In vitro model for lytic replication, latency, and transformation of an oncogenic alphaherpesvirus.

Marek's disease virus (MDV) is an alphaherpesvirus that causes deadly T-cell lymphomas in chickens and serves as a natural small animal model for virus-induced tumor formation. In vivo, MDV lytically replicates in B cells that transfer the virus to T cells in which the virus establishes latency. MDV also malignantly transforms CD4+ T cells with a T(reg) signature, ultimately resulting in deadly lymphomas. No in vitro infection system for primary target cells of MDV has been available due to the short-lived nature of these cells in culture. Recently, we characterized cytokines and monoclonal antibodies that promote survival of cultured chicken B and T cells. We used these survival stimuli to establish a culture system that allows efficient infection of B and T cells with MDV. We were able to productively infect with MDV B cells isolated from spleen, bursa or blood cultured in the presence of soluble CD40L. Virus was readily transferred from infected B to T cells stimulated with an anti-TCRαVß1 antibody, thus recapitulating the in vivo situation in the culture dish. Infected T cells could then be maintained in culture for at least 90 d in the absence of TCR stimulation, which allowed the establishment of MDV-transformed lymphoblastoid cell lines (LCL). The immortalized cells had a signature comparable to MDV-transformed CD4+ α/ß T cells present in tumors. In summary, we have developed a novel in vitro system that precisely reflects the life cycle of an oncogenic herpesivrus in vivo and will allow us to investigate the interaction between virus and target cells in an easily accessible system.

Role of the short telomeric repeat region in Marek's disease virus replication, genomic integration, and lymphomagenesis.

UNLABELLED: Marek's disease virus (MDV) is a cell-associated alphaherpesvirus that causes generalized polyneuritis and T-cell lymphomas in chickens. MDV is able to integrate its genome into host telomeres, but the mechanism of integration is poorly understood. The MDV genome harbors two arrays of telomeric repeats (TMR) at the ends of its linear genome: multiple telomeric repeats (mTMR), with a variable number of up to 100 repeats, and short telomeric repeats (sTMR), with a fixed number of 6 repeats. The mTMR have recently been shown to play an important role in MDV integration and tumor formation; however, the functions of the sTMR have remained unknown. In this study, we demonstrate that deletion of the sTMR in the MDV genome abrogates virus replication, while extensive mutation of the sTMR does not, indicating that the presence of the sTMR but not the sTMR sequence itself is important. Furthermore, we generated a panel of truncation mutants to determine the minimal length of the sTMR and observed a direct correlation between sTMR length and MDV replication. To address the role of sTMR in MDV replication, integration, and tumorigenesis, sTMR sequences were replaced by a scrambled repeated sequence (vsTMR_mut). vsTMR_mut replicated comparably to parental and revertant viruses in vitro. In vivo, however, a significant reduction in disease and tumor incidence was observed in chickens infected with vsTMR_mut that also correlated with a reduced number of viral integration sites in tumor cells. Taken together, our data demonstrate that the sTMR play a central role in MDV genome replication, pathogenesis, and MDV-induced tumor formation. IMPORTANCE: Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects chickens and causes high economic losses in the poultry industry. MDV integrates its genetic material into host telomeres, a process that is crucial for efficient tumor formation. The MDV genome harbors two arrays of telomeric repeats (TMR) at the ends of its linear genome that are identical to host telomeres and that are termed mTMR and sTMR. mTMR have been recently shown to be involved in MDV integration, while the functions of sTMR remain unknown. Here, we demonstrate that the presence and length of sTMR sequence, but not the exact nucleotide sequence, are crucial for MDV replication. Furthermore, the sTMR contribute to the high integration frequency of MDV and are important for MDV pathogenesis and tumor formation. As a number of herpesviruses harbor arrays of telomeric repeats (TMR), MDV serves as a model to determine the role of the herpesvirus TMR in replication, integration, and pathogenesis.