Abstract
Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus-induced lymphoma.
Highlights
Telomerase is a multi-component ribonucleoprotein complex that governs the maintenance of telomeres, protein-associated hexameric sequence repeats at the end of linear chromosomes, and ensures chromosomal integrity and cellular survival [1,2]
Led by these previous observations, we hypothesized that expression of viral telomerase RNA (vTR) encoding a mutated template sequence (AATCCCAATC to ATATATATAT), termed AU5, could have an effect on avian cancer cells that is similar to that described previously for mammalian cells [23,24]
We here report on effects of a mutation in the template sequence (CR1) of vTR encoded by Marek’s disease virus (MDV) on virus replication and tumorigenesis in a natural virus-host model
Summary
Telomerase is a multi-component ribonucleoprotein complex that governs the maintenance of telomeres, protein-associated hexameric sequence repeats at the end of linear chromosomes, and ensures chromosomal integrity and cellular survival [1,2]. The telomerase complex consists of two core components, telomerase reverse transcriptase (TERT) and telomerase RNA (TR). Vertebrate TRs exhibit a universally conserved secondary structure comprised of four structural domains (Fig. 1): the pseudoknot (core) domain containing the template sequence in conserved region (CR) 1 (CR1), the CR4 and CR5 domains with a highly conserved stem-loop structure (CR4-5), the H/ACA box domain, and the CR7 domain [4]. The H/ACA box and CR7 domains confer TR stability [4,5,6]
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