Herpesviruses mimic zygotic genome activation to promote viral replication

Abstract

Zygotic genome activation (ZGA) is crucial for maternal to zygotic transition at the 2-8-cell stage in order to overcome silencing of genes and enable transcription from the zygotic genome. In humans, ZGA is induced by DUX4, a pioneer factor that drives expression of downstream germline-specific genes and retroelements. Here we show that herpesviruses from all subfamilies, papillomaviruses and Merkel cell polyomavirus actively induce DUX4 expression to promote viral transcription and replication. Analysis of single-cell sequencing data sets from patients shows that viral DUX4 activation is of relevance in vivo. Herpes-simplex virus 1 (HSV-1) immediate early proteins directly induce expression of DUX4 and its target genes, which mimics zygotic genome activation. Upon HSV-1 infection, DUX4 directly binds to the viral genome and promotes viral transcription. DUX4 is functionally required for infection, since genetic depletion by CRISPR/Cas9 as well as degradation of DUX4 by nanobody constructs abrogates HSV-1 replication. Our results show that DNA viruses including herpesviruses mimic an embryonic-like transcriptional program that prevents epigenetic silencing of the viral genome and facilitates herpesviral gene expression.

Fig. 1. DUX4 induction after herpesviral infection.

A Western blot of primary HFF cells infected with HSV-1 for 24 h and 293T cells infected with HSV-1 for 18 h, both at a MOI 5. ICP0 was used as marker for infection. Representative experiment out of n = 3. B RNA-seq. data of the DUX4 and neighboring FRG1 and FRG2 loci in WI38 9 hpi, HFF 4 hpi and HEK 293T cells 18 hpi. The Precision Run-On Sequencing (Pro-Seq) data show RNA pol II occupancy on the FRG1, FFRG2 and DUX4 locus in infected and uninfected cells. ICP4 ChIP-Seq data show ICP4 occupation at the DUX4 locus in cells infected with HSV-1. C Western blot of iSLK rKSHV 219 cells induced with 1 µg/ml Doxycycline for 1, 2 or 3 days. K8α run on a different gel and was used as control for KSHV reactivation. Representative experiment out of n = 3. D Western blot of 293T cells infected with HSV-1 for 18 h at a MOI of 5 and analyzed for expression of ZSCAN4. Glycoprotein D (gD) served as a control for viral infection. Representative experiment out of n = 3. E HFF cells infected with HCMV for 6 d with MOI 1. Western blot analysis of DUX4. HCMV glycoprotein B (gB) was used as marker for infection. Representative experiment out of n = 3. F RNA-seq. analysis of DUX4 target genes after HSV-1 or Ad5 infection. Ad5 RNA-seq. data was taken from BioProject PRJEB57806. Source Data are provided as a Source Data file.

Fig. 2. Herpesviral immediate-early proteins induce DUX4 expression.

A Western Blot of DUX4 and HSV-1 proteins ICP0, ICP27, VP16 and glycoprotein D (gD) in HFF cells infected with HSV-1 harvested at different hpi (MOI of 10). Representative experiment out of n = 3. B DUX4 expression kinetics of newly synthesized RNA (4su-sequencing) in HFF cells infected with HSV-1 wt and HSV-1 delta vhs virus. Reanalysis of data from Friedel et al.. C Western Blot of DUX4 and HSV-1 proteins in primary HFF infected with HSV-1 and HSV-1 mutants (HSV-1 deltaICP0 (ICP4-YFP), HSV-1 deltaICP27, HSV-1 deltaICP34.5/ICP47) harvested at 16 hpi (MOI 10). One representative experiment out of n = 5. D Western blot analysis of DUX4 and HSV-1 protein in 293T cells transfected with HSV-1 IE proteins ICP0, ICP0 FXE, ICP4 (ICP4-YFP), ICP0 + ICP4 (ICP4-YFP) and ICP0 FXE + ICP4 (ICP4-YFP) for 48 h or infected with HSV-1 for 18 h (MOI of 10). ICP0 FXE is a mutant with a deletion in the RING domain, which inhibits Ubiquitin E3 ligase activity. EV: empty vector control. One representative experiment out of n = 3. E qRT-PCR analysis of cellular genes DUX4, TRIM43 as well as the viral genes ICP0 and gC in HDF-TERT cells untreated or treated with PAA and infected with HSV-1 (MOI of 0.1). Values are biological replicates and presented as mean fold induction +/-SD (normalized to HPRT RNA) relative to uninfected control cells. Source Data are provided as a Source Data file.

Fig. 3. Direct binding of DUX4 to the HSV-1 genome.

A CUT&RUN with a DUX4-specific antibody in HFF1 cells infected with HSV-1 at 12 hpi performed in duplicates. One sample with an H3K27me3-specific antibody was included as control. Data was normalized to Drosophila Spike-in DNA. Lines labeled with KOS indicate the DUX4 motif sites in the HSV-1 genome determined with FIMO. B Comparison of DUX4 consensus binding sequence from DUX4 CUT&Tag in HSV-1 infected cells (upper panel), DUX4 HSV-1 ChIP-Seq (middle panel) and with the previously published DUX4 consensus sequences (Geng et al.) in the lower panel. C, D Electrophoretic Mobility Shift Assay (EMSA) of different 600 bp fragments of fluorescently labelled viral DNA containing either one DUX4 binding motif (B1/B2) or no DUX4 binding motif (NB), incubated with purified DUX4 protein (DUX4). B1 shows a T- > C nucleotide exchange at position 9 of the DUX4 binding motif in the KOS strain. Representative experiment out of n = 4. E Reanalysis of ATAC-seq and histone modifications at known DUX4 binding sites of THP-1 cells infected with HSV-1 at 1 h, 2 h, 4 h and 8 h post infection. Histone modification and ATAC-seq were taken from Gao et al. and Hennig et al. respectively^, and DUX4 binding sites from the DUX4 ChIP-Sequencing after HSV-1 infection. F Venn diagram showing overlap of genes that are significantly upregulated in the 8-cell stage of human development, HSV-1 infection and FSHD. G Expression pattern of embryonic genes during embryonic genome activation compared to expression pattern in DUX4 overexpressing and HSV-1 infected cells. Embryonic data is normalized to the average expression value of each gene. Data from DUX4 overexpression and HSV-1 infection is relative to mock. Shown are genes which have DUX4 binding sites in the proximity of the TSS (500 bp downstream, 1000 bp upstream of the annotated promoter). (OO: oozyte, PRO: pronucleus, ZY: zygote, 2 C: 2-cell state, 4 C: 4-cell state, 8 C: 8-cell state, MO: morula). Source Data are provided as a Source Data file.

Fig. 4. Viral DUX4 expression in patient samples.

A Immunohistochemistry staining of liver biopsies from hepatitis patients with HSV-1, HBV and HCV. Consecutive slices were stained with antibodies specific for DUX4 (left panel) and HSV-1 (right panel). B Reanalysis of sequencing datasets from the International Cancer Genome Consortium (ICGC). Datasets were classified according to a DUX4 score (Expression of DUX4 and DUX4-target genes, y-axis) and viral abundances (x-axis). Bladder Urothelial Carcinoma (BLCA), Breast invasive carcinoma (BRCA), Cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), Chronic Lymphocytic Leukemia (CLLE), Colon adenocarcinoma (COAD), Diffuse large B cell lymphoma (DBLC), Esophageal Adenocarcinoma (ESAD), Glioblastoma multiforme (GBM), Head and Neck squamous cell carcinoma (HNSC), Kidney renal clear cell carcinoma (KIRC), Kidney renal papillary cell carcinoma (KIRP), Liver hepatocellular carcinoma (LIHC), Liver Cancer (LIRI), Lung adenocarcinoma (LUAD), Lung squamous cell carcinoma (LUSC), Malignant Lymphoma (MALY), Ovarian serous cystadenocarcinoma (OV), Pancreatic Cancer (PACA), Prostate adenocarcinoma (PRAD), Rectum adenocarcinoma (READ), Renal Cancer (RECA), Sarcoma (SARC), Skin Cutaneous Melanoma (SKCM), Stomach adenocarcinoma (STAD), Thyroid carcinoma (THCA), Uterine Corpus Endometrial Carcinoma (UCEC). C Reanalysis of single cell sequencing datasets from patients with EBV-positive Nasopharynx carcinoma (NPC) from Liu et al.. Left panel: UMAP projection of number of EBV-specific reads per cell, pool of 10 donors. Right panel: UMAP projection of number of DUX4-target gene specific reads pool of 10 donors. D Reanalysis of single cell sequencing datasets from patients with HPV-positive head and neck cancer from Kürten et al.. Left panel: UMAP projection of number of HPV-specific reads per cell, pool of 10 donors, 5 donors HPV-positive and 5 donors HPV-negative. Right panel: UMAP projection of number of DUX4-target gene specific reads, pool of 10 donors, 5 donors HPV positive and 5 donors HPV-negative. E Reanalysis of single cell sequencing datasets from patients with Merkel cell polyomavirus (MCPyV) from Das et al.. Left panel: UMAP projection of number of MCPyV-specific reads per cell, pool of 11 donors. Right panel: UMAP projection of number of DUX4-target gene specific reads, pool of 11 donors. F Quantification of DUX4-reads and HPV-reads in single cells in cell clusters from D. Statistical analysis was done using the two-sided Kruskal-Wallis test with adjustment for multiple comparison.

Fig. 5. DUX4 expression is critical for HSV-1 and HSV-2 replication.

A DUX4 ko HAP1 cells were generated with CRISPR/Cas9 and DUX4 specific sgRNAs. HSV-1 protein expression in HAP1 wt cells and HAP1 cells with DUX4 knockout. Cells were infected with HSV-1-GFP (MOI of 2) for 20 h and analyzed by western blot. HSP90 was used as loading control. Representative experiment out of n = 3. B mRNA expression (RNA-seq.) of all host genes of HAP1-DUX4-ko cells plotted against HAP1 wt cells, both infected with HSV-1-GFP (MOI of 1) for 8 h. Depicted in red are known DUX4 target genes. C Heatmap of mRNA-expression of selected viral transcripts from HSV-1 (MOI of 1) infected wt and DUX4 ko HAP1 cells 8 h post infection. Marked are viral genes with immediate-early, early and late expression kinetics. D GFP-expression of HAP1 wt and HAP1 DUX4-ko cells infected with HSV-1-GFP (MOI of 0.1) at day 1–4 post infection, measured by flow cytometry (upper panel). In parallel, viral titers were assessed by plaque assays on Vero cells (lower panel). One representative experiment out of n = 3. Values shown are technical replicates. E GFP-expression of HAP1 wt and HAP1 DUX4-ko cells infected with HSV-2-GFP (MOI of 0.05) at day 1–4 post infection, measured by flow cytometry (upper panel). In parallel, viral titers were assessed by plaque assays on Vero cells (lower panel). One representative experiment out of n = 3. Values shown are technical replicates. F Western blot of 293T WT cells and 293T DUX4-ko cells infected with HSV-1 for 18 h. Comparison of HSV-1 protein expression in wt cells and cells with complete DUX4-ko. Actin was used as loading control. Representative experiment out of n = 4. G qRT-PCR analysis of the replication of HSV-1 GFP (F-strain) and HSV-1 KOS at 0,6 and 12 h after infection of HAP1 wt and HAP1 DUX4-ko cells (MOI of 1). Representative experiment out of n = 3. Values shown are technical replicates. H qRT-PCR analysis of the DUX4 target genes TRIM43 as well as the viral genes ICP0 and ICP27 in cells treated with 2,6 nM, 9,8 nM A485 or DMSO and infected with HSV-1 (MOI of 3). Values shown are the biological replicates (n = 3) and presented as mean fold induction +/- SD (normalized to HPRT RNA) relative to uninfected control cells. Source Data are provided as a Source Data file.

Fig. 6. Degradation of DUX4 during HSV-1 infection using the mini-TRIMAway assay.

A Principle of the mini-TRIMAway assay: A DUX4 specific nanobody linked to the TRIM21 ring targets DUX4 for proteosomal degradation. B AlphaFold-Multimer generated prediction of the interaction of the two DUX4 directed nanobody clones Nb2-4 and Nb2-19 to the DUX4 protein. C Western blot analyzing DUX4 levels after simultaneous induction of DUX4 and induction of the nanobody targeting DUX4 for degradation (nb 2–4 or nb 2–19) or control nanobody (nb ctrl). HEK293T were induced with 0,1 µg/ml Doxycycline and analyzed 24 h post induction. HSP90 was used as loading control. D Microscope analysis of the mini-TRIMAway assay for DUX4 degradation after HSV-1 infection at day 1–3 post infection. The corresponding nanobody (nb 2-4, nb 2-19 or nb ctrl) was induced with 2 µg/ml doxycycline and infected with HSV-1 GFP (MOI 0.05). Representative experiment out of n = 3. E Measurement of GFP-expression by flow cytometry of the mini-TRIMAway assay for DUX4 degradation after HSV-1 infection at day 1-3 post infection. The corresponding nanobody (nb 2–4, nb 2–19 or nb ctrl) was induced with 2 µg/ml doxycycline and infected with HSV-1 GFP (MOI 0.05). Representative experiment out of n = 3. Values shown are technical replicates. Source Data are provided as a Source Data file.