The HUSH epigenetic repressor complex silences PML nuclear body-associated HSV-1 quiescent genomes

Abstract

Herpes simplex virus 1 (HSV-1) latently infected neurons display diverse patterns in the distribution of the viral genomes within the nucleus. A key pattern involves quiescent HSV-1 genomes sequestered in promyelocytic leukemia nuclear bodies (PML NBs) forming viral DNA-containing PML-NBs (vDCP NBs). Using a cellular model that replicates vDCP NB formation, we previously demonstrated that these viral genomes are chromatinized with the H3.3 histone variant modified on its lysine 9 by trimethylation (H3.3K9me3), a mark associated with transcriptional repression. Here, we identify the HUSH complex and its effectors, SETDB1 and MORC2, as crucial for the acquisition of H3K9me3 on PML NB-associated HSV-1 and the maintenance of HSV-1 transcriptional repression. ChIP-seq analyses show H3K9me3 association with the entire viral genome. Inactivating the HUSH-SETDB1-MORC2 complex before infection significantly reduces H3K9me3 on the viral genome, with minimal impact on the cellular genome, aside from expected changes in LINE-1 retroelements. Depletion of HUSH, SETDB1, or MORC2 alleviates HSV-1 repression in infected primary human fibroblasts and human induced pluripotent stem cell-derived sensory neurons (hiPSDN). We found that the viral protein ICP0 induces MORC2 degradation via the proteasome machinery. This process is concurrent with ICP0 and MORC2 depletion capability to reactivate silenced HSV-1 in hiPSDN. Overall, our findings underscore the robust antiviral function of the HUSH-SETDB1-MORC2 repressor complex against a herpesvirus by modulating chromatin marks linked to repression, thus presenting promising avenues for anti-herpesvirus therapeutic strategies.

Keywords: HUSH; Microbiology; epigenetics.

Fig. 1.

Detection of proteins of the HUSH–SETDB1–MORC2 entity in vDCP NBs. FISH-IF to detect proteins of the HUSH–SETDB1–MORC2 (green) entity and PML (blue/gray) together with HSH-1 genomes. hFC were infected or not with lqHSV-1 (m.o.i.= 3, 100% of cells infected) and harvested at 4 dpi to perform immuno-FISH. Endogenous SETDB1 and MPP8 were readily detectable with native antibodies supporting the FISH procedure. All other proteins were detected through the expression of an ectopic tagged version. FLAG-MPP8 was also detected as a control for the other HUSH complex components. Insets represent nuclei. Zooms point out vDCP NBs. (A–E) Detection of endogenous SETDB1, endogenous MPP8, FLAG tagged MPP8, v5 tagged periphilin (PPHLN1), and v5 tagged MORC2, respectively, in noninfected (NI) and infected (I) cells. (Scale bars, 5 µm.) (A’–E) Quantification of the colocalization of the protein with PML NBs for the respective samples in NI and I cells. Means are indicated in the graphs. (A”–E”), Quantification of the average number of HSV-1 spots per nucleus (dark blue) and of the colocalization of the protein with HSV-1 and PML (light blue) for the respective samples in I cells. N = number of events counted. Means are indicated in the graphs in %.

Fig. 2.

Interaction of the proteins of the HUSH–SETDB1–MORC2 entity with the PML NB-associated quiescent HSV-1 genome. (A) Schematic view of the ChIP procedure to detect interactions of proteins with viral genomes. Control hFC or hFC expressing a tagged version of proteins of interest were infected with lqHSV-1 (m.o.i.= 3) and ChIP-qPCR was performed 24 h pi. All experiments were performed at least three times. Measurements were taken from distinct samples. Details about analyzed viral loci (names and sequences) are indicated in the Materials and Methods section. Quantification data are mean values (–SD). P-values * <0.05; ** <0.01 (one-tailed paired Student’s t test). (B) Interaction of endogenous MPP8 with viral genome loci. Pro = promoter; CDS = coding sequence. (C, D, E) MPP8endo colocalization with lqHSV-1 in hFC transfected with a siRNA CTRL (siCTRL) or siRNA against PML (siPML) and infected with lqHSV-1 for 48 h (m.o.i. = 3). (C) WB of PML in siCTRL or siPML-treated hFC. The quantification of PML levels relative to tubulin (Tub) is depicted below the WB. (D) Representative images showing the PML signal by immunofluorescence (gray) to exemplify the efficiency of the siPML on PML NBs disappearance (also see ref. 31). (E) Quantification of the colocalization of MPP8endo with lqHSV-1 following immuno-FISH to detect MPP8, lqHSV-1, and PML. Distribution of HSV-1 genome signals/nucleus (dark blue) and colocalization of MPP8endo with lqHSV-1/nucleus (light blue) are shown. Means of colocalization between MPP8endo and lqHSV-1 are shown (in %). N = total number of viral genomes analyzed. (F) Quantification of PML colocalization with lqHSV-1 in hFC transduced with lentiviruses expressing control shRNA (shCTRL) or shRNA against proteins of interest and infected with lqHSV-1 for 48 h (m.o.i. = 3). Distribution of the numbers of colocalization between PML and lqHSV-1/nucleus are shown. Means of colocalization between PML and lqHSV-1 are shown (in %). N = total number of viral genomes analyzed.

Fig. 3.

Inactivation of proteins of the HUSH–SETDB1–MORC2 entity reduces H3K9me3 mark on the PML NB-associated quiescent HSV-1 genome. (A) Schematic view of the ChIP-qPCR/ChIP-seq procedure to detect enrichment of H3K9me3 on viral genomes after depletion or not by shRNAs of proteins of interest. Details about analyzed viral loci (names and sequences) are indicated in the Materials and Methods section. (B–F) WB on the target proteins to confirm the efficiency of the specific shRNAs. The quantification of protein levels relative to housekeeping proteins is depicted below the WBs. (G) ChIP-qPCR showing mean fold enrichments on the different viral loci of H3K9me3 in specific shRNA-depleted cells compared to cells expressing a control shRNA. Experiments were performed at least three times. H3K9me3 signal was standardized over total H3 present on each locus. Measurements were taken from distinct samples. Quantification data are mean values (±SD). For quantification details on each loci, see expanded SI Appendix, Fig. S6. P-values * <0.05; ** <0.01; *** <0.001 (one-tailed paired Student’s t test). (H–N) Data from H3K9me3 ChIP-seq experiments in infected hFC treated with shCTRL or a shRNA against the indicated proteins of interest. (H) Genome browser snapshot of the H3K9me3 enrichment normalized on input across the entire quiescent HSV-1 genome. (I) Profile plot (Top) and heatmaps (Bottom) showing the density of H3K9me3 signal on HSV-1 genes. (J) Box plots showing the quantification of H3K9me3 reads on HSV-1 genes (in Reads Per Million). P-values * <0.05; ** <0.01; **** <0.0001 (nonparametric Wilcoxon rank-sum test). (K) Representative genome browser snapshot of the H3K9me3 enrichment normalized on input across a gene-rich region of chromosome 21. (L) Profile plot of the H3K9me3 density on large H3K9me3-rich regions defined in ref. . (M) Profile plots of the H3K9me3 density on a specific family of LINE-1 (L1PA6) regulated by HUSH. (N) ChIP-Seq mean signal coverage of H3K9me3 on L1PA6 across the different samples. The Y axis was adjusted to a 0 to 5 range. Adjusted P-values ** <0.01; *** <0.001; *** <0.001 (Wilcoxon rank-sum test), ns: nonsignificant.

Fig. 4.

The HUSH–SETDB1–MORC2 entity maintains the silencing of PML NB-associated quiescent HSV-1. (A) Schematic view of the experimental procedure to determine the restrictive activity of the individual proteins of the HUSH–SETDB1–MORC2 entity. hFC expressing inducible shRNA control (shCTRL) or shRNA against a protein of interest were infected with lqHSV-1 (m.o.i.= 3) at 38.5 °C to establish quiescence. 5 d later doxycycline was added for 9 d shifting the temperature at 32 °C to allow virus replication. Cells were used to perform RT-qPCR to quantify expression of viral lytic genes or immuno-FISH to detect viral RC. (B–E) RT-qPCR on representative HSV-1 lytic genes following treatment with a shCTRL, or a shRNA inactivating SETDB1, PPHLN1, MPP8, and MORC2, respectively. All experiments were performed at least three times. Measurements were taken from distinct samples. Quantification data are mean values (±SD). P-values * <0.05; ** <0.01; *** <0.001 (one-tailed paired Student’s t test). (F) Immuno-FISH allowing the detection of PML (green), HSV-1 (red), and nuclei (gray, DAPI) in cell monolayers after induction of the shRNA CTRL, or targeting HUSH components (MPP8 and PPHLN1), or targeting SETDB1 or MORC2. (Scale bar, 5 µm.) RC: replication compartment. (G) Titration on U2OS cells of infectious viral progeny production from the above experiments using shRNA CTRL, or a shRNA inactivating each of the protein of the HUSH–SETDB1–MORC2 entity. Up: procedure; Down: viral plaques with supernatant from shMORC2 hFC shown as an example. (Scale bar, 100 µm.) (H) numerations of viral plaques from the above conditions.

Fig. 5.

ICP0 induces proteasomal-dependent degradation of MORC2 and reactivation of HSV-1 from quiescently infected neurons. (A and B) HFC were infected or not with wild-type HSV-1 (HSV) or a mutant HSV-1 unable to express ICP0 (HSVΔICP0), in the presence (+) or not (−) of the proteasome inhibitor MG132. (A) Detection of ICP0 expression and of PML. The star depicts the ICP0- and proteasome-dependent degradation of the SUMOylated forms of PML as first shown in ref. . (B) Detection of proteins of the HUSH complex, MORC2, and SETDB1. Tub or actinin were used as loading controls. Data are representative of at least two independent experiments that showed similar results. The quantification of protein levels relative to housekeeping proteins is depicted below the WBs. (C) Schematic view of the experimental procedure in hiPSDN infected with lqHSV-1 at 38.5 °C. 4 d after infection with lqHSV-1 neurons were transduced with lentiviruses control (CTRL) or expressing ICP0 or ICP0RFmut, and temperature was shifted to 32 °C to allow virus replication. Multiple analyses were performed at 3 d posttransduction (dpt). (D) RT-qPCR on representative HSV-1 lytic genes following expression of ICP0 (Left) or its nonfunctional mutant ICP0RFmut (Right). All experiments are in triplicates. All quantification data are mean values (±SD). Measurements were taken from distinct samples. P-values * <0.05; ** <0.01; *** <0.001 (one-tailed paired Student’s t test). (E) Detection of viral plaques on U2OS cell monolayers in contact with supernatants from lqHSV-1 infected neurons transduced for 3 dpt with lentivirus CTRL or expressing ICP0 or ICP0RFmut. Crystal violet (grayscale) images for plaques (circled in red) visualization are shown. (Scale bar, 500 µm.) (F) Numeration of the plaques detected in (E). (G) Immunofluorescent detection of ICP4 (red) and NF200 (green) showing viral RC in neurons. Arrows point out two RCs in the same nucleus of a single neuron. (Scale bar, 10 µm.)

Fig. 6.

The HUSH–SETDB1–MORC2 entity acts as a restriction pathway in neurons infected with HSV-1. (A) Schematic view of the experimental procedure to determine the restriction activity of the HUSH–SETDB1–MORC2 entity in hiPSDN infected with lqHSV-1 at 38.5 °C. 4 d after infection with lqHSV-1 neurons were transduced with lentiviruses expressing a shRNA control, or a shRNA against MORC2, and temperature was shifted to 32 °C to allow virus replication. Multiple analyses were performed 5 and 7 d posttransduction (dpt). (B) RT-qPCR at 5dpt (Left) and 7dpt (Right) on representative HSV-1 lytic genes following treatment with a shRNA CTRL (shCTRL), or a shRNA inactivating MORC2. All experiments are in triplicates. Measurements were taken from distinct samples. All quantification data are mean values (±SD). P-values * <0.05; ** <0.01 (one-tailed paired Student’s t test). (C) Detection of viral plaques in U2OS cells monolayers in contact with supernatants from shCTRL i) or shMORC2 ii) treated lqHSV-1 infected neurons. Crystal violet (grayscale) images for plaque visualization are shown. (Scale bar, 100 µm.) (D) Numeration of viral plaques from C. P-values * <0.05 (one-tailed paired Student’s t test). Measurements were taken from distinct samples. Crystal violet images (grayscale) of U2OS cell monolayers are shown above each condition. (Scale bar, 500 µm.) (E) Model of epigenetic restriction of nuclear incoming HSV-1 DNA by the HUSH–SETDB1–MORC2 entity through the apposition of trimethyl marks on H3K9 on the chromatinized viral genome ultimately leading to PML NB-associated quiescent HSV-1 genomes. Made with BioRender.