Replication and recombination in cells infected with Herpes simplex virus type 1

Abstract

The studies presented in this thesis have been performed in order to improve our understanding of the molecular background of HSV-1 DNA metabolism. Specifically, it attempts to address the role of homologous recombination in the viral life cycle and to identify host enzymes that take part in viral DNA metabolism. We investigated the effects of ICRF-193, an inhibitor of topoisomerase II, on the replication of HSV-1. Then we examined mechanisms of recombination in cells infected with HSV-1 to establish the mechanism of circularization of HSV-1 genome. Finally, we investigated the coupling between HSV-1 DNA replication and homologous recombination as well as requirements for cellular gene products in homologous recombination. Plasmids containing HSV-1 oriS were constructed as the substrates for viral replication and recombination. Uninfected and HSV-1 infected cells with different genetic backgrounds were used. Transient replication assays were utilized to detect virus-dependent DNA replication and recombination. Furthermore, two other assays were developed for detecting homologous recombination: recombination-dependent expression of luciferase and recombination between HSV-1 tsS and tsK strains. To study the enzymatic background of homologous recombination HSV-1 amplicons were used to express recombination genes in human cells.We found that HSV-1 replication was specifically inhibited by ICRF-193 early in infection suggesting that topoisomerase II decatenates early replication intermediates. We have seen that linear DNA provided with directly repeated HSV-1 a sequences at the ends replicated as efficiently as circular DNA. The results indicate that linear HSV-1 DNA might be circularized by a sequences-mediated homologous recombination before replication. The reaction was not dependent on XPF/ERCC4 protein, suggesting that it does not proceed by a single strand-annealing pathway. Homologous recombination occurred also in absence of HSV-1 dependent DNA replication and viral gene products, which implies that DNA replication and homologous recombination may be coordinated but mechanistically independent events. We also demonstrated that homologous recombination was cell type dependent. Linear DNA with non-homologous ends induced p53 dependent apoptosis and failed to recombine in Balb/c 3T3 cells. It appears that homologous recombination is controlled by p53. Furthermore, homologous recombination may protect HSV-1-infected cells from initiating apoptosis and stimulate viral DNA replication.Finally, homologous recombination of replicating HSV-1 genomes was profoundly reduced by overexpression of a non-functional RAD51 in human primary fibroblasts. Our results suggest that RAD51 dependent homologous recombination is an important, but not necessarily exclusive, mechanism for promoting recombination of replicating HSV-1 DNA.