Functional Regulation Of The Structure-Specific Endonuclease Fen1 By The Human Cytomegalovirus Protein Ie1 Suggests A Role For The Re-Initiation Of Stalled Viral Replication Forks

Flap endonuclease 1 (FEN1) is a member of the family of structure-specific endonucleases implicated in regulation of DNA damage response and DNA replication. So far, knowledge on the role of FEN1 during viral infections is limited. Previous publications indicated that poxviruses encode a conserved protein that acts in a manner similar to FEN1 to stimulate homologous recombination, double-strand break (DSB) repair and full-size genome formation. Only recently, cellular FEN1 has been identified as a key component for hepatitis B virus cccDNA formation. Here, we report on a novel functional interaction between Flap endonuclease 1 (FEN1) and the human cytomegalovirus (HCMV) immediate early protein 1 (IE1). Our results provide evidence that IE1 manipulates FEN1 in an unprecedented manner: we observed that direct IE1 binding does not only enhance FEN1 protein stability but also phosphorylation at serine 187. This correlates with nucleolar exclusion of FEN1 stimulating its DSB-generating gap endonuclease activity. Depletion of FEN1 and inhibition of its enzymatic activity during HCMV infection significantly reduced nascent viral DNA synthesis demonstrating a supportive role for efficient HCMV DNA replication. Furthermore, our results indicate that FEN1 is required for the formation of DSBs during HCMV infection suggesting that IE1 acts as viral activator of FEN1 in order to re-initiate stalled replication forks. In summary, we propose a novel mechanism of viral FEN1 activation to overcome replication fork barriers at difficult-to-replicate sites in viral genomes.Author summaryHuman cytomegalovirus (HCMV) infection is usually asymptomatic in healthy individuals. However, newborns and immunocompromised patients are suffering from life-threatening diseases upon infection. After decades of research, vaccines are still not available and the use of antiviral therapeutics is limited. During the last years, drugs targeting the cellular DNA damage response (DDR) are increasingly applied in cancer therapy. Interestingly, HCMV utilizes the cellular DDR for its replication thereby implicating DDR components as promising targets for antiviral intervention. In this study, we found that the DDR enzyme flap endonuclease 1 (FEN1) is activated by the HCMV protein IE1 in a unique manner that depends on a direct protein-protein interaction. Moreover, we could demonstrate that FEN1, which is tremendously stabilized by IE1, supports efficient viral DNA replication. We propose a novel mechanism whereby a viral protein manipulates the cellular enzyme FEN1 to facilitate the multiplication of viral DNA genomes. Small molecule inhibitors of FEN1 might thus serve as a new antiviral therapy option.