Herpesviruses are a large group of DNA viruses that cause disease in many organisms [1, 2]. The ability of herpesviruses to occupy diverse biological niches, the wide range of cell types targeted for primary infection and latency, along with acute and chronic infection symptoms, contribute to the significance and relevance of herpesvirus research. In particular, human herpesviruses are a major public health concern due to the prevalence of such viruses within and among populations worldwide and the associated pathologies. Two of the eight disease-causing human herpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma virus (KSHV), are associated with neoplastic transformation. A closely related avian herpesvirus, Marek's disease virus (MDV; family Herpesviridae, genus Mardivirus, species Gallid herpesvirus 2) also exhibits oncogenic properties inducing lymphoid tumors in chickens.

MDV rapidly infects B cells and subsequently transforms T cells during pathogenesis of the highly contagious neoplastic disease of chickens known as Marek's disease (MD). Since discovery [3], MD has been extensively studied and utilized as a valuable biomedical model for human herpesvirus infection and viral induced malignancies [4, 5]. MDV is of great concern in agriculture, causing large economic losses as well as impacting animal well-being. Despite current control through the use of vaccines, viral evolution leads to increasing virulence and vaccine breaks on a cyclical basis [6, 7].

A fascinating biological property that MDV shares with all herpesviruses is the ability to persist indefinitely in the host and evade immune response by establishing a latent infection. The molecular mechanisms controlling lytic replication and latent persistence of herpesviruses have been the subject of intense study in recent years, aided largely by advances made in the field of viral genetics. Although new information has emerged from these studies regarding mechanisms of the lytic cycle, factors controlling latency and tumorigenesis remain poorly understood. MDV presents a unique model system in which to explore the mechanisms of herpesvirus latency and virus-induced tumor formation.

Another intriguing aspect of herpesvirus infection is that both episomal and linear-integrated forms of the virus are present in the host [8]. One consideration is that latency, and potential reactivation of the virus, could be achieved in part through genomic interactions between the host and virus, evasion through integration and recurring infection through emergence/release. Evidence for herpesvirus integration into human chromosomes was reported for Human Herpesvirus 6 (HHV-6) and EBV [9–14]. Since Delecluse and Hammerschmidt (1993) first reported on the potential for MDV to exist in an integrated state, it has been a subject of debate as to whether the virus integrates stably (and where) into the chicken genome or is merely associated and how such associations might be germane to pathogenesis and oncogenesis. Since that early report, complete genome sequences have been reported for several MDV strains and BAC vectors containing the full-length MDV genome have been cloned [15–20]. The sequencing provided data supportive of the hypothesis that MDV interacts with the chicken genome, as it was discovered that the circular viral genome encodes several orthologous host genes including the telomerase RNA (vTR) gene as well as telomeric repeats (TTAGGG) [17, 18, 21].

To resolve whether and where MDV integrates in the host chicken genome we employed high-resolution multi-color fluorescence in situ hybridization (FISH) using the tools developed from both the MDV and chicken sequencing efforts. We investigated the details of integration on both intra-and inter-chromosomal levels by mapping the location of MDV integrations. In depth analysis of MDV integration sites allowed us to investigate several specific objectives, including the clonal composition of MDV tumors and integration site preference of the virus. We show here that MDV specifically integrates into the telomeric regions of chicken chromosomes in MDV-induced clonal lymphomas.

Delecluse and colleagues provided initial evidence for viral integration of MDV into chicken chromosomes [30, 31]. At the time, this work was necessarily limited in scope and resolution by the lack of sequence information and probes for both the MDV and the chicken chromosomes. Having the advantage of genomics tools and high resolution cytogenetics, we are able to now show that the MDV genome is present in an integrated state at the telomeres of specific chicken chromosomes of tumors resulting from MDV-infection. MDV was observed on both sister chromatids indicating the viral DNA replicates in a semi-conservative fashion along with the host genomic DNA. We also noted that signal intensity varied among integration locations, suggestive of different numbers of integrated copies. The sequencing of the chicken genome coupled with the availability of chromosome specific BACs to identify chicken microchromosomes [24, 25, 32] created the opportunity for mapping of integrations and allowed us to address several crucial questions related to integration site preference and lineages of transformation events.

Preferences of Viral Integration: Subchromosomal and Chromosomal

MDV integrates at the chicken telomeres

Our initial working hypothesis was that MDV preferentially integrates into the chicken mega-telomeres, which are ultra-long telomeric arrays ranging in size from 100 s of Kb to several Mb [24, 28, 33–35]. Although our data did not support this hypothesis, the MDV genome was found exclusively at the telomeric regions of the host chromosomes. Viral integration into the interstitial telomeric sequences was not observed. We hypothesize that the terminal ends of chromosomes may provide an environment more conducive to integration, perhaps through a specialized chromatin confirmation (e.g., the t-loop of the telomere as it is replicated, and/or the actual telomerase extension apparatus). Interestingly, HHV-6 was found integrated into the subtelomeric region of human chromosomes and, similar to MDV, possesses telomere repeat arrays within its genome [9, 36]. The telomeric array repeats were hypothesized to assist the HHV-6 integration mechanism through homologous recombination [9].

In exploring our initial hypothesis regarding MDV preference for integration into mega-telomeres we found additional evidence for extensive genotypic variation in telomere array sizes within and among chicken genetic lines and cell lines [28, 35]. Although variation for the number of mega-telomere loci observed among the birds in this study was to be expected from an F1 generation (see methods), the variation among tumors within birds was not expected. Inter-tumor variation (within an individual) could be a manifestation of gain or loss of telomeric repeat sequence due to genomic instability. Another consideration is the unknown effect that integration of MDV DNA may have upon the telomere sequence itself.

MDV integration: random versus targeted?

Unlike HHV-6, which typically integrates into one or two chromosomes [10, 14, 37] MDV integrates into more chromosomes, typically five (and as many as nine). We found integrations across all size categories of chromosomes, i.e., both macro-and microchromosomes (Figure 2C). A few chromosomes stand out as interesting candidates for future studies, as highlighted by Figure 5. In the two instances that MDV was found integrated on GGA 9, both homologs were involved. GGA 9 encodes the single copy chicken telomerase RNA (cTR) component of the telomerase enzyme [36]. Sequencing of the MDV genome showed that it also encodes two copies of TR (viral (v)TR) with high sequence identity (85-88%) to cTR [16, 17, 38, 39]. In comparison to cTR, vTR was more efficient at restoring telomerase activity in telomerase negative cell systems [39]. Furthermore, when vTR was deleted from the MDV genome, the vTR-deleted virus showed reduced capabilities in causing lymphomas, suggesting that vTR is a key element in promoting T-cell lymphomagenesis [40]. Whether MDV is specifically utilizing vTR to aid in integration by taking advantage of telomerase activity at the telomeres requires further study, and would be a novel role for TR (chicken or viral).

Several other chromosomes frequently exhibited MDV integrations, for example GGA 4, 6, 12, and 20, were found in common among tumors between three or more birds (Figure 5). Interestingly, GGA 4 encodes the chemokine interleukin-8 (IL-8) gene sequence [41] and MDV sequencing showed the virus also encodes an IL-8 gene [16, 42]. The presence of host sequences in the virus genome is noteworthy because a plausible mechanism for MDV to obtain such chicken-derived genes (vTR and vIL-8) and sequences (e.g., TTAGGG repeats) is by integration into the chicken chromosomes, and then subsequent emergence with exogenous host sequence. Since telomeric integration appears to be the preferred location, we propose that viral integration near host genes is a rare event. However, if a "captured" host gene sequence provides a selective advantage to the virus, e.g., vTR's ability to promote oncogenesis or perhaps facilitate integration, it would persist in future generations of virus.

Clonality of Late-Stage Lymphomas: Lineage relationships among tumors within birds

Another objective of this study was to improve our understanding regarding clonality of MDV-induced lymphomas. That is, are the multiple tumors of a bird derived from a single transformation event (monoclonal origin) or do the tumors arise from independent events (polyclonal origin)? Clonality of tumors has been addressed by immunoglobulin analysis, T-cell receptor gene rearrangements, X-chromosome methylation patterns, various molecular markers for somatic mutations, and analysis of viral integration [reviewed in [43–48]]. Viral integration sites and viral sequencing have proved to be useful markers when assessing clonality of tumors [49]. Here we utilized the mapped MDV integrations to gain insight into tumor lineage(s).

Mapping lineage analysis indicates that four (of six) birds had multiple tumors of monoclonal origin, inclusive of one individual (Bird 1) showing a derived cell population (Figure 6), which is not unexpected given lack of genome stability in cancers and/or the possibility that the virus could be mobile. The remaining two birds (Bird 3 and 12) displayed tumors consist with a mixed or polyclonal origin (Figure 6). The most conservative analysis of the three tumors from Bird 3 places them in a polyclonal category, that is, three separate transformation events occurred to form each tumor. Alternatively, the tumors from Bird 3 could be related, which raises the possibility that the virus is capable of remerging from its integrated state and moving to new integrated locations. Alternatively, tumors 3-T1 and 3T-2 could be of monoclonal origin given the loss of one of the integration sites (GGA 13). However, the third tumor (3-T3) showed only two MDV integration sites (GGA 12 and 18) in common with the other two tumors, suggestive of a separate transformation event. The tumors from Bird 12 may also represent a polyclonal set of tumors, indicating tumors 12-T1 and 12-T2 have different founder cells altogether (Figure 6). However, it must be noted that 12-T1 and 12-T2 both have three unidentified MDV integration sites and the possibility remains that those three sites could be in common.

The indications for predominately, but not exclusively, monoclonal tumor origins, as well as derived cell populations within the MDV lymphomas, illustrates the complexity of tumor biology and the potentially diverse strategies employed by the herpesvirus. Understanding the timeframe during pathogenesis when MDV integrates into the chicken genome will be essential to assessing both the utility of integration profiles as a marker for clonality and the significance of integration on the latency cycle. In addition, further investigation of the effect of viral integration on cellular transformation is necessary. Integration may have a profound effect on transformation, or it may simply serve as a mechanism of maintaining the latent genome. We are currently investigating the stages at which MDV integrates, from early infection to late oncogenesis, and have preliminary indications that MDV integrates prior to transformation and tumorigenesis. Further research on the molecular mechanism for telomere-targeting of MDV integration should provide new insight regarding the role of telomeres in aspects of host genome stability and also viral evolution.