RecQ1 helicase unwinds G-Quadruplexes at oriLyt to facilitate KSHV Lytic DNA Replication

KSHV utilizes distinct origins of DNA replication (origin of replications) during the latent and lytic phases of its life cycle. Lytic DNA replication requires the participation of cellular and viral proteins to initiate replication at a specific region in the KSHV genome, oriLyt. These regions contain multiple secondary structures and binding sites for the viral and cellular proteins. We discovered the presence of G-quadruplex (G4s) sites in the oriLyt region. G-quadruplexes are secondary structures in nucleic acid sequences that are considered regulators of multiple biological processes, such as transcription, translation, and replication initiation. Importantly, herpesviruses have a high density of PQS (putative quadruplex formation sites) in their genome, including in the regulatory regions, which control replication and transcription. The binding of RecQ1 to the oriLyt region of KSHV and its ability to unwind the G4 structures led us to speculate that G-quadruplex sites play an important role in lytic DNA replication. In this study, we confirmed the formation of stable G-quadruplexes through biochemical and biophysical assays. We further demonstrated that RecQ1 associates with the G4 sites of the oriLyt. The functional significance of RecQ1-mediated unwinding of G4 sites was confirmed by the inhibition and depletion of RecQ1 activity and protein levels through a pharmacologic inhibitor and short-hairpin, respectively. Furthermore, the detection of replication initiation through single-molecule analysis of the replicated DNA (SMARD) approach demonstrated that G4 stabilization leads to a subdued initiation of replication at the oriLyt. This confirmed the role of the G-quadruplex in regulating viral DNA replication, which can be used for controlling virus growth. Significance Biological processes originating from the DNA and RNA can be regulated by the secondary structures present in the stretch of nucleic acids, and the G-quadruplexes are shown to regulate transcription, translation, and replication. In this study, we identified the presence of multiple G-quadruplex sites in the region (oriLyt) of KSHV DNA, which is essential for the replication of DNA during the lytic cycle. We demonstrated the roles of these G-quadruplexes through multiple biochemical and biophysical assays in controlling replication and efficient virus production. We demonstrated that KSHV achieves this by recruiting RecQ1 (helicase) at these G-quadruplex sites for efficient viral DNA replication. Analysis of the replicated DNA through nucleoside labeling and immunostaining showed a reduced initiation of DNA replication in cells with a pharmacologic stabilizer of G-quadruplexes. Overall, this study confirmed the role of the G-quadruplex in regulating viral DNA replication, which can be exploited for controlling viral DNA replication.