A number of viruses, including members of the Herpesviridae, can cause tumors in their hosts. Tumorigenesis requires the continued survival of once transformed cells, which is critically dependent on telomerase activity to avoid cellular senescence and apoptosis. Telomerase is a ribonucleoprotein complex with its core constituents being telomerase reverse transcriptase (TERT) and telomerase RNA (TR). Telomerase dysfunction can have tumor-promoting functions independently of net telomere elongation and maintenance of chromosomal integrity, which is consistent with the short telomeres usually present in malignantly transformed tumor cells. Marek's disease virus (MDV) is a herpesvirus of chickens that causes a highly malignant T cell lymphoma. MDV harbors two copies of a viral TR gene (vTR) that exhibits 88% sequence identity to chicken TR (chTR) and was presumably pirated from the host genome. Our long-term goal is to elucidate in detail the mechanisms of the tumor-promoting effects of telomerase. In this proposal we shall test the hypothesis that the structural integrity of vTR is critical for transformation and metastasis, as well as integration of the MDV DNA into the host genome. The hypothesis will be tested by two specific aims:
Specific Aim 1 : To test the effect of mutations in conserved regions of vTR on MDV-induced tumor formation and dissemination. Using an infectious clone of the highly oncogenic MDV strain RB-1B (pRB-1B), we shall replace vTR or its promoter with the chicken homologues, and also introduce mutations into vTR that have been shown to render the molecule nonfunctional. We shall target the template region (conserved region 1, CR1) specifying the telomeric repeat sequence TTAGGG and the so-called H box, in which a single point mutation was identified in avirulent MDV vaccine strain CVI988. Recombinant MDV carrying the mutations will be tested in vivo for lytic replication, establishment of latency and tumorigenesis.
Specific Aim 2 : To analyze the contributions of vTR and telomeric repeats to MDV genome integration and host cell chromosome stability. MDV, like other herpesviruses such as Epstein-Barr virus (EBV) or human herpesviruses 6 and 7 (HHV-6, HHV-7), is capable of integrating its DNA genome into host chromosomes without any obvious hotspot of integration. The MDV genome contains telomeric repeats at both ends of the genome, suggesting that viral telomeres may be utilized for specific integration into homologous sequences within chicken chromosomes. Mutant MDV lacking telomeric repeats will be generated and their ability for integration and induction chromosome aberrations will be analyzed by using fluorescent in situ hybridization using MDV probes and chicken chromosome paints. Virus integration sites will be determined by cloning and sequencing of cloned cellular DNA, which are positive for MDV genomic termini by colony PCR. The relevance of the proposed research to public health is that it will provide a better understanding of the role of telomerase in cancer development and could lead to novel approaches for the treatment of a variety of tumors by specifically targeting telomerase RNA. It will also address the mechanism of herpesvirus integration into the host genome and its importance for latent infection and transformation of lymphocytes.
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