Human Cytomegalovirus (HCMV) is a double-stranded DNA virus that establishes life-long infection in the human host. The overarching objective of our work is to define critical virus-host interactions important for virus replication and latency which provide targets for antiviral strategies aimed at limiting viral pathogenesis. HCMV encodes a single DNA polymerase (UL54). As herpesviruses encode their own DNA polymerase it has been broadly presumed that they do not require host polymerases for the replication of their genomes. However herpesvirus genomes are complex with high-GC content and repeat sequences that constrain the B-family DNA polymerases such as UL54. Through our collaborative effort we demonstrated a striking role for specialized host translesion polymerases (TLS pols) in HCMV genome replication and stability. TLS pols function in lesion bypass at the replication fork or in single-stranded DNA gap filling or homology-directed repair that occurs post-synthesis (behind the fork). TLS pols also maintain fragile site stability during unperturbed DNA synthesis. TLS pols include the Y-family polymerases eta (h) iota (i) kappa (k) and Rev 1 as well as the error-prone B-family polymerase zeta (z). Strikingly we found that Y-family TLS pols (hik and Rev1) and pol z are important to maintain HCMV genome stability. Further our results indicate that pols h i and k generate single nucleotide variants across the viral genome. These findings indicate important roles for host TLS pols in ensuring viral genomic integrity and potentially in generating viral genome diversity. We also found that depletion of TLS pols differentially impacts viral genome synthesis and replication. Defining how HCMV maintains genomic stability and the significance of host TLS pols and DNA damage repair (DDR) pathways on the viral lifecycle is important for understanding mechanisms of virus replication and latency. Further exciting new data indicates a role for host TLS pols in the evolution of resistance to nucleoside antiviral therapies such as ganciclovir. We hypothesize that HCMV actively recruits TLS pols and coopts corresponding DDR pathways to maintain genome integrity and regulate viral replication and latency. Aim 1 will determine the mechanisms by which HCMV recruits host TLS pols and other DDR repair factors to viral replication compartments and the subdomains in which they function. Aim 2 will define the mechanisms by which host TLS pols and other DDR repair factors act on viral sequences to ensure genome stability and contribute to antiviral resistance. Aim 3 will determine the significance of host DDR pathways to viral latency. These aims are driven by our published work and exciting preliminary data identifying virus-host interactions that control host TLS pols and DDR pathways. Our multi-PI collaborative work establishes the importance of host TLS pols for the stability and diversity of viral genomes and would not be possible without the combined expertise of Drs. Goodrum and Bosco. Further this study offers the unique possibility of illuminating new insights into the biology of TLS pols in human cells using the HCMV genome as a model system.