Inhibition of the histone demethylase LSD1 blocks alpha-herpesvirus lytic replication and reactivation from latency

Abstract

Reversible methylation of histone tails serves as either a positive signal recognized by transcriptional assemblies or a negative signal that result in repression. Invading viral pathogens that depend upon the host cell's transcriptional apparatus are also subject to the regulatory impact of chromatin assembly and modifications. Here we show that infection by the alpha-herpesviruses, herpes simplex virus (HSV) and varicella zoster virus (VZV), results in the rapid accumulation of chromatin bearing repressive histone H3 Lys9 methylation. To enable expression of viral immediate early (IE) genes, both viruses use the cellular transcriptional coactivator host cell factor-1 (HCF-1) to recruit the lysine-specific demethylase-1 (LSD1) to the viral immediate early promoters. Depletion of LSD1 or inhibition of its activity with monoamine oxidase inhibitors (MAOIs) results in the accumulation of repressive chromatin and a block to viral gene expression. As HCF-1 is a component of the Set1 and MLL1 histone H3 Lys4 methyltransferase complexes, it thus coordinates modulation of repressive H3 Lys9 methylation levels with addition of activating H3 Lys4 trimethylation marks. Strikingly, MAOIs also block the reactivation of HSV from latency in sensory neurons, indicating that the HCF-1 complex is a crucial component of the reactivation mechanism. The results support pharmaceutical control of histone modifying enzymes as a strategy for controlling herpesvirus infections.

Figure 1. LSD1 is critical for viral activator mediated transcription of VZV IE and HSV IE model promoters

(a) The VZV IE model promoter-reporter is illustrated with Sp1, TATA, and IE62 binding sites. ChIP assay showing H3K4 and H3K9 methylation and (b) activator/coactivator occupancy at the VZV IE model promoter in the presence and absence of the VZV IE62 activator. IgG, control immunoglobulin, me-1, monomethyl; me-2, di-methyl; me-3, tri-methyl. (c) Western blot of LSD1 and control (tubulin) showing depletion of LSD1 (LSD-1, LSD-2) relative to cells transfected with control scrambled RNAi (C). VZV IE promoter-luciferase reporter activity in cells transfected with IE62 and LSD1 or control RNAi(s) relative to cells transfected with no RNAi. LSD1 depletions ranged from 42–57%. (d) ChIP assay showing H3K4 and H3K9 methylation and activator/coactivator occupancy on the model VZV IE promoter in cells transfected with control shRNA (HCF-1⁺) or HCF-1 shRNA (HCF-1⁻). Occupancy is expressed as the ratio of that in HCF-1-depleted cells to that in control HCF-1⁺ cells. (e) The HSV-1 ICP0 promoter-reporter is schematically illustrated with the enhancer core (EC) element that nucleates the assembly of the HCF-1 protein enhancer complex, CCAAT, TATA, and Sp1 binding sites. ICP0 promoter-luciferase reporter activity in cells transfected with HCF-1, or LSD1 RNAi relative to control (GFP) RNAi. (f) Activity of ICP0 promoter-luciferase reporter in cells expressing control β-galactosidase (LacZ), wild-type LSD1, or an LSD1 catalytic mutant (LSD1 K661A).

Figure 2. An HCF-1/LSD1 complex is essential for α-herpesvirus IE gene transcription

(a) ChIP assay showing H3K4 and H3K9 methylation and activator/coactivator occupancy on the VZV genomic IE62 promoter and coding sequences at 4 hrs post infection. (b) ChIP assay showing H3K4 and H3K9 methylation and activator/coactivator occupancy on the VZV IE promoter in cells transfected with HCF-1 shRNA (HCF-1⁻) relative to that in cells transfected with control shRNA (HCF-1⁺). (c) Western blot of LSD1, IE62, and control (tubulin) in VZV infected MCF7 cells inducibly expressing LSD1 shRNA in the presence and absence of doxycycline induction. S15, ribosomal subunit mRNA. (d) ChIP assay showing H3K4 and H3K9 methylation and coactivator occupancy on the genomic HSV ICP0 promoter at 4 hours post HSV-1 infection. H3, histone H3. (e) ChIP assay showing HCF-1 and LSD1 occupancy on an HSV IE promoter in cells transfected with control or HCF-1 siRNA (HCF-1⁻). (f) Western blot of HSV IE proteins (ICP4, ICP27), LSD1, and control TATA-binding protein (TBP) in control cells (GFP RNAi) and cells depleted for LSD1 (LSD1-1, LSD1-2). (g) The levels of the HSV-1 IE protein ICP4 in cells depleted for LSD1 and transfected with plasmids expressing no protein (−), control GFP, wild-type LSD1, an LSD1 catalytic mutant (K661A), or an LSD1 mutant lacking the amine oxidase domain (LSD1ΔAO). The results of 2-tailed t tests are shown representing 4 independent experiments. (h) ChIP assay showing histone H3 and H3K9 methylation on the HSV ICP0 promoter in cells depleted of LSD1 (LSD1 RNAi) and control cells (GFP RNAi). The data is normalized to the levels of H3 at the cellular GAPDH promoter in the appropriate GFP RNAi cells or LSD1 RNAi cells. Western blot showing depletion of LSD. (i) HCF-1 western blot of HCF-1, LSD1, Set1, and control IgG immunoprecipitates (top panel). Western blot of the Set1/MLL1 HMT core subunit RbBP5 from LSD1, HCF-1, and control IgG immunoprecipitates (bottom panel). (j) Model of the HCF-1-Set1-LSD1 complex representing HCF-1 coupled demethylase (LSD1) and methyltransferase (Set1) activities. RbBP5, Ash2L, and WDR5 are core subunits of the Set1 HMT complex.

Figure 3. Inhibition of LSD1 with MAOIs blocks α-herpesviral lytic gene expression

(a) Western blot showing Pargyline (P) mediated inhibition of VZV IE gene expression (IE62) in cells infected with VZV for 4 hrs relative to control DMSO (D). LSD1, Tubulin, and TBP control proteins are shown. qRT-PCR of IE62 and control (s15, actin) mRNA levels in cells infected with VZV for 4 hrs in the presence of increasing amounts of Pargyline or Tranylcypromine (TCP). The results are graphed as the percent of levels in control treated cells. (b) Western blot showing inhibition of HSV IE protein expression (ICP4, ICP27) at 4 hours post HSV-1 infection in cells treated with increasing concentrations of either Pargyline or TCP. Control proteins (Sp1, TBP) are shown. (c) qRT-PCR analyses of mRNA levels of HSV IE genes and controls (Sp1, TBP, s15) in cells treated with selected concentrations of Pargyline or TCP. (d) Viral yields from cells infected with 0.1 plaque forming units (PFU) HSV-1 per cell in the presence of 2 mM TCP or control DMSO for 24 hrs. (e) ChIP assay showing histone H3 and H3K9 methylation on the HSV-1 IE0 promoter in the presence of 3 mM Pargyline or 2 mM TCP at 4 hrs post HSV-1 infection. The results are shown as ratios of occupancy in drug treated cells to those in control DMSO treated cells. The data is normalized to the ratio of total H3 in drug treated/DMSO treated cells at the cellular actin promoter. me-1, mono-methyl; me-2, di-methyl; H3, total histone H3.

Figure 4. Inhibition of LSD1 with MAOIs blocks HSV-1 reactivation from latency

(a) Viral yield from ganglia explanted in the absence (DMSO) or presence of 2 mM TCP for 2 days or 4 days. Day 2 P = 0.0043; day 4 P = 0.0011; n=6 for each sample set. (b) Viral yield from paired explanted ganglia in the absence (DMSO) or presence of 2 mM TCP for 2 days. P = 0.0002; n=16 for each sample set. (c) Viral yield from explanted ganglia in the presence or absence (DMSO) of various concentrations of TCP. P < 0.05 for 1.0, 1.5, and 2.0 mM TCP while P > 0.05 for 0.5 mM TCP; n=5 for control, 0.5, 1.0, and 2.0 mM; n=6 for 1.0 mM. (d) Viral yield from explanted ganglia in the absence (DMSO) or presence (TCP) of 2 mM TCP for 2 days followed by incubation in the absence for 3 days (TCP-R). P < 0.025 for TCP vs TCP-R; n=4 for TCP; n=6 for TCP-R. Details of all statistical analyses are in Supplementary Methods. (e) Immunofluorescent staining of HSV-1 latently infected ganglia explanted for 48 hrs in the presence of control (DMSO), 100 uM ACV, or 2 mM TCP. ICP8, HSV single stranded DNA binding protein. For each condition, the number of HSV-1⁺ and HSV-1⁻ ganglia is shown (P =.00002). (f) Nested RT-PCR analyses of HSV ICP27 IE mRNA from ganglia explanted in the presence of 2 mM TCP or control ACV for 12 hrs. cDNA samples were normalized to the levels of the cellular Sp1 mRNA as determined by qPCR. HSV Stnd represents is the signal from an equivalent amount of control cDNA produced from HSV infected 3T3 cells at 6.4×10⁻⁵ pfu/cell. Quantitation was as described in Supplementary Methods. −RT, +RT denote the absence or presence of the reverse transcriptase in the cDNA synthesis reaction. (g) An HCF-1 complex couples LSD1 with Set1/MLL1 to promote α-herpesvirus IE gene transcription. Upon infection, the genomes of infecting α-herpesviruses are subject to the accumulation of repressive chromatin (H3K9 methylation and HP1 occupancy). For productive infection, α-herpesviruses recruit an HCF-1-dependent modification complex containing LSD1 and the H3K4 HMTs Set1/MLL1 to promote the installation of positive transcriptional marks. Failure to recruit this complex results in continued accumulation of nucleosomes bearing repressive H3K9 methylation that silences the viral genome. RNAPII, RNA polymerase II; HP1, heterochromatin protein 1.