Denis Rasschaert

mdv1-miR-M7-5p, located in the newly identified first intron of the latency-associated transcript of Marek's disease virus, targets the immediate-early genes ICP4 and ICP27.

Mareks disease virus serotype 1 (MDV-1) is an oncogenic alphaherpesvirus causing fatal T-cell lymphoma in chickens. MDV latency is characterized by the production of latency-associated transcripts (LATs), a family of non-protein-coding spliced RNAs. A cluster of four microRNAs (cluster mdv1-miR-M8-M10) was identified, but not formally mapped, at the predicted LAT 5 end. We established a LAT cDNA library from latently MDV-infected cell line MSB-1. We identified 22 highly variable LATs, which were due to the extensive alternative splicing of a total of 14 introns. RACE PCR confirmed the predicted 3 end and allowed identification of the 5 end, 400 nt upstream of the previously predicted LAT end. The LATs share their transcription start site with the microRNA-expressing transcript described previously, localizing the microRNAs to the first LAT intron and identifying the LATs as the primary transcripts of the microRNAs. We identified MDV immediate-early (IE) genes ICP4 and ICP27 as putative targets of mdv1-miR-M7-5p, the third microRNA of the cluster mdv1-miR-M8-M10. Endogenously expressed mdv1-miR-M7-5p in MSB-1 cells reduced luciferase activity significantly when microRNA-responsive elements from ICP4 or ICP27 were cloned in the 3 UTR of the firefly luciferase gene. ICP27 protein levels were decreased by 70u200a% when the mdv1-miR-M7-5p precursor was co-expressed with an ICP27 expression plasmid. Additionally, we showed a negative correlation between the decreased expression of mdv1-miR-M7-5p and an increase in ICP27 expression during virus reactivation. Our results suggest that, by targeting two IE genes, MDV microRNAs produced from LAT transcripts may contribute to establish and/or maintain latency.

Kinetic expression analysis of the cluster mdv1-mir-M9-M4, genes meq and vIL-8 differs between the lytic and latent phases of Marek's disease virus infection.

Mareks disease virus (GaHV-2) is an alphaherpesvirus that induces T-cell lymphoma in chickens. The infection includes both lytic and latent stages. GaHV-2 encodes three clusters of microRNAs (miRNAs) located in the internal (I)/terminal (T) repeat (R) regions. We characterized transcripts encompassing the mdv1-mir-M9-M4 and mir-M11-M1 clusters located in the IR(L)/TR(L) region, upstream and downstream from the meq oncogene, respectively. By 5- and 3-RACE-PCR and targeted RT-PCR, we showed that mdv1-mir-M9-M4 could be transcribed from an unspliced transcript or from at least 15 alternatively spliced transcripts covering the IR(L)/TR(L) region, encompassing the meq and vIL-8 genes and localizing the mdv1-mir-M9-M4 cluster to the first intron at the 5-end. However, all these transcripts, whether spliced or unspliced, seemed to start at the same transcriptional start site, their transcription being driven by a single promoter, prmiRM9M4. We demonstrated alternative promoter usage for the meq and vIL-8 genes, depending on the phase of GaHV-2 infection. During the latent phase, the prmiRM9M4 promoter drove transcription of the meq and vIL-8 genes and the mdv1-mir-M9-M4 cluster in the first intron of the corresponding transcripts. By contrast, during the lytic phase, this promoter drove the transcription only of the mdv1-mir-M9-M4 cluster to generate unspliced mRNA, the meq and vIL-8 genes being transcribed principally from their own promoters. Despite the expression of meq and the mdv1-mir-M9-M4 cluster under two different transcriptional processes during the latent and lytic phases, our data provide an explanation for meq expression and mdv1-mir-M4-5P overexpression in miRNA libraries from GaHV-2-infected cells, regardless of the phase of infection.

Alternative splicing and nonsense-mediated decay regulate telomerase reverse transcriptase (TERT) expression during virus-induced lymphomagenesis in vivo

BackgroundTelomerase activation, a critical step in cell immortalization and oncogenesis, is partly regulated by alternative splicing. In this study, we aimed to use the Mareks disease virus (MDV) T-cell lymphoma model to evaluate TERT regulation by splicing during lymphomagenesis in vivo, from the start point to tumor establishment.ResultsWe first screened cDNA libraries from the chicken MDV lymphoma-derived MSB-1 T- cell line, which we compared with B (DT40) and hepatocyte (LMH) cell lines. The chTERT splicing pattern was cell line-specific, despite similar high levels of telomerase activity. We identified 27 alternative transcripts of chicken TERT (chTERT). Five were in-frame alternative transcripts without in vitro telomerase activity in the presence of viral or chicken telomerase RNA (vTR or chTR), unlike the full-length transcript. Nineteen of the 22 transcripts with a premature termination codon (PTC) harbored a PTC more than 50 nucleotides upstream from the 3 splice junction, and were therefore predicted targets for nonsense-mediated decay (NMD). The major PTC-containing alternatively spliced form identified in MSB1 (ie10) was targeted to the NMD pathway, as demonstrated by UPF1 silencing. We then studied three splicing events separately, and the balance between in-frame alternative splice variants (d5f and d10f) plus the NMD target i10ec and constitutively spliced chTERT transcripts during lymphomagenesis induced by MDV indicated that basal telomerase activity in normal T cells was associated with a high proportion of in-frame non functional isoforms and a low proportion of constitutively spliced chTERT. Telomerase upregulation depended on an increase in active constitutively spliced chTERT levels and coincided with a switch in alternative splicing from an in-frame variant to NMD-targeted variants.ConclusionsTERT regulation by splicing plays a key role in telomerase upregulation during lymphomagenesis, through the sophisticated control of constitutive and alternative splicing. Using the MDV T-cell lymphoma model, we identified a chTERT splice variant as a new NMD target.

ICP27 protein of Marek's disease virus interacts with SR proteins and inhibits the splicing of cellular telomerase chTERT and viral vIL8 transcripts

All herpesviruses have a post-transcriptional regulatory protein that prevents precursor mRNA splicing and leads to the shutting off of host protein synthesis. The ICP27 protein of herpes simplex virus 1 (HSV-1) is the prototype of these proteins. Mareks disease virus (MDV-1), an alphaherpesvirus that induces lymphoma in birds, also has an ICP27 protein that is produced in lytic MDV-1-infected cells. We characterized this protein. We demonstrated ICP27 production in latently infected MSB-1 cells, but only on MDV-1 reactivation. ICP27 was found predominantly in specific structures within the nucleus. The ICP27 of MDV-1 colocalized and interacted with SR proteins. We demonstrated inhibitory effects of MDV-1 ICP27 on the splicing of both the viral vIL8 and cellular chTERT (telomerase reverse transcriptase) genes. Thus, the ICP27 of MDV-1 plays a similar role to the ICP27 of HSV-1 and may be involved in MDV-1 replication and the development of Mareks disease.

Vaccination against Marek's disease reduces telomerase activity and viral gene transcription in peripheral blood leukocytes from challenged chickens.

We investigated whether telomerase activity and viral gene transcription were associated with protection against the RB-1B strain of Mareks disease virus (MDV) in chickens vaccinated with Rispens CVI988 or the herpes virus of turkey (HVT). Telomerase activity in peripheral blood leukocytes (PBLs) seemed to be an appropriate marker of lymphoma and levels of viral transcription were correlated with the virulence of MDV strains. Vaccinated protected birds had lower levels of telomerase activity and RB-1B viral gene transcription than unvaccinated chickens infected with RB-1B. The decrease in RB-1B viral transcription was more marked in chickens vaccinated with CVI988 than in those vaccinated with HVT. Indeed, RB-1B viral transcription was not detectable after 14 days post-challenge. In conclusion, telomerase activity and gene transcription in challenge MDV strains are potential new reliable criteria of protection in vaccinated chickens.

Small RNA cloning and sequencing strategy affects host and viral microRNA expression signatures

The establishment of the microRNA (miRNA) expression signatures is the basic element to investigate the role played by these regulatory molecules in the biology of an organism. Mareks disease virus 1 (MDV-1) is an avian herpesvirus that naturally infects chicken and induces T cells lymphomas. During latency, MDV-1, like other herpesviruses, expresses a limited subset of transcripts. These include three miRNA clusters. Several studies identified the expression of virus and host encoded miRNAs from MDV-1 infected cell cultures and chickens. But a high discrepancy was observed when miRNA cloning frequencies obtained from different cloning and sequencing protocols were compared. Thus, we analyzed the effect of small RNA library preparation and sequencing on the miRNA frequencies obtained from the same RNA samples collected during MDV-1 infection of chicken at different steps of the oncoviral pathogenesis. Qualitative and quantitative variations were found in the data, depending on the strategy used. One of the mature miRNA derived from the latency-associated-transcript (LAT), mdv1-miR-M7-5p, showed the highest variation. Its cloning frequency was 50% of the viral miRNA counts when a small scale sequencing approach was used. Its frequency was 100 times less abundant when determined through the deep sequencing approach. Northern blot analysis showed a better correlation with the miRNA frequencies found by the small scale sequencing approach. By analyzing the cellular miRNA repertoire, we also found a gap between the two sequencing approaches. Collectively, our study indicates that next-generation sequencing data considered alone are limited for assessing the absolute copy number of transcripts. Thus, the quantification of small RNA should be addressed by compiling data obtained by using different techniques such as microarrays, qRT-PCR and NB analysis in support of high throughput sequencing data. These observations should be considered when miRNA variations are studied prior addressing functional studies.

Alternative splicing of a viral mirtron differentially affects the expression of other microRNAs from its cluster and of the host transcript

ABSTRACT Interplay between alternative splicing and the Microprocessor may have differential effects on the expression of intronic miRNAs organized into clusters. We used a viral model — the LAT long non-coding RNA (LAT lncRNA) of Mareks disease oncogenic herpesvirus (MDV-1), which has the mdv1-miR-M8-M6-M7-M10 cluster embedded in its first intron — to assess the impact of splicing modifications on the biogenesis of each of the miRNAs from the cluster. Drosha silencing and alternative splicing of an extended exon 2 of the LAT lncRNA from a newly identified 3′ splice site (SS) at the end of the second miRNA of the cluster showed that mdv1-miR-M6 was a 5′-tailed mirtron. We have thus identified the first 5′-tailed mirtron within a cluster of miRNAs for which alternative splicing is directly associated with differential expression of the other miRNAs of the cluster, with an increase in intronic mdv1-miR-M8 expression and a decrease in expression of the exonic mdv1-miR-M7, and indirectly associated with regulation of the host transcript. According to the alternative 3SS used for the host intron splicing, the mdv1-miR-M6 is processed as a mirtron by the spliceosome, dispatching the other miRNAs of the cluster into intron and exon, or as a canonical miRNA by the Microprocessor complex. The viral mdv1-miR-M6 mirtron is the first mirtron described that can also follow the canonical pathway.

The chicken miR-150 targets the avian orthologue of the functional zebrafish MYB 3'UTR target site

BackgroundThe c-myb proto-oncogene is the founding member of a family of transcription factors involved principally in haematopoiesis, in diverse organisms, from zebrafish to mammals. Its deregulation has been implicated in human leukaemogenesis and other cancers. The expression of c-myb is tightly regulated by post-transcriptional mechanisms involving microRNAs. MicroRNAs are small, highly conserved non-coding RNAs that inhibit translation and decrease mRNA stability by binding to regulatory motifs mostly located in the 3UTR of target mRNAs conserved throughout evolution. MYB is an evolutionarily conserved miR-150 target experimentally validated in mice, humans and zebrafish. However, the functional miR-150 sites of humans and mice are orthologous, whereas that of zebrafish is different.ResultsWe identified the avian mature miRNA-150-5P, Gallus gallus gga-miR-150 from chicken leukocyte small-RNA libraries and showed that, as expected, the gga-miR-150 sequence was highly conserved, including the seed region sequence present in the other miR-150 sequences listed in miRBase. Reporter assays showed that gga-miR-150 acted on the avian MYB 3UTR and identified the avian MYB target site involved in gga-miR-150 binding. A comparative in silico analysis of the miR-150 target sites of MYB 3UTRs from different species led to the identification of a single set of putative target sites in amphibians and zebrafish, whereas two sets of putative target sites were identified in chicken and mammals. However, only the target site present in the chicken MYB 3UTR that was identical to that in zebrafish was functional, despite the additional presence of mammalian target sites in chicken. This specific miR-150 site usage was not cell-type specific and persisted when the chicken c-myb 3UTR was used in the cell system to identify mammalian target sites, showing that this miR-150 target site usage was intrinsic to the chicken c-myb 3UTR.ConclusionOur study of the avian MYB/gga-miR-150 interaction shows a conservation of miR-150 target site functionality between chicken and zebrafish that does not extend to mammals.

Oncogenic Marek's disease herpesvirus encodes an isoform of the conserved regulatory immediate early protein ICP27 generated by alternative promoter usage.

Herpesvirus gene expression is temporally regulated, with immediate early (IE), early (E) and late (L) genes. ICP27, which is involved in post-transcriptional regulation, is the only IE gene product conserved in all herpesviruses. We show here that the ICP27 transcript of the oncogenic Mareks disease virus shares the same polyadenylation signal as the bicistronic glycoprotein K-ICP27 transcript and is regulated by alternative promoter usage, with transcription from its own promoter (pICP27) or that of gK (pgK). The pgK can generate a spliced ICP27 transcript yielding an N-terminal-deleted ICP27 isoform (ICP27ΔN) that, like ICP27, co-localizes with the SR protein in infected cells, but with a diffuse nuclear distribution. The pICP27 includes functional responsive elements (REs) for SP1, AP1 and CREB, is essentially active during the lytic phase and leads to exclusive expression of the native form of ICP27. The alternative promoter, pgK, including active REs for GATA, P53 and CREB, preferentially generates the gK transcript during the lytic phase and the spliced ICP27 transcript (ICP27ΔN) during the latent phase. An analysis of the DNA methylation marks of each promoter showed that pgK was systematically demethylated, whereas pICP27 was methylated during latency and demethylated during the lytic stage. Thus, MDV ICP27 gene expression is dependent on alternative promoters, the usage of which is regulated by DNA methylation, which differs between viral stages.

The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells

The minimal vertebrate telomerase enzyme is composed of a protein component (telomerase reverse transcriptase, TERT) and an RNA component (telomerase RNA, TR). Expression of these two subunits is sufficient to reconstitute telomerase activity in vitro, while the formation of a holoenzyme comprising telomerase-associated proteins is necessary for proper telomere length maintenance. Previous reports demonstrated the high processivity of the human telomerase complex and the interspecies compatibility of human TERT (hTERT). In this study, we tested the function of the only known viral telomerase RNA subunit (vTR) in association with human telomerase, both in a cell-free system and in human cells. When vTR is assembled with hTERT in a cell-free environment, it is able to interact with hTERT and to reconstitute telomerase activity. However, in human cells, vTR does not reconstitute telomerase activity and could not be detected in the human telomerase complex, suggesting that vTR is not able to interact properly with the proteins constituting the human telomerase holoenzyme.