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

Abstract

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.

Cross-Species Analysis of Innate Immune Antagonism by Cytomegalovirus IE1 Protein

“…To address this issue, the effect of hIE1_rH mutants on further cellular proteins was examined. Schilling et al showed that IE1 interacts with human flap endonuclease 1 (hFEN1), stabilising the protein to raise its activity in a pro-viral manner [ 18 ]. First, the interaction of rIE1 and IE1_rH mutants with hFEN1 aa 176-380 was analyzed by NanoBRET measurements ( Figure 5 A,B).…”

“…In addition to PML, hFEN1 could recently be identified as a binding partner of hIE1 that is hijacked for promoting viral replication [ 18 ]. NanoBRET analyses and cycloheximide stabilization assays revealed that rIE1 is neither able to interact with hFEN1 176-380 nor does it mediate hFEN1 protein stabilization ( Figure 5 A, bar 1).…”

“…Since mutants that carry helix 1 or helices 1/2 of rIE1 lost their interaction with hFEN1, we assume that the region comprising aa 1-82 of hIE1 may constitute the binding surface for FEN1 or may form an important part of it. In the previous analysis, we detected that mutation of residues located in helices H7 and H9 of hIE1 also interferes with FEN1 binding [ 18 ]. One potential explanation for this may be that IE1 dimerization is a prerequisite for hFEN1 interaction, and this may be affected by mutations in helices H7 and H9.…”

“…Furthermore, besides expression plasmids with Flag-tagged hIE1, hIE1 core, rIE1, and rIE1 core [ 14 ], an expression plasmid with a firefly luciferase gene under the control of the UL112 promotor (pHM 142, [ 32 ]) and an expression plasmid for IE2 (pHM 134, [ 33 ]) were used for the gaussia luciferase assay. Additionally, Schilling et al generated the Flag-tagged FEN1 176-380 [ 18 ] used for cloning the expression plasmids for the NanoBRET assay and the stabilization assay.…”

“…Schilling et al detected that the hIE1 core interacts with the flap endonuclease one protein (FEN1) that plays a role in the DNA damage response pathway. Due to this interaction, hIE1 stabilizes FEN1 and has a pro-viral effect on HCMV lytic replication [ 18 ].…”