The intrinsic antiviral defense to incoming HSV-1 genomes includes specific DNA repair proteins and is counteracted by the viral protein ICP0

Abstract

Cellular restriction factors responding to herpesvirus infection include the ND10 components PML, Sp100 and hDaxx. During the initial stages of HSV-1 infection, novel sub-nuclear structures containing these ND10 proteins form in association with incoming viral genomes. We report that several cellular DNA damage response proteins also relocate to sites associated with incoming viral genomes where they contribute to the cellular front line defense. We show that recruitment of DNA repair proteins to these sites is independent of ND10 components, and instead is coordinated by the cellular ubiquitin ligases RNF8 and RNF168. The viral protein ICP0 targets RNF8 and RNF168 for degradation, thereby preventing the deposition of repressive ubiquitin marks and counteracting this repair protein recruitment. This study highlights important parallels between recognition of cellular DNA damage and recognition of viral genomes, and adds RNF8 and RNF168 to the list of factors contributing to the intrinsic antiviral defense against herpesvirus infection.

Figure 1. DNA repair proteins accumulate at sites associated with incoming HSV-1 genomes.

(A) HFF cells were infected with ICP0-null HSV-1 at an MOI of 0.1 for 1 hr. Cells were fixed at 24 hpi, stained for ICP4, and localization of γH2AX, Mdc1, 53BP1 and BRCA1 was assessed in asymmetrically infected cells at edges of plaques. Nuclei were stained with DAPI as shown in the merged image. (B) HFF cells were infected with wild-type HSV-1 at an MOI of 0.001 for 1 hr and processed as in A.

Figure 2. Sites of DNA repair protein accumulation are distinct from viral genomes, and independent of ND10 proteins.

(A) HFF cells were infected with ICP0-null HSV-1 at an MOI of 0.1 for 1 hr. Cells were fixed at 24 hpi, stained for ICP4, and localization of γH2AX, 53BP1 and PML was assessed in asymmetrically infected cells. The lower panel represents enlarged images to show co-localization. (B) HepaRG cells depleted for PML, Sp100, or control shRNA treated cells were infected with wild-type virus at an MOI of 0.001 or ICP0-null HSV-1 at an MOI of 0.1 for 1 hr. Cells were fixed at 24 hpi, stained for ICP4, and localization of 53BP1 was assessed.

Figure 3. 53BP1 accumulation at sites associated with incoming HSV-1 genomes is dependent on RNF8 and RNF168.

(A) Control HepaRG cells or cells in which RNF8 had been depleted using shRNA were infected with wild-type virus at an MOI of 0.001 or ICP0-null HSV-1 at an MOI of 0.1 for 1 hr. Cells were fixed at 24 hpi, stained for ICP4, and 53BP1 localization was assessed in asymmetrically infected cells. (B) RIDDLE cells or RIDDLE cells complemented with HA-tagged RNF168 were infected and analyzed as in A. (C) HepaRG cells containing tet-inducible RNF8-GFP were induced with 0.1 µg/ml tet for 8 hrs and infected with ICP0-null HSV-1 at an MOI of 0.1. RIDDLE cells reconstituted with HA-tagged RNF168 were infected with ICP0-null HSV-1 at an MOI of 0.1. Cells were fixed at 16–24 hpi, stained for ICP4, and localization of RNF8-GFP or HA-RNF168 was assessed.

Figure 4. The requirements for 53BP1 recruitment parallel the cellular response to DNA damage.

(A) H2AX-/- MEFs or matched control MEFs were infected with wild-type virus at an MOI of 0.001 or ICP0-null HSV-1 at an MOI of 0.1 for 1 hr. Cells were fixed at 24 hpi, stained for ICP4, and 53BP1 localization was assessed in asymmetrically infected cells. (B) A-T cells or cells in which ATM had been reconstituted were infected and processed as in A. (C) A-TLD-1 cells or A-TLD-1 cells in which Mre11 had been reconstituted were infected and processed as in A.

Figure 5. Conjugated ubiquitin accumulation at sites associated with incoming viral genomes is dependent on RNF8 and RNF168.

(A) RNF8-/- MEFs or matched control MEFs were infected with ICP0-null HSV-1 at an MOI of 0.1 for 1 hr. Cells were fixed at 24 hpi, stained for ICP4, and localization of conjugated ubiquitin (FK2) was assessed in asymmetrically infected cells. (B) RIDDLE cells or RIDDLE cells expressing HA-tagged RNF168 were infected and analyzed as in A.

Figure 6. SUMO accumulation at sites associated with incoming viral genomes is not dependent on RNF8 or RNF168.

(A) Control cells or cells in which RNF8 had been depleted using shRNA were infected with ICP0-null HSV-1 at an MOI of 0.1 for 1 hr. Cells were fixed at 24 hpi, stained for ICP4, and localization of SUMO1 or SUMO2/3 was assessed in asymmetrically infected cells. (B) RIDDLE cells or RIDDLE cells with HA-tagged RNF168 reconstituted were infected and analyzed as in A. (C) Control HFF cells or cells in which RNF8 had been depleted using shRNA were infected as in A and assessed for localization of PML. (D) RIDDLE cells or RIDDLE cells with HA-tagged RNF168 reconstituted were infected and analyzed as in C.

Figure 7. H2AX is beneficial for HSV-1 replication while RNF8 represses viral genomes.

(A) H2AX-/- MEFs or matched controls were infected with wild-type or ICP0-null virus. Relative probabilities of plaque formation were calculated by counting the numbers of plaques on the different cell lines at each separate dilution of virus. Infections were carried out at least in duplicate and the experiment was repeated four times. Data are represented as mean +/- SEM. (B) RNF8-/- MEFs expressing RNF8 or empty vector were infected with WT or ICP0-null virus at an MOI of 0.01. ICP27 transcripts were detected at 2 or 5 hpi and normalized to a cellular control. Data were analyzed by comparing transcription in the presence of RNF8 to transcription in the absence of RNF8 and therefore represent fold repression by RNF8 for each virus. Experiments were performed in duplicate and averaged. Results are representative of three independent experiments and the error is one standard deviation of the duplicate samples. (C) Model showing the parallels between sites of cellular DNA damage and sites associated with incoming HSV-1 genomes and the role of central role of ICP0, RNF8 and RNF168 in disrupting both structures.