Inhibition of the Super Elongation Complex Suppresses Herpes Simplex Virus Immediate Early Gene Expression, Lytic Infection, and Reactivation from Latency

Abstract

Induction of herpes simplex virus (HSV) immediate early (IE) gene transcription promotes the initiation of lytic infection and reactivation from latency in sensory neurons. IE genes are transcribed by the cellular RNA polymerase II (RNAPII) and regulated by multiple transcription factors and coactivators. The HCF-1 cellular coactivator plays a central role in driving IE expression at multiple stages through interactions with transcription factors, chromatin modulation complexes, and transcription elongation components, including the active super elongation complex/P-TEFb (SEC-P-TEFb). Here, we demonstrate that the SEC occupies the promoters of HSV IE genes during the initiation of lytic infection and during reactivation from latency. Specific inhibitors of the SEC suppress viral IE expression and block the spread of HSV infection. Significantly, these inhibitors also block the initiation of viral reactivation from latency in sensory ganglia. The potent suppression of IE gene expression by SEC inhibitors indicates that transcriptional elongation represents a determining rate-limiting stage in HSV IE gene transcription and that the SEC plays a critical role in driving productive elongation during both phases of the viral life cycle. Most importantly, this supports the model that signal-mediated induction of SEC-P-TEFb levels can promote reactivation of a population of poised latent genomes.IMPORTANCE HSV infections can cause pathologies ranging from recurrent lesions to significant ocular disease. Initiation of lytic infection and reactivation from latency in sensory neurons are dependent on the induced expression of the viral immediate early genes. Transcription of these genes is controlled at multiple levels, including modulation of the chromatin state of the viral genome and appropriate recruitment of transcription factors and coactivators. Following initiation of transcription, IE genes are subject to a key regulatory stage in which transcriptional elongation rates are controlled by the activity of the super elongation complex. Inhibition of the SEC blocks both lytic infection and reactivation from latency in sensory neurons. In addition to providing insights into the mechanisms controlling viral infection and reactivation, inhibitors of critical components such as the SEC may represent novel antivirals.

Keywords: AFF4; HCF-1; HSV reactivation; herpes simplex virus; latency; super elongation complex; transcription; transcription elongation.

FIG 1

Inhibitors of the SEC suppress HSV IE gene expression. (A) KL-1 and KL-2 interfere with the interaction of CCNT1/P-TEFb with SEC scaffold subunit AFF4. (B to D) HFF cells were treated with the indicated concentrations of KL-1 (B) or KL-2 (C) and infected with HSV (MOI = 1) for 2.5 h. (B and C) mRNA levels of viral IE genes (ICP4, ICP22, ICP27, and ICP0) and a control cellular gene (HPRT) relative to those in vehicle-treated cells. Sample mRNA levels were normalized based on the levels of cellular GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNAs. Data are means ± SEM of results from at least two experiments. (D) (Left) Western blot of viral IE (ICP4 and ICP27) and cellular control (GAPDH) protein levels. (Right) Quantitation of protein levels in cells treated with KL-1 or KL-2 relative to those in cells treated with vehicle. Data are means ± SEM of results from four replicates (analysis of variance [ANOVA] with Dunnett’s post hoc test). (E) HFF cells were treated with vehicle or 8 μM KL-1 or KL-2 and infected with HSV (MOI = 1) for 2 h. Nuclear HSV DNA levels in cells treated with KL-1 or KL-2 relative to those in cells treated with vehicle are indicated. ns, not significant (ANOVA with Dunnett’s post hoc test).

FIG 2

Inhibitors of the SEC suppress the spread of HSV lytic infection. (A to C) HFF and MRC5 cells were infected with HSV (MOI = 0.025) for 8 h and then treated with vehicle, acyclovir (ACV), or 5 or 10 μM KL-1 or KL-2 for 16 h. (A) HFF cells were stained with anti-UL29 (green) and phalloidin-647 (F-actin, red). Scale bars, 100 μm (left 2 columns) and 20 μm (right two columns). (B and C) Viral yields from HFF-infected cells (B) and MRC5-infected cells (C). Data are means ± SEM of results from 4 replicates (ANOVA with Dunnett’s post hoc test). (D) Viral yields from HFF cells treated with vehicle or 5 or 10 μM KL-1 or KL-2 and infected with HSV (MOI = 3) for 12 h. Data are means ± SEM of results from 8 replicates (ANOVA with Dunnett’s post hoc test).

FIG 3

Inhibitors of the SEC reduce the number of transcriptionally active viral genomes. (A and B) HFF cells were treated with vehicle or with KL-1 (10 μM) or KL-2 (10 μM) and infected with HSV (MOI = 5) for 2 h. (A) Cells were stained with anti-ICP4 (viral transcriptional foci; red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar = 3 μm. Three representative images are shown for each condition. (B) The number of transcriptionally active foci per cell. Data are means ± SEM (n > 136 cells per group) (ANOVA with Dunnett’s post hoc test).

FIG 4

KL-2 reduces the recruitment of the SEC to viral IE promoters. (A and B) HFF cells were treated with vehicle or 8 μM KL-2 and infected with HSV (MOI = 2) for 2 h. (A) ChIP assay data showing the levels of the SEC (AFF4) associated with viral IE (ICP0 and ICP4), control viral (LAT), or control cellular (HSPA8) genes in cells treated with vehicle or KL-2. Data are means ± SEM of results from 4 experiments (paired two-tailed t tests). (B) Ratio of AFF4 occupancy levels in KL-2-treated versus vehicle-treated cells.

FIG 5

KL-2 suppresses the initiation of viral reactivation from latency. (A) Viral yields from latently infected trigeminal ganglia explanted in the presence of vehicle, JQ1 (1 μM), KL-2 (10 μM), or ACV (100 μM) for 48 h. Data are from individual ganglia (n ≥ 9; Wilcoxon matched-pairs 2-tailed signed rank test). (B) mRNA levels of viral (ICP27, UL30, and gC) genes and cellular control mHprt genes in latently infected trigeminal ganglia explanted in the presence of vehicle, JQ1 (1 μM), JQ1/iCDK9 (1 μM/150 nM), or JQ1/KL-2 (1 μM/5 μM) for 6 h. (C) mRNA levels of viral (ICP27) and cellular control (mHPRT) genes in latently infected trigeminal ganglia explanted in the presence of vehicle, iCDK9 (150 nM), or KL-2 (5 μM) for 12 h. (B and C) Sample mRNA levels were normalized based on the levels of cellular murine GAPDH (mGapdh) mRNA. Data are means ± SEM of results from 2 experiments (n ≥ 2 pools of five ganglia per group.

FIG 6

SEC recruitment to viral IE genes during initiation of viral reactivation from latency. (A) Data from ChIP assays showing the levels of the SEC (mAff4) associated with viral (ICP4 and UL36) and cellular (mHspa1a) genes in latently infected trigeminal ganglia at 0 h post-explant (latent) and 6 h post-explant (reactivation). Data are means ± SEM of results from 2 experiments; each immunoprecipitation was performed with a pool of 10 ganglia. (B) mRNA levels of mAff4 and mHspa1a in explanted ganglia relative to latently infected ganglia. mRNA levels were normalized based on the levels of cellular mGapdh mRNA. Data represent means of results from 2 pools of 4 ganglia each.

FIG 7

HSV infection increases the levels of AFF4 protein in an ICP0-dependent manner. (A) (Left) Western blot of AFF4 and control cellular proteins (HEXIM1, BRD4, CDK9, and GAPDH) and viral IE proteins (ICP4, ICP0, and ICP27) in extracts from mock-infected HFF cells or cells infected with wild-type HSV (MOI = 5) for 4 h. (Right) Quantitation of protein levels in HSV-infected relative to mock-infected cells. Data are means ± SEM of results from 4 experiments (unpaired two-tailed t tests). (B) mRNA levels of AFF4 and cellular control genes (GAPDH and HPRT) in cells infected with HSV (MOI = 5) relative to those in mock-infected cells. Data are means ± SEM of results from at least 2 experiments and 4 to 10 replicates (ANOVA with Dunnett’s post hoc test). (C) (Left) Western blot of AFF4 and control cellular proteins (HEXIM1, BRD4, CDK9, and GAPDH) and viral IE proteins (ICP4, ICP0, and ICP27) in extracts from mock-infected cells or cells infected with wild-type (WT) HSV or ΔICP0 HSV (MOI = 5) for 4 h. (Right) Quantitation of protein levels in HSV-infected relative to mock-infected cells. Data are means ± SEM of results from 2 experiments and 3 to 6 replicates (ANOVA with Dunnett’s post hoc test). (D) (Left) Western blot of AFF4 and control cellular proteins (BRD4 and GAPDH) and viral IE proteins (ICP0 and ICP27) in extracts from mock-infected cells or cells infected with wild-type (WT) HSV or ΔICP0 HSV (MOI = 5) for 4 h in the absence or presence of 10 μM MG132. (Right) Quantitation of protein levels in HSV-infected relative to mock-infected cells. Data are means ± SEM of results from 3 replicates (unpaired two-tailed t tests).

FIG 8

Model of epigenetic modulation and SEC-mediated induction of viral IE gene expression during initiation of HSV reactivation. Stress signaling promotes viral reactivation at multiple rate-limiting stages. Signaling induces the transport of HCF-1 to the nucleus of latently infected neurons where HCF-1 complexes containing histone H3K9 demethylases (LSD1 and JMJD2s) and histone H3K4 methyltransferases (SETD1A and MLLs) are involved in promoting the transition of heterochromatic viral genomes to accessible euchromatic genomes. This transition can be blocked by inhibitors (LSDi and ML324) of these HCF-1-associated H3K9 demethylases, resulting in suppression of viral reactivation. Signaling also results in the release of P-TEFb from the 7SK-snRNP complexes, which increases the levels of active SEC-P-TEFb. Along with HCF-1, recruitment of SEC-P-TEFb to viral IE gene promoters stimulates productive expression of IE genes and reactivation of a population of accessible genomes. Compounds such as BET inhibitors (JQ1) enhance the levels of active P-TEFb and induce reactivation, while SEC inhibitors (KL-1/2) disrupt the active SEC and suppress reactivation. Following induction, the accumulation of the viral IE ICP0 protein may further stabilize the SEC, enhance active SEC-P-TEFb levels, and contribute to reactivation. Note that while part of this model reflects the described roles of HCF-1 and its associated chromatin modulation factors in viral reactivation, the epigenetic regulation of latent viral genomes is complex. Other repressive histone marks (e.g., H3K27me3 [72, 77–81]) are also associated with latent viral genomes, and it is likely that additional pathways and components contribute to the epigenetic regulation of latency and reactivation.