNA, is the most abundant transcript expressed in latent Kaposi's sarcoma-associated herpesvirus infection, and yet it is also induced during lytic replication. The portion of the transcript that includes the microRNA and the kaposin A sequence has been shown to have tumorigenic potential. Genome coordinate 117990, which is within this transcript, has been found to be heterogeneous, primarily in RNAs but also among viral DNA sequences. This sequence heterogeneity affects an amino acid in kaposins A and C and the microRNA. The functional effects of this sequence heterogeneity have not been studied, and its origin has not been definitively settled; both RNA editing and heterogeneity at the level of the viral genome have been proposed. Here, we show that transcripts containing A at position 117990 are tumorigenic, while those with G at this position are not. Using a highly sensitive quantitative assay, we observed that, in PEL cells under conditions where more than 60% of cDNAs derived from K12 RNA transcripts have G at coordinate 117990, there is no detectable G in the viral DNA sequence at this position, only A. This result is consistent with RNA editing by one of the host RNA adenosine deaminases (ADARs). Indeed, we observed that purified human ADAR1 efficiently edits K12 RNA in vitro. Remarkably, the amount of editing correlated with the replicative state of the virus; editing levels were nearly 10-fold higher in cells treated to induce lytic viral replication. These results suggest that RNA editing controls the function of one segment of the kaposin transcript, such that it has transforming activity during latent replication and possibly another, as-yet-undetermined, function during lytic replication.


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INTRODUCTION
 
Human herpesvirus 8 (HHV-8) is the etiologic agent believed to be responsible for the AIDS-associated malignancies Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) (12, 13, 32). During the latent phase of viral replication, expression of RNA and protein is restricted to a limited number of genes; among the most abundant latent transcripts is that which includes the K12 (kaposin) open reading frame (ORF) (46, 55). Transcription and translation of the K12 region, which is unique to HHV-8, is complex (7, 28, 38, 46). As many as three proteins, kaposins A to C, may be produced from this transcript (33, 34, 46, 55). Moreover, miR-K10, one of several microRNAs (miRNAs), known to be produced by HHV-8, is derived from the middle of the kaposin A ORF (8, 19, 39, 47, 48). The K12 RNA transcript is not only abundant during the latent phase of virus replication (46, 55) but is also strongly induced after stimulation of lytic viral replication (49, 50) by activation of a separate strong promoter (7, 28, 38). Thus, the K12 transcript is a class II gene (49) and may be functional during both the latent and the lytic phases of the viral replication cycle.

Expression of the K12 transcript, in particular the kaposin A ORF, transforms cells in culture (24, 33, 34, 51) and induces tumor formation in athymic nude mice (33). This transforming ability is consistent with that attributed to latent genes of other gammaherpesviruses (reviewed in reference 16). Whether this activity is due to the kaposin A protein, miR-K10, or both is not yet clear. McCormick and Ganem (30) have reported that kaposin B, which is produced from an unusual ORF in the same mRNA transcript (46), induces cytokine production. It has been suggested that this induction could also play a role in the latent phase of HHV-8 infection (30). No function has been determined for kaposin C, which contains the same C-terminal sequences as kaposin A, nor is it known whether the additional N-terminal sequences present in this protein affect its transforming potential.

Several reports have noted sequence heterogeneity at position 117,990 (sequence numbering according to Russo et al. [45]) in K12 RNAs isolated from PEL tumors and cell lines persistently infected with HHV-8 (18, 28, 39). Most reported cDNA sequences contain G at this position, while most viral DNA sequences contain A. This sequence heterogeneity could be functionally significant: it affects the protein coding sequence in the kaposin A ORF—glycine versus serine at amino acid 38—and changes position 2 at the 5' end of miR-K10. Sequences at the 5' end of miRNAs may help determine which particular mRNAs are affected by a given miRNA (22, 27).

The origin of the sequence variability at position 117,990 has not been definitively settled. Pfeffer et al. (39) observed that 10 of 10 cloned PCR products obtained from viral genomic DNA isolated from BCBL1 cells contained adenosine at this position, while 12 of 14 cloned reverse transcription-PCR (RT-PCR) products obtained from RNA in the same cells exhibited G. These authors therefore suggested that the heterogeneity could be due to RNA editing via deamination of the adenosine at coordinate 117990 by one of the host RNA adenosine deaminases (ADARs) (39). On the other hand, an earlier report from Li et al. (28) indicated that heterogeneity at this position occurred at the level of the viral genome. Seven of seven cloned PCR products obtained from viral genomic DNA isolated from a PEL tumor contained G at position 117990 (28). However, cDNAs obtained from the same tumor, as well as the DNA of a PEL cell line also derived from the same tumor, contained an A at this position (28). These authors thus suggested that heterogeneity exists at the level of the viral genome and that RNA editing was not necessary to explain the observed sequence variability. One limitation of these previous studies is that the analysis of relatively small numbers of cloned sequences constrains the accuracy with which one can determine the relative amounts of the two sequence variants.

Here we report the analysis of the sequence heterogeneity at position 117990 in viral RNA and DNA isolated from two different PEL cell lines. Using a highly sensitive assay for sequence heterogeneity, as well as analysis of editing of the K12 RNA in vitro, we find compelling evidence that heterogeneity in the K12 transcript is due to RNA editing, most likely by the host enzyme ADAR1. Remarkably, we observed that editing is functionally significant: editing eliminated the tumorigenic activity of the kaposin transcript. Moreover, the level of editing is regulated by the replicative state of the virus: editing levels were low in untreated cells harboring latent virus infection and increased nearly 10-fold upon treatment with either phorbol ester or sodium butyrate to activate lytic virus replication. These results suggest that editing controls the function of the kaposin A/miR-K10 portion of the K12 transcript such that it has transforming activity during latent replication and another, as-yet-undetermined, function during lytic replication.


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MATERIALS AND METHODS
 
Cell culture. PEL cell lines BC3 (2) and BCBL1 (41) were obtained from the American Type Culture Collection and the AIDS Research and Reference Reagent Program, respectively. Cells were grown in RPMI 1640 supplemented with glutamine and either 20% fetal bovine serum (BC3) or 10% fetal bovine serum and 10 µM 2-mercaptoethanol (BCBL1). Cells were maintained at between 1.2 x 105 and 1 x 106 per ml. Prior to treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or with sodium butyrate (Sigma), cells were pelleted at 1,000 x g for 5 min, resuspended in fresh medium at a density of 2 x 105 per ml, and then divided into aliquots. TPA treatment was at 25 nM for 48 h; sodium butyrate treatment was at 3 mM for 48 h.

Rat3 cells were grown in Dulbecco modified Eagle medium supplemented with 2 mM glutamine and 10% fetal bovine serum. Focal transformation assays were conducted essentially as described previously (33), except that lower passage Rat3 cells (passage 4, kindly supplied by Peter Medveczky, University of South Florida) were used, and Effectene (QIAGEN) was used for transfections, according the suppliers' recommendations. Cells were transfected in 35-mm-diameter petri dishes, in triplicate, and then subcultured into 100-mm-diameter dishes at 48 h posttransfection. Morphologically transformed foci were counted at 3 weeks posttransfection. Stably transfected cells were selected by the addition of Geneticin (G418) at a concentration of 400 µg/ml and were subsequently maintained at a concentration of 200 µg/ml.

Plasmid constructs. The kaposin A ORF (genome sequence from 118101 to 117922) was amplified by PCR and cloned between the NcoI and BamHI sites of the pCMV-Tag1 expression vector (Stratagene, La Jolla, CA) to generate the plasmid pCMV-Kap-Tag. The position in the ORF corresponding to coordinate 177990 was changed from A to G by site-directed mutagenesis. Sequence analysis (MWG Biotech, High Point, NC) verified the proper construction of both plasmids.

Tumorigenicity assay. The tumorigenic potential of Rat3 cells stably transfected with kaposin-expressing constructs was tested in athymic Ncr nu/nu mice as previously described (33). Pooled cells (5 x 106/100 µl) were injected subcutaneously behind the neck. Tumor volumes were measured every 7 days beginning on day 14. Animals with tumors were sacrificed at 28 days due to tumor burden. All animal experiments for the present study were conducted under protocols reviewed and approved by the Georgetown University Institutional Animal Care and Use Committee.

Nucleic acid preparation and sequence analysis. Viral DNA was harvested from BCBL1 and BC3 cells by using a Wizard genomic DNA purification kit (Promega). Total cellular RNA was harvested from 5 x 105 cells by using the QiaShredder and RNeasy miniprep kits (QIAGEN) according to the supplier's recommendations. RNA samples were treated with DNase (Invitrogen) to remove traces of copurified viral genomic DNA and then subjected to RT using random hexamers (40). A 35-cycle PCR was performed by using Amplitaq Gold (Applied Biosystems) with either these cDNAs or viral DNA samples as templates and using primers SZ5 and either SZ5 or K12R1. The primers and their corresponding sequences were as follows: SZ4, 5'-ATGGATAGAGGCTTAACGGTGTTTGTGGC-3'; SZ5, 5'-CGCGCCCGTTGCAACTCGTGTCCTGAATG-3'; K12F3, 5'-GATACCACCACTCGTTTGCCAGTTGG-3'; and K12R1, 5'-GGAGGGCACGCTAGCTTCAGTG-3'. The effectiveness of DNase treatment on RNA samples was verified by the absence of PCR products after PCR amplification without prior RT. All PCR assays included negative control samples to verify the absence of contamination of reagents. PCR products amplified with primers SZ4 and SZ5 were first purified by using the QiaQuick PCR purification kit (QIAGEN) and then sequenced directly (MWG Biotech, High Point, NC).

Analysis of sequence variation at position 117,990. A modified forward PCR primer, K12F3 (see above), was designed such that amplification of PCR products with G at position 117990 created an XcmI restriction site; this site was not present in products with an A at this position. PCR products were labeled with [{alpha}-32P]dCTP added prior to the final cycle of the PCR (40) to avoid the possibility that during the latter cycles of PCR amplification mixtures of PCR products containing A and G could produce heteroduplex products that would not be fully digested by the restriction enzyme (9). Sequence variation was indicated by the appearance of an XcmI site in the labeled amplification product and was quantified by electrophoresis, followed by radioanalytic imaging with a Molecular Dynamics Storm phosphorimager.

ADAR1 expression and purification. A baculovirus transfer vector was designed for expression of the p110 form of human ADAR1 with an N-terminal FLAG tag. Briefly, using PCR, a BamHI site and FLAG epitope tag were inserted at the N terminus of the coding region of the p110 form of ADAR1 in the expression plasmid pDL701 (52) (kindly provided by David Lazinski, Tufts University). The resulting construct, pFLAG-ADAR1, contains a FLAG epitope tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) after the methionine residue of ADAR1. The 3-kb BamHI/XbaI fragment from pFLAG-ADAR1containing the coding sequence for the FLAG-tagged p110 form of ADAR1 was inserted between the BamHI and XbaI sites of the baculovirus transfer vector pVL1393 (Pharmingen, San Diego, CA).

Sf9 cells were grown to a density of 2 x 106 cells/ml and infected with ADAR1 recombinant baculovirus at a multiplicity of infection of 10 to 20 according to supplier's recommendations. At 72 h postinfection, ~109 cells were collected by centrifugation at 5,000 rpm. Cells were resuspended in 1 ml of BD Pharmingen insect cell lysis buffer and 1x complete protease inhibitor mixture (Sigma, St. Louis, MO). After a 45-min incubation at 4°C, protein extract was cleared of debris by centrifugation at 25,000 rpm for 45 min. FLAG-tagged ADAR1 was purified by column chromatography essentially as described previously (26). Briefly, an anti-FLAG M2 monoclonal antibody agarose gel affinity column (Sigma) was equilibrated with BD Pharmingen insect cell lysis buffer. Total infected cell extract was passed over 250 µl of resin four times. The column was then washed with three 1-ml volumes of BD Pharmingen lysis buffer; ADAR protein was eluted with 200 µg of 3x FLAG peptide/ml diluted in lysis buffer.

In vitro RNA editing by ADAR1. The 283-bp kaposin A ORF was inserted into the NotI site of the plasmid vector pCMV-Tag1 (Stratagene) such that the T7 promoter produces a sense transcript. DC1S and MD-III-2 (29) are miniaturized derivatives of hepatitis delta virus (HDV) genotype I and III isolates, respectively, that include the sequences required for forming the secondary structures of the amber/W editing sites particular to these virus genotypes (10). Analysis of editing by ADAR1 in vitro was performed essentially as described previously (29), except that purified ADAR1 was used. Briefly, after transcription from linearized plasmid with T7 RNA polymerase and gel purification, RNA (1.2 fmol) was incubated with purified ADAR1 (2 to 8 nM) for 30 min at 37°C. After extraction and precipitation, editing was assessed as indicated above for K12 RNA and as previously described (29) for HDV RNAs.


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RESULTS
 
Analysis of sequence heterogeneity by direct sequence analysis of RT-PCR and PCR products. Determination of sequence heterogeneity by analysis of cloned PCR products is limited by statistical sampling errors, particularly with small sample sizes, and can be further skewed by the occurrence of sibling clones. Thus, to better determine whether sequence heterogeneity at position 117990 occurs at the level of the viral DNA or as a result of posttranscriptional modification such as RNA editing, we directly sequenced PCR and RT-PCR products derived from HHV-8 DNA and RNA, respectively (Fig. 1). Products for analysis were obtained from BC3 and BCBL1 cells, two PEL cell lines that harbor HHV-8 (2, 41). Peaks for both adenosine and guanosine are clearly present in the electropherogram for the sequence of the PCR-amplified cDNA at position 117,990, indicating that the K12 RNA sequence is heterogeneous at this position in both cell lines (Fig. 1). However, the sequence of the genomic DNA shows only adenosine at this position. The DNA sequence result is consistent with the results of Pfeffer et al. (39), who found that 10 of 10 clones derived from PCR products obtained from genomic DNA had an A at position 117990.


Figure 1
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  		FIG. 1. Electropherograms showing direct sequencing of PCR and RT-PCR products. HHV-8 viral DNA and RNA were harvested from TPA-stimulated BCBL1 and BC3 cells and subjected to PCR and RT-PCR amplification using the primers SZ4 and SZ5 as described in Materials and Methods. Colors: G, black; A, green; C, blue; T/U, red. The sequence shown is in the same orientation as that of the kaposin coding sequence. A dashed line indicates the location of position 117990.

Sensitive and quantitative analysis of sequence heterogeneity at position 117990. The results in Fig. 1 could be interpreted as evidence for RNA editing at position 117990. However, neither the analysis of 10 clones (39) nor our direct sequence results completely rule out the possibility that some fraction of HHV-8 genomes contain G at position 117990, as suggested by Li et al. (28), and that these genomes contribute disproportionately to the population of K12 RNA transcripts. That Pfeffer et al. (39) found no sequence heterogeneity at position 117990 in 10 clones derived from HHV-8 genomic DNA does not indicate that no heterogeneity exists; rather, according to the binomial distribution, this finding places a statistical upper limit of ca. 25% on the abundance of genomes containing G at this position. Our direct sequence analysis places a slightly lower upper limit of 10 to 20% on the abundance of genomes with a G at coordinate 117990 because direct sequencing is unlikely to detect heterogeneity below ca. 10 to 20%.

In order to establish an even lower limit for the level of sequence heterogeneity in the HHV-8 genome, we developed a quantitative assay based on restriction digestion of PCR products that is similar to an assay used for analysis of RNA editing in HDV (40). Because the sequence heterogeneity at position 117990 does not affect any restriction digestion sites in cDNAs derived from the K12 RNA, we introduced two sequence changes in the forward PCR primer K12F3 such that PCR products that have a G at 117990 contain an XcmI restriction site that is not present in those with an A at this position (Fig. 2A). The XcmI recognition site is a 6-bp palindrome interrupted by nine nonspecific bases. This spacing allowed us to position the two sequence changes introduced to create the site six bases or more from the 3' end of the primer such that PCR amplification is efficient; moreover, the end of primer K12F3 is positioned six bases away from position 117990, and its identity does not affect the efficiency of the PCR amplification. Using samples with defined levels of heterogeneity, we found that this assay accurately measures the fraction of 117990 G at levels at least as low as 1%.


Figure 2
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  		FIG. 2. Sensitive quantitative assay for sequence heterogeneity at position 117990. (A) Schematic showing the primary K12 RNA transcript (thin horizontal line), the locations of position 117990, and the primers used for PCR amplification (left and right arrows). Primer K12F3 contains two changes (underlined) that create an XcmI restriction site in PCR products derived from templates with a G at position 117990. Incubation of PCR products containing an A at position 117990 with XcmI yields an undigested 119-bp DNA, whereas PCR products with a G at position 117990 are digested into 93- and 26-bp fragments. (B) The upper panels show the electrophoretic separation of 32P-labeled PCR products either digested with XcmI (+) or undigested (–). The 119- and 93-bp digestion products are indicated as 117990 A and 117990 G, respectively; the 26-bp product is not visible. The results are shown for products obtained from DNA and RNA harvested from BCBL1 and BC3 cells. The lower panel shows digested PCR products obtained from BCBL1 and BC3 cell DNA compared to that of a control template containing 99% A 117990 and 1% G 117990. Brightness and contrast were adjusted identically using the program ImageJ such that the 119-bp digestion product for the 1% G control sample is readily visible; quantification of this sample indicated 0.9% G.

As for the direct sequence analysis (Fig. 1), the quantitative assay clearly showed sequence heterogeneity at position 117990 in the K12 transcript in both PEL cell lines; indeed, more than half of the cDNAs derived from K12 RNA transcripts contained a G at position 117990 (Fig. 2). However, no heterogeneity was detectable in the viral genome (Fig. 2B, upper panels). Comparison of the phosphorimager scan of the digested PCR product from the viral DNA with that of a control template preparation containing 99% A and 1% G at position 117990 indicates that if the viral DNA at this position is heterogeneous, the heterogeneity must be much less than 1% (Fig. 2B, lower panels). Thus, we conclude that the sequence of the viral DNA at position 117990 is effectively homogeneous in both of the PEL cell lines analyzed.

K12 RNA is edited by ADAR1 in vitro. Pfeffer et al. (39) suggested that RNA editing via adenosine deamination could account for the observation that K12 cDNAs contained both A and G at position 117990, but the HHV-8 DNA contained only an A. (Editing changes the targeted adenosine to inosine; however, due to the different base-pairing tendencies of adenosine and inosine, the edited positions appear as guanosines after RT-PCR amplification.) Two host RNA adenosine deaminases, ADAR1 and ADAR2, are known to edit specific adenosines in RNA to inosine (reviewed in reference 3). Quantitative RT-PCR analysis of RNA derived from PEL cells indicated that the mRNA for ADAR1 is 10- to 20-fold more abundant than that for ADAR2. This result is consistent with the general pattern of ADAR1 and ADAR2 expression: ADAR1 is expressed in most tissues and cell types, whereas ADAR2 expression is highest in the brain (23, 31). We therefore analyzed whether human ADAR1, expressed and purified from baculovirus-infected Sf9 cells, could edit position 117990 in vitro using a segment of the K12 transcript as substrate (Fig. 3). We observed that incubation of this RNA with increasing amounts of ADAR1 resulted in increasing amounts of editing at position 117990. Indeed, the K12 transcript was an even better substrate for editing than the antigenomic RNA of HDV, which uses the host RNA editing activity as an essential part of its replication strategy (10, 21). Moreover, 10-fold-higher amounts of RNA were edited to similar levels (not shown). Editing of the K12 substrate RNA in vitro by ADAR1 was highly specific for position 117,990. Direct sequencing of 140 bp of a 175-bp PCR-amplified cDNA fragment derived from RNA edited in vitro indicated that none of the additional 24 adenosines were edited, while the extent of editing at position 117990 was more than 80%. This result is consistent with sequencing results obtained from K12 RNAs isolated from cells: only position 117990 exhibited sequence heterogeneity.


Figure 3
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  		FIG. 3. Kaposin RNA is efficiently edited by ADAR1 in vitro. (A) Kaposin A RNA (1.2 fmol) was incubated with increasing amounts of purified human ADAR1 expressed in Sf9 cells. ADAR1 concentrations were, from left to right: 0, 1, 2, 4, 8, and 16 nM. RNAs were subjected to RT-PCR amplification and digestion with XcmI as described in the text. (B) Editing of kaposin RNA (K12) and two HDV RNAs (MDIII-2 and DC1S) by purified ADAR1. Reaction conditions were identical for all three RNAs.

Editing eliminates the transforming activity of the kaposin A RNA. The HHV-8 K12 transcript has been shown to have oncogenic activity when expressed in fibroblasts (24, 33, 51) and may play a role in the transformed phenotype of KS and PEL cell tumors. The region of the K12 transcript analyzed for oncogenic potential in these studies includes both the ORF for kaposin A and microRNA miR-K10. The activities of both the protein and the miRNA could be affected by editing at position 117990. In the protein sequence, editing changes serine 38 to glycine; in miRNA-K10, editing occurs at position 2, near the 5' end, and could change the specific mRNAs that might be targets for this miRNA (22, 27).

In order to determine whether editing of the transcript might affect its transforming activity, we compared the abilities of kaposin expression constructs containing either A or G at position 117990 to (i) induce focus formation in transfected Rat3 cells and (ii) produce tumors in nude mice upon injection of Rat3 cells stably transfected with these constructs. For these experiments, we used the kaposin expression construct pCMV-Kap-Tag, which contains A at position 117990, and a mutated version of this construct containing G at this position. As previously reported (33), transfection of Rat3 cells with a kaposin expression construct containing an A at the position corresponding to 117990 produced morphologically altered, highly refractile foci not seen in untransfected cells, nor in cells transfected with the empty expression vector pCMV-Tag1 (Fig. 4A). In contrast, the construct containing 117990 G produced no foci in three independent transfection experiments, indicating that the sequence change reduced the transforming activity of the transcript. We extended this analysis of transforming activity by investigating the ability of Rat3 cells stably transfected with A- and G-containing constructs to produce tumors in nude mice. As previously reported (33), nude mice injected with Rat3 cells stably transfected with a kaposin expression construct with A at position 117990 developed tumors that appeared 2 weeks postinjection and continued to grow rapidly (Fig. 4B); all five mice injected with cells expressing this form of the kaposin transcript developed tumors. Remarkably, and consistent with the focus formation results (Fig. 4A), no tumors were observed in any of the mice injected with Rat3 cells stably transfected with a kaposin expression construct with G at position 117990. The same negative result was obtained for cells stably transfected with the empty control vector plasmid pCMV-Tag1. We conclude from these results that RNA editing alters the functional activity of the transcript: unedited transcripts are oncogenic, and edited transcripts are not.


Figure 4
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  		FIG. 4. The transforming ability of kaposin transcripts is lost when position 117,990 is changed to G. (A) Focus formation in Rat3 cells. Low-passage Rat3 cells were transfected with pCMV-Tag1 (vector), pCMV-Kap-Tag (Kap), or pCMV-Kap-Tag in which position 117990 was changed from an A to a G (Kap 117,990G). Foci were counted at 21 days posttransfection. (B) Tumorigenicity of kaposin variants in nu/nu mice. Rat3 cells were stably transfected with the kaposin transcript expression construct pCMV-Kap-Tag designed to produce RNAs containing either A at coordinate 117990 ({triangleup}) or G at coordinate 117990 ({square}) or with the empty vector pCMV-Tag1 ({circ}). Cells were injected into nu/nu mice, and the tumor volume was measured beginning on day 14.

Editing of K12 RNA is induced by activation of lytic replication. The results shown in Fig. 1 and 2, which, together with Fig. 3, indicate that the K12 RNA transcript is edited at position 117990, were obtained for RNAs isolated from cells stimulated with phorbol ester to undergo lytic HHV-8 replication. As shown in Fig. 4, unedited transcripts are oncogenic, whereas edited transcripts are not. Thus, in a population of cells treated with TPA to undergo lytic replication, a majority of K12 transcripts are edited and have little or no transforming activity. Because transforming activity is typically a central feature of latent gammaherpesvirus infection, we considered that the level of editing of the kaposin transcript, and hence its transforming activity, might be affected by the replication state of HHV-8 in PEL cells. We therefore compared the level of editing in untreated BC3 cells and in BC3 cells in which lytic replication was activated by treatment with TPA (Fig. 5). The levels of editing in untreated cells were substantially lower than in cells treated with TPA (8% versus 65%), suggesting that editing of the kaposin transcript is dramatically increased by the switch from latent to lytic replication. In order to rule out potential nonspecific effects of TPA treatment, we also stimulated lytic replication by treatment with 3 mM sodium butyrate, which also activates lytic HHV-8 replication. Consistent with the results obtained with TPA treatment, treatment with 3 mM sodium butyrate also stimulated editing at position 117990; in this case the editing levels were 76%, even higher than the levels observed after TPA treatment.


Figure 5
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  		FIG. 5. Editing at coordinate 117990 is strongly induced by induction of lytic replication. BC3 cells seeded at 2 x 105 per ml were either left untreated or treated with 25 nM TPA or 3 mM sodium butyrate. RNAs were harvested at 48 h and analyzed for sequence heterogeneity at position 117990 as described in the text.


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DISCUSSION
 
We have shown that the sequence heterogeneity at genome coordinate 117990 in the K12 region of HHV-8 is due to RNA editing of the K12 RNA, most likely by the host RNA adenosine deaminase ADAR1. We reached this conclusion after analyzing viral DNA and RNA in two PEL cell lines, BC3 and BCBL1. Using a highly quantitative and sensitive assay that can readily detect sequence heterogeneity below 1%, we found no detectable guanosine at position 117990 in the viral DNA from either cell line, while at the same time the level of guanosine at this position in K12 cDNA approached 70% (Fig. 2). Moreover, the K12 RNA functions very efficiently as a substrate for editing by the host enzyme ADAR1 in vitro. We did not analyze the ability of ADAR2 to edit this RNA, but given that ADAR1 RNA levels are 10- to 20-fold higher in these cells, it seems more likely that ADAR1 is responsible for editing. Definitive determination of which enzyme is responsible for editing the K12 transcript in PEL cells will require further studies, such as knockdown of ADAR1 and ADAR2 expression by the expression of short hairpin RNAs (15). Our results unambiguously confirm the earlier suggestion, based on analysis of cloned PCR products from BCBL1 cells, that the G at position 117990 in K12-derived cDNAs occurs due to RNA editing (39). We cannot explain the results of Li et al. (28), who observed a G at this position in viral DNA isolated from a PEL tumor. Perhaps the reported sequence was peculiar to that particular tumor. Analysis of additional PEL and KS tumors might help to resolve whether there is any heterogeneity at the level of the viral DNA at this location.

RNA editing provides a mechanism for modulating or altering the function or expression level of a protein (4, 6, 11, 20, 44) or microRNA (22, 54). For HHV-8, editing of the K12 transcript eliminates its tumorigenic activity (Fig. 4). Editing changes a serine to a glycine in the kaposin A protein and in the microRNA modifies the second position, which could function to target a specific mRNA(s). Kawahara et al. (22) recently demonstrated that editing of an adenosine in the 5' region of human miR-376 redirected the miRNA from one set of mRNA targets to another. It remains to be determined to what extent the tumorigenic activity of this transcript is due to the kaposin A protein, miR-K10, or both. Editing would also produce the same amino acid change in the kaposin C protein; although a specific function has not yet been attributed to kaposin C, its function could conceivably be altered by editing as well.

Even though the effect of lytic activation on editing demonstrated in Fig. 5 is substantial, it is possible that the coupling between editing and lytic replication is even tighter than shown. Because the virus undergoes lytic activation in a fraction of cells even without treatment (1) and because the K12 transcript is induced during lytic replication, edited K12 transcripts from this small subset of cells could be responsible for the low level of editing observed in untreated cells. Furthermore, because neither TPA nor butyrate activate lytic replication in a majority of treated cells (1), it is possible that unedited transcripts from cells harboring latent HHV-8, despite TPA or butyrate treatment, decrease the total amount of editing measured. Pfeffer et al. (39) reported high levels of editing (12 of 14 clones) in K12 RNAs isolated from BCBL1 cells. It is not clear how that result compares with our data, since it was not clearly stated whether the cells from which the RNAs were obtained were treated with TPA. Also, BCBL1 cell cultures contain a high fraction of cells in which HHV-8 is replicating lytically and could therefore exhibit higher levels of editing in the absence of chemical induction than other PEL cells. To gain a better measure of the effects of lytic activation on editing of the K12 transcript, it might be useful to sort cell populations (of different PEL cell lines) by the type of HHV-8 replication supported (see, for example, reference 1) and then examine the editing in these specific cells.

Regardless of the degree of coupling between editing and lytic replication, the coordinated regulation of K12 RNA editing with the activation of lytic replication (Fig. 5) is particularly interesting in light of the classification of K12 as a class II transcript (50). Thus, PEL cells harboring latent HHV-8 produce a kaposin transcript that is predominantly unedited and that has tumorigenic activity, while PEL cells in which HHV-8 is undergoing lytic replication produce an increased amount of the transcript that is edited and has little or no tumorigenic activity. The differential tumorigenic activities of the latent and lytic K12 RNAs is attractive in light of the general replication strategy of gammaherpesviruses, which produce tumorigenic transcripts during latency (reviewed in reference 16) as a means of maintaining a population of virally infected cells, but might not need such an activity during lytic replication. Because the K12 transcript is induced during lytic replication, we suppose that the induced, edited transcript has a different function that remains to be determined.

It is important to note that, while the K12 transcript can transform Rat3 cells, the process of cell transformation by HHV-8 is complex, and it is not yet clear how this activity is related to HHV-8 tumorigenesis. A number of studies have implicated other HHV-8 transcripts, including lytic genes, in the process (reviewed in reference 16). Thus, the interplay of several genes may be involved. One difficulty in advancing insight has been the inability to readily transform infected cultured endothelial cells, the target cell infected in KS (see, for example, references 14 and 25). Furthermore, it is not known whether the same viral transcripts are involved in KS and PEL tumorigenesis. In the present study, we analyzed editing of the K12 transcript in PEL cells. It will be interesting to see whether the K12 transcript is also edited in HHV-8-infected endothelial cells and in KS tumors and whether the same correlation between lytic activation and K12 editing that we observed in BC3 cells also exists in such cells. Regarding the difficulty in experimentally transforming cultured cells with HHV-8, we note that editing of the K12 transcript could vary in different cell types and culture conditions. Our results suggest that analysis of K12 transcript editing could be important for fully understanding studies of HHV-8 tumorigenicity.

The mechanism whereby editing of the K12 transcript increases during lytic activation requires further study. One possibility is that the level of ADAR1 is induced during lytic replication. Expression of one form of ADAR1 is increased by interferon treatment (17, 37) and inflammation (53). Lytic activation of HHV-8 is accompanied by increased production of numerous cytokines (reviewed in references 35 and 36). Preliminary analysis of ADAR1 and ADAR2 mRNA levels after TPA treatment using quantitative RT-PCR indicated no changes (data not shown). Nevertheless, because TPA treatment activates lytic replication in only a fraction of cells (ca. 20 to 30%) (1), it remains possible that ADAR expression is induced by host or viral cytokines that are stimulated during lytic replication or is directly affected by viral lytic genes (5, 42, 43). To more definitively determine whether ADAR expression is increased during lytic activation may require isolation of activated cells (1) prior to RNA analysis. Another possible explanation for increased editing during lytic activation is that editing somehow occurs more efficiently on the transcript that is made during lytic replication. The lytic K12 transcript starts from a different promoter and is not spliced, whereas the latent transcript is spliced (7, 38). Perhaps these differences affect the access of ADAR1, which is primarily located in the nucleus (37), to the latent and lytic K12 transcripts.

Editing of the HHV-8 K12 RNA is similar in some respects to editing that occurs during replication of HDV (10), for which editing levels also vary according to the stage of replication. HDV uses this host enzymatic activity to produce a protein variant that is required for virus particle formation; editing thus performs an essential function in the HDV replication cycle. Further studies are necessary to determine the specific role of K12 transcript editing in the life cycle of HHV-8.


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ACKNOWLEDGMENTS
 
This study was supported in part by grants R01AI42324 (J.L.C.) and R01CA78210 (L.J.R.) from the National Institutes of Health. The Lombardi Cancer Research Center Animal Care Facility, supported by Public Health Service grant P30 CA51008-09, provided assistance with animal studies.

We thank Peter Medveczky, University of South Florida, for the Rat3 cells and David Lazinski, Tufts University, for ADAR1 expression plasmid pDL701. We thank Renxiang Chen for helpful comments on the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Georgetown University Medical Center, 3900 Reservoir Rd., NW, Washington, DC 20007. Phone: (202) 687-1052. Fax: (202) 687-1800. E-mail: caseyj{at}georgetown.edu Back

{triangledown} Published ahead of print on 3 October 2007. Back


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Journal of Virology, December 2007, p. 13544-13551, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.01521-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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