Inhibition of histone methyltransferase SETD8 represses DNA virus replication

Abstract

Multiple diseases, such as cancer and neural degeneration diseases, are related with the latent infection of DNA viruses. However, it is still difficult to clean up the latent DNA viruses and new anti-viral strategies are critical for disease treatment. Here, we screen a pool of small chemical molecules and identify UNC0379, an inhibitor for histone H4K20 methyltransferase SETD8, as an effective inhibitor for multiple DNA viruses. UNC0379 not only enhances the expression of anti-viral genes in THP-1 cells, but also repress DNA virus replication in multiple cell lines with defects in cGAS pathway. We prove that SETD8 promotes DNA virus replication in a manner dependent on its enzyme activity. Our results further indicated that SETD8 is required for PCNA stability, one factor critical for viral DNA replication. Viral infection stimulates the interaction between SETD8 and PCNA and thus enhances PCNA stability and viral DNA replication. Taken together, our study reveals a new mechanism for regulating viral DNA replication and provides a potential strategy for treatment of diseases related with DNA viruses.

Keywords: DNA replication; DNA virus; PCNA; SETD8; UNC0379.

Graphical abstract

Fig. 1

UNC0379 inhibitsHSV-1replication. (A) HFF cells were infected with HSV-1 and treated with inhibitors of histone modification enzymes.12 h later, expression of viral US11 gene were measure by quantitative RT-PCR. (B) U2OS cells were treated with UNC0379 for 12 h at the indicated concentrations and cell viability was measured by MTT assay. (C) U2OS cells were infected with HSV-1 and treated with UNC0379 for 10 h at the indicated concentrations. The amount of viral DNA was measured by quantitative PCR. (D) U2OS cells were infected with HSV-1 for 10 h and treated with 5 μM UNC0379 for the indicated time. The amount of viral DNA was measured by quantitative PCR. (E–F) HFF cells were infected with HSV-1 and treated with 5 μM UNC0379 for 10 h. The amounts of viral DNA (F) and viral genes expression (E) were measure by quantitative PCR. (G) Virus plaque assay with the supernatants of Vero cells infected with HSV-1 (MOI = 0.5) for 10 h with or without UNC0379. The results in all experiments represent the means (±SD) of at least three independent experiments. Statistical analysis was performed using an unpaired Student's t-test. ∗ means p-value ≤ 0.05, ∗∗ for p-value ≤ 0.01, ∗∗∗ for p-value ≤ 0.001.

Fig. 2

Setd8 deficiency inhibitsHSV-1replication. (A–E) U2OS cells were transfected with SETD8 siRNAs and infected with HSV-1 for 10 h. The amounts of viral DNA (A), viral genes expression (B) and SETD8 expression (D&E) were measured by quantitative PCR and immunoblot. Virus plaque assay (C) was performed with the supernatants of U2OS cells infected with HSV-1 (MOI = 0.5) for 10 h with or without SETD8 knockdown. (F–J) Vero cells were transfected with Flag-SETD8 and infected with HSV-1 for 10 h. The viral DNA amount (F), viral genes expression (G), virus plaque assay (H) and SETD8 expression (I&J) were analyzed as above. (K–M) U2OS cells were transfected with SETD8 siRNA, treated with or without 5 μM UNC0379 and infected with HSV-1 for 10 h. Viral genes expression (K) and SETD8 expression (L&M) were measured. (N–P) SETD8 was knocked down in U2OS cells with siRNA and then wild type SETD8 or R265G mutant was expressed. Viral genes expression (N) and SETD8 expression (O&P) were measured after HSV-1 infection for 10 h. The results in all experiments represent the means (±SD) of at least three independent experiments. Statistical analysis was performed using an unpaired Student's t-test. ∗ means p-value ≤ 0.05, ∗∗ for p-value ≤ 0.01, ∗∗∗ for p-value ≤ 0.001.

Fig. 3

UNC0379 promotesanti-viralgene expression inTHP-1. (A) THP1 cells were infected with HSV-1 and treated with 5 μM UNC0379 for 10 h. RNA-seq was performed and the heat map shows the expression (FPKM) of all the DEGs compared with negative control. Four clusters of genes were identified. (B) Bar plot shows the function and pathway analysis of cluster 3, gradient color filled with p-value. (C&D) THP1 cells (C) and U2OS cells (D) were infected with HSV-1 and treated with or without 5 μM UNC0379 for 10 h. The immune genes expression was measured by quantitative RT-PCR. The results represent the means (±SD) of at least three independent experiments. Statistical analysis was performed using an unpaired Student's t-test. ∗ means p-value ≤ 0.05, ∗∗ for p-value ≤ 0.01, ∗∗∗ for p-value ≤ 0.001.

Fig. 4

PCNA is required forHSV-1replication. (A) Immunoblot assay of PCNA in Vero cells after HSV-1 infection and UNC0379 treatment for 10 h. (B) Immunoblot assay of PCNA and SETD8 in U2OS cells after SETD8 knockdown and HSV-1 infection. (C–G) Vero cells were transfected with Flag-PCNA plasmid for 48 h and infected with HSV-1 for 10 h. The amounts of viral DNA (E), viral genes expression (F), virus titer (G) and PCNA expression (H&I) were measured. The results in all experiments represent the means (±SD) of at least three independent experiments. Statistical analysis was performed using an unpaired Student's t-test. ∗ means p-value ≤ 0.05, ∗∗ for p-value ≤ 0.01, ∗∗∗ for p-value ≤ 0.001.

Fig. 5

PCNA rescuesHSV-1repression by SETD8 deficiency. (A–D) Vero cells were transfected with Flag-PCNA plasmids for 48 h and followed by HSV-1 infection and 5 μM UNC0379 treatment for 10 h. The amounts of viral DNA (A), viral genes expression (B) and PCNA expression (C&D) were assayed by quantitative PCR and immunoblotting. (E–G) U2OS cells were transfected SETD8 siRNA and PCNA plasmid. The amounts of viral DNA (E), viral genes expression (F) and PCNA expression (G) were measured after HSV-1 infection for 10 h. (H–K) Vero cells were transfected with PCNA K248A mutant plasmid for 48 h and followed by 5 μM UNC0379 treatment and HSV-1 infection for 10 h. The amounts of viral DNA (H), viral genes expression (I) and PCNA expression (J&K) were measured. (L) Immunoblot assay of PCNA and SETD8 in U2OS cells after SETD8 overexpression and HSV-1 infection. The number below the blots indicates the relative amount of Flag-Setd8/actin or PCNA/actin. (M) FLAG-SETD8 were expressed in 293T cells. After 48 h, cells were infected with or without HSV-1 for 10 h. Cells were IPed with anti-FLAG, and immunoprecipitants were blotted with anti-FLAG and anti-PCNA antibodies, respectively. The number below the blots indicates the relative amount of PCNA/Flag-Setd8. The results in all experiments represent the means (±SD) of at least three independent experiments. Statistical analysis was performed using an unpaired Student's t-test. ∗ means p-value ≤ 0.05, ∗∗ for p-value ≤ 0.01, ∗∗∗ for p-value ≤ 0.001.

Fig. 6

UNC0379 treatment repressesHSV-1in vivo. (A) Survival (Kaplan-Meier curve) of mice intraperitoneal injected with HSV-1 (1 × 10⁸ PFU per mouse) with (n = 15) or without UNC0379 treatment (2 mg/kg, intraperitoneal injection). Animal survival was monitored for 12 days. (B–C) The viral DNA amounts of lung (B) and viral titers of brain (C) from mice intravenously injected with HSV-1 (2.5 × 10⁶) and UNC0379 (2 mg/kg) for 48 h (n = 5). (D) The primary BMDM cells isolated from the mouse bone were infected with HSV-1 and treated with UNC0379 for 10 h. The amount of viral DNA was measured. (E–G) SETD8 was knocked down in BMDM cells by siRNA and cells were infected with HSV-1 for 10 h. The amounts of viral DNA (E) and SETD8 expression (F&G) were measured. (H–J) The amounts of viral DNA (H) and PCNA expression (I&J) in BMDM cells transfected with PCNA plasmid for 48 h and treated with 5 μM UNC0379 and HSV-1 for 10 h. The results in all experiments represent the means (±SD) of at least three independent experiments. Statistical analysis was performed using an unpaired Student's t-test. ∗ means p-value ≤ 0.05, ∗∗ for p-value ≤ 0.01, ∗∗∗ for p-value ≤ 0.001.