Role of the Short Telomeric Repeat Region in Marek's Disease Virus Replication, Genomic Integration, and Lymphomagenesis

Abstract

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.

Vaccination and Host Marek's Disease-Resistance Genotype Significantly Reduce Oncogenic <b><i>Gallid alphaherpesvirus 2</i></b> Telomere Integration in Host Birds

“…ic repeats [Kaufer et al, 2011;Greco et al, 2014]. In the MDV infection cycle, oncogenic strains transition from a cytolytic replication stage, during which the viral genome takes an episomal and extrachromosomal form, to a latent stage, characterized by evasion of host immune responses Adldinger and Calnek, 1973;Baigent and Davison, 2004;Osterrieder et al, 2006;Arumugaswami et al, 2009;Gimeno et al, 2011] with the latter having a temporal overlap with MDV-host telomere integration events [Robinson et al, 2010[Robinson et al, , 2014 and decline in the quantity of extrachromosomal MDV genomes [Kaschka-Dierich et al, 1979;Delecluse and Hammerschmidt, 1993].…”

Epigenetics and Genetics of Viral Latency

“…Precisely why these viruses have captured cellular telomere repeats and use these chromosomal sites for integration during latency is not known, but it is possible that telomeres provide a unique recombinogenic environment conducive for viral integration and mobilization during reactivation. For MDV, telomere integration enhances tumor formation (Greco et al, 2014). It is not yet clear whether these integrated viral genomes can reactivate efficiently to complete their viral productive cycles or if these integrations are pathogenic aberrations.…”

Telomeres and Telomerase: Role in Marek’s Disease Virus Pathogenesis, Integration and Tumorigenesis

“…MDV integration can occur in all chicken chromosome classes, including macro-, intermediate- and micro-chromosomes without a preference for the either the p or q arm [ 22 ]. The presence of the virus genome in host telomeres has been confirmed by various techniques including fluorescent in situ hybridization (FISH) and pulsed-field gel electrophoresis (PFGE) [ 21 , 23 , 24 , 25 , 26 ]. Integration into host telomeres has also been shown for other herpesviruses, which will briefly be addressed within this review.…”

“…This phenotype was comparable to the mutant viruses harboring the mutated TMR, indicating that the mTMR plays a major role in the MDV genome integration into host telomeres. In addition, it has recently been demonstrated that the sTMR has a dual function in the MDV life cycle [ 23 ]. Deletion of sTMR abrogated MDV replication, as these sequences serve as a spacer between the packaging signal pac-1 and the DR-1 cleavage site to ensure proper cleavage and packaging of the viral genome into the capsid.…”