Abstract
Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes a devastating neoplastic disease in chickens. MDV has been shown to integrate its genome into the telomeres of latently infected and tumor cells, which is crucial for efficient tumor formation. Telomeric repeat arrays present at the ends of the MDV genome facilitate this integration into host telomeres; however, the integration mechanism remains poorly understood. Until now, MDV integration could only be investigated qualitatively upon infection of chickens. To shed further light on the integration mechanism, we established a quantitative integration assay using chicken T cell lines, the target cells for MDV latency and transformation. We optimized the infection conditions and assessed the establishment of latency in these T cells. The MDV genome was efficiently maintained over time, and integration was confirmed in these cells by fluorescence in situ hybridization (FISH). To assess the role of the two distinct viral telomeric repeat arrays in the integration process, we tested various knockout mutants in our in vitro integration assay. Efficient genome maintenance and integration was thereby dependent on the presence of the telomeric repeat arrays in the virus genome. Taken together, we developed and validated a novel in vitro integration assay that will shed light on the integration mechanism of this highly oncogenic virus into host telomeres.
Highlights
IntroductionThe causative agent of this lymphoproliferative disease is Marek’s disease virus (MDV)
Expression cassette driven by the HSV-1 thymidine kinase (TK) promoter was inserted into the minimal fertility factor of the wild type and previously generated viral telomere mutants: ∆multiple telomeric repeats (mTMR) containing a complete deletion of the mTMRs (TTAGGG)27; sTMRmut in which the short telomeric repeats (sTMR) repeats (TTAGGG)6 were replaced by scrambled repeats (ACGACA)6 ; and TE2 in which both the sTMR and mTMR were replaced by scrambled repeats (ACGACA)n (Figure 1) [12,24]
Previous studies showed that viral telomeric repeats based assays
Summary
The causative agent of this lymphoproliferative disease is Marek’s disease virus (MDV) This virus causes major losses in the poultry industry, despite widespread vaccination for decades and extensive research on MDV virulence factors, host resistance and more efficient vaccines. Upon infection of the host, MDV establishes latency primarily in CD4+ T cells, allowing the virus to persist in the host for life [9] These infected CD4+ T cells can become transformed, resulting in deadly lymphomas [10]. Both latently infected and MDV-induced tumor cells harbor the integrated virus genome in the telomeres of one or multiple host chromosomes [11,12,13].
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