G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV

Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P.; Robertson, Erle S.; Schildkraut, Carl L.; Verma, Subhash C.

Our analysis of replication fork pausing and reduction in viral genome copies were further substantiated by the results from a transient DNA replication assay and Gardella gel electrophoresis assay to determine whether that reduction in viral genome copies is due to inhibition of viral DNA replication. We found that BCBL-1 cells treated with 20 M PhenDC3 indeed show a significant reduction in transient replication of TR containing plasmids. These data further substantiated and strengthened our findings that, PhenDC3 could cause pausing or stalling of the bi-directional progress of the replication machinery at or near the outer boundary of the terminal repeats. These data also suggest that treatment with PhenDC3 leads to a reduction in the replication of KSHV genome. In complete agreement with these findings, our Gardella gel electrophoresis assay further demonstrated a significant reduction in both circular and linear DNA copies of the KSHV genome compared to DMSO treated, control cells. Taken together, our data suggest that PhenDC3 stabilizes the G-quadruplexes that could potentially form in the GC-rich TRs region, thereby blocking the progression of replication forks. This likely leads to the reduction in viral genome replication, which ultimately affects the maintenance of the KSHV episomes in the infected cells. Previous studies have determined the roles of G-quadruplex stabilizing compounds on viral gene expression (31) , but this is the first report to our knowledge that G-quadruplex stabilizing compounds stalled the progression of replication forks of the viral episomes inhibiting KSHV latency, thus identifying a potential target for therapeutically blocking KSHV-associated malignancies.