Terminal Dna Sequences Of Varicella-Zoster And Marek'S Disease Virus: Roles In Genome Replication, Integration, And Reactivation
One of the major obstacles in varicella-zoster virus (VZV) research has been the lack of an efficient genetic system. To overcome this problem, we generated a fulllength, infectious bacterial artificial chromosome (BAC) system of the P-Oka strain (pP-Oka), which facilitates generation of mutant viruses and allowed light to be shed on the role in VZV replication of the ORF9 gene product, a major tegument protein, and ORFS/L (ORF0), a gene with no known function and no direct orthologue in other alphaherpesviruses. Mutation of the ORF9 start codon in pP-Oka, abrogated pORF9 expression and severely impaired virus replication. Delivery of ORF9 in trans via baculovirus-mediated gene transfer partially restored virus replication of ORF9 deficient viruses, confirming that ORF9 function is essential for VZV replication in vitro. Next we targeted ORFS/L and could prove that the ORFS/L gene product is important for efficient VZV replication in vitro. Furthermore, we identified a 5' region of ORFS/L that is essential for replication and plays a role in cleavage and packaging of viral DNA. To elucidate the mechanisms of Marek's disease virus (MDV) integration and tumorigenesis, we investigated two sequence elements of the MDV genome: vTR, a virus encoded telomerase RNA, and telomeric repeats present at the termini of the virus genome. We demonstrate that vTR serves two distinct functions in MDVinduced tumor formation. The first is dependent on an increase of telomerase activity mediated by vTR, which contributes to the survival of rapidly dividing transformed cells and is crucial for rapid onset of lymphoma formation in infected animals. The second function of vTR is independent of telomerase action and is required for tumor formation and metastasis. This function is likely mediated by vTR interaction with RPL22, a cellular factor involved in T-cell development and virus-induced transformation. Finally, our studies on herpesvirus telomeric repeats provide the first conclusive evidence that herpesvirus telomeric repeats mediate integration into host telomeres, are critical for efficient tumor formation, and support efficient reactivation of latent virus.
dissertation or thesis