Slug, a Stress-Induced Transcription Factor, Stimulates Herpes Simplex Virus 1 Replication and Transactivates a cis-Regulatory Module within the VP16 Promoter

Abstract

Stress-mediated activation of the glucocorticoid receptor (GR) and specific stress-induced transcription factors stimulate herpes simplex virus 1 (HSV-1) productive infection, explant-induced reactivation, and immediate early (IE) promoters that drive expression of infected cell protein 0 (ICP0), ICP4, and ICP27. Several published studies concluded the virion tegument protein VP16, ICP0, and/or ICP4 drives early steps of reactivation from latency. Notably, VP16 protein expression was induced in trigeminal ganglionic neurons of Swiss Webster or C57BL/6J mice during early stages of stress-induced reactivation. If VP16 mediates reactivation, we hypothesized stress-induced cellular transcription factors would stimulate its expression. To address this hypothesis, we tested whether stress-induced transcription factors transactivate a VP16 cis-regulatory module (CRM) located upstream of the VP16 TATA box (-249 to -30). Initial studies revealed the VP16 CRM cis-activated a minimal promoter more efficiently in mouse neuroblastoma cells (Neuro-2A) than mouse fibroblasts (NIH-3T3). GR and Slug, a stress-induced transcription factor that binds enhancer boxes (E-boxes), were the only stress-induced transcription factors examined that transactivated the VP16 CRM construct. GR- and Slug-mediated transactivation was reduced to basal levels when the E-box, two 1/2 GR response elements (GREs), or NF-κB binding site was mutated. Previous studies revealed GR and Slug cooperatively transactivated the ICP4 CRM, but not ICP0 or ICP27. Silencing of Slug expression in Neuro-2A cells significantly reduced viral replication, indicating Slug-mediated transactivation of ICP4 and VP16 CRM activity correlates with enhanced viral replication and reactivation from latency. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latency in several types of neurons. Periodically cellular stressors trigger reactivation from latency. Viral regulatory proteins are not abundantly expressed during latency, indicating cellular transcription factors mediate early stages of reactivation. Notably, the glucocorticoid receptor (GR) and certain stress-induced transcription factors transactivate cis-regulatory modules (CRMs) essential for expression of infected cell protein 0 (ICP0) and ICP4, key viral transcriptional regulatory proteins linked to triggering reactivation from latency. Virion protein 16 (VP16) specifically transactivates IE promoter and was also reported to mediate early stages of reactivation from latency. GR and Slug, a stress-induced enhancer box (E-box) binding protein, transactivate a minimal promoter downstream of VP16 CRM, and these transcription factors occupy VP16 CRM sequences in transfected cells. Notably, Slug stimulates viral replication in mouse neuroblastoma cells suggesting Slug, by virtue of transactivating VP16 and ICP4 CRM sequences, can trigger reactivation in certain neurons.

Keywords: HSV-1; Slug; VP16; stress response.

FIG 1

DEX stimulates VP16 protein expression during explant-induced reactivation. TG were dissected from C57BL/6J (A) or Swiss Webster (B) female mice 30 days post-ocular infection with HSV-1 strain McKrae and explanted in MEM containing 2% stripped FBS with or without DEX for 8 h. IHC was performed using a VP16 primary antibody (1:100 dilution) (Abcam). Black arrows denote neurons expressing VP16 in the nucleus.

FIG 2

Quantification of VP16⁺ TG neurons during explant-induced reactivation. The number of VP16⁺ TG neurons was estimated from ~500 total neurons for each treatment shown in Fig. 1. The results show a comparison of samples from latently infected TG versus TG explants incubated with MEM containing 2% stripped FBS with or without DEX treatment for 8 h. Sections were obtained from 5 mice/treatment. Asterisks denote significant difference as determined by a Student's t test: ***, P < 0.005; ****, P < 0.0005; N.D., none detected; NS, not significant.

FIG 3

Analysis of VP16 CRM activity in Neuro-2A or NIH-3T3 cells. (A) Nucleotide sequence of VP16 CRM and location of motifs that have the potential to be transactivated by GR and other stress-induced transcription factors. Shaded nucleotides denote a transcription factor binding site that matches the respective consensus binding site. Each transcription factor binding site was mutated by replacement of the binding site with an EcoRI restriction enzyme site (5′-GAATTC-3′). (B) Schematic of wt and mutant VP16 CRM constructs. Transcription factor binding sites have the same color scheme as described in panel A. (C) Neuro-2A or NIH-3T3 cells were cultured and transfected as described in Materials and Methods. Cells were transfected with plasmids encoding Renilla luciferase (0.05 μg DNA) and the wt VP16 CRM construct or designated mutant constructs (0.5 μg DNA). Cells were harvested 48 h posttransfection, and luciferase activity was measured. Basal transcriptional activity in the empty luciferase vector (pGL4.24[luc2/minP]) was normalized to a value of 1, and fold activation values for the other constructs were calculated. The results are the average from six separate experiments, and error bars denote standard errors. Asterisks denote significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001) between results for VP16 wt CRM relative to empty vector and relative to the denoted VP16 mutant constructs. #, not significant. Student's t test was used to analyze all results.

FIG 4

Transactivation of wt and mutant VP16 CRMs by GR and Slug. Neuro-2A (A) or NIH-3T3 (B) cells were transfected with a plasmid encoding Renilla luciferase (0.05 μg DNA), the wt VP16 CRM construct or the denoted mutant construct (0.5 μg DNA), a plasmid that expresses the mouse GR protein (1.0 μg DNA), and/or Slug (1.0 μg DNA) as described in Materials and Methods. Empty vector was included in certain samples to maintain the same amount of DNA in each sample. At 24 h posttransfection, MEM was changed, and the denoted cultures were treated with water-soluble DEX (10 μM). Cells were harvested 48 h posttransfection, and luciferase activity was measured. Basal transcriptional activity of cells transfected with wt VP16 CRM construct with empty expression vectors was normalized to a value of 1, and fold activation values for other samples were calculated. The results are the average from three separate experiments, and error bars denote standard errors. Asterisks denote significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001) between results for all VP16 mutant enhancer constructs (Slug ± GR ± DEX) relative to the wt (blue columns) and between wt constructs as indicated. #, P > 0.05. Student’s t test was used to analyze the results.

FIG 5

KLF4-mediated transactivation of wt and mutant VP16 CRMs. Neuro-2A (A) or NIH-3T3 (B) cells were cultured and transfected as described in Materials and Methods. Cells were transfected with a plasmid encoding Renilla luciferase (0.05 μg DNA), the wt VP16 CRM construct or designated mutant constructs (0.5 μg DNA), a plasmid that expresses the mouse GR protein (1.0 μg DNA), and KLF4 (1.0 μg DNA). Empty vector was included in certain samples to maintain the same amount of DNA in each sample. Certain cultures were treated with water-soluble DEX (10 μM) 24 h after transfection. Cells were harvested 48 h posttransfection and luciferase activity measured. Basal transcriptional activity in cells transfected with VP16 wt CRM construct with just empty expression vector was normalized to a value of 1, and fold activation values for other samples were calculated. The results are the average from 3 separate experiments, and error bars denote the standard errors. Asterisks denote significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001) between results of all VP16 mutant enhancer constructs (KLF4 ± GR ± DEX) relative to wt VP16 CRM (blue columns). #, P > 0.05. Student's t test was used to analyze the results.

FIG 6

KLF15-mediated transactivation of wt and mutant VP16 CRMs. Neuro-2A (A) or NIH-3T3 (B) cells were cultured as described in Materials and Methods. Cells were transfected with plasmid encoding Renilla luciferase (0.05 μg DNA), the wt VP16 enhancer construct, or designated mutant constructs (0.5 μg DNA), a plasmid that expresses the mouse GR protein (1.0 μg DNA), and KLF15 (1.0 μg DNA). Empty vector was included in certain samples to maintain the same amount of DNA in each sample. At 24 h posttransfection, the medium was changed, and the denoted cultures were treated with water-soluble DEX (10 μM). Cells were harvested 48 h posttransfection and luciferase activity measured. Basal transcriptional activity in cells transfected with VP16 wt enhancer construct with just empty expression vector was normalized to a value of 1, and fold activation values for other samples were calculated. The results are the average from three separate experiments, and error bars denote the standard errors. Asterisks denote significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001) between results for all VP16 mutant enhancer constructs (KLF15 ± GR ± DEX) relative to the wt (blue columns). #, P > 0.05. Student's t test was used to analyze the results.

FIG 7

PLZF-mediated transactivation of wt and mutant VP16 enhancers. Neuro-2A (A) or NIH-3T3 (B) cells were grown in MEM containing 2% charcoal-stripped FBS. Cells were transfected with plasmid encoding Renilla luciferase (0.05 μg DNA), the wt VP16 enhancer construct or the designated mutant constructs (0.5 μg DNA), a plasmid that expresses the mouse GR protein (1.0 μg DNA), and PLZF (1.0 μg DNA). Empty vector was included in certain samples to maintain the same amount of DNA in each sample. At 24 h posttransfection, the medium was changed, and certain cultures were treated with water-soluble DEX (10 μM). Cells were harvested 48 h posttransfection and luciferase activity measured. Basal transcriptional activity of cells transfected with VP16 wt CRM construct with just empty expression vector was normalized to a value of 1, and fold activation values for other samples were calculated. The results are the average from three separate experiments, and error bars denote the standard errors. Asterisks denote significant differences (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001) between results for all VP16 CRM mutant constructs (PLZF ± GR ± DEX) relative to the wt (blue columns). #, P > 0.05. Student's t test was used to analyze the results.

FIG 8

GR and Slug occupy VP16 CRM sequences. Neuro-2A cells were transfected with the wt VP16 CRM construct and the empty expression vector of Slug (A [Empty]) or in combination with GR (B), Slug (C), or GR plus Slug (D) expression plasmids. ChIP was performed using specific antibodies directed against GR (αGR), Slug (αSlug), or a nonspecific IgG isotype control (isotype) to immunoprecipitate DNA specifically associated with the designated protein. Immunoprecipitated DNA was amplified by PCR and analyzed using Image Lab software. The data presented are the DNA signal as a percentage of the input sample using a nonspecific IgG isotype control (white columns), GR antibody (black columns), or Slug antibody (hatched columns). Protein occupancy of the enhancer fragment is demonstrated as significant signal enrichment by GR or Slug antibody relative to the isotype, denoted by asterisks (**, P < 0.005; ***, P < 0.0005). Statistics were performed using Student's t test. The data presented represent four independent experiments.

FIG 9

Slug stimulates productive infection. Slug or scrambled negative-control siRNA was transfected into Neuro-2A cells at the designated concentrations of siRNA using Lipofectamine 3000. Cells were incubated in MEM containing 2% charcoal-stripped FBS at 37°C for 48 h prior to a trypan blue assay using the Bio-Rad TC20 automated cell counter to measure cell viability. Data are shown as the mean ± standard error of the mean (SEM) for duplicate wells from triplicate experiments. Neuro-2A cells were grown and transfected with increasing concentrations of Slug siRNA (a cocktail of siRNAs A, B, and C as shown in panels A and C) or scrambled negative-control siRNA (B and D). At 24 h posttransfection, cells were infected with HSV-1 at an MOI of 1 for 1 h at 37°C in 5% CO₂ with rocking every 15 min. The medium was replaced, and infected cells were incubated for 24 h. Infectious virus in cultures was measured by plaque assays. Data are shown as the mean ± SEM for duplicate wells from triplicate experiments. Asterisks denote significant differences (**, P ≤ 0.01; ***, P ≤ 0.001) relative to untreated (UT) cells that were not transfected. (E) Neuro-2A cells were transfected with 1.0 nM siRNA. At 48 h posttransfection, cells were collected and processed for Western blot analysis. β-Actin was used as a loading control. Lane 1 contained cells that were not transfected with any siRNA (denoted UT), lane 2 was transfected with the scrambled siRNA control (denoted Neg), and lanes 3 to 5 were transfected with 1 nM Slug siRNA (A, B or C). For each lane, 50 μg of protein was loaded. Cell lysate was probed with Slug polyclonal antibody. The bottom panel in panel E was overexposed to visualize Slug bands not readily detected by the lighter exposure. (F) siRNA knockdown of Slug was quantified using ImageJ. Data are shown as the mean ± SEM from two experiments normalized to UT controls. *, P ≤ 0.05, **, P ≤ 0.01, and ns, not significant, using unpaired Student’s t test. (G) Neuro-2A cells were mock infected or infected with HSV-1 (MOI = 1 PFU/cell) for 1 h. At the designated times after infection (hours), cells were collected and lysed with RIPA buffer. Western blot analysis was performed using the Slug polyclonal antibody. As a loading control, β-actin levels were examined. For each lane, 50 μg protein was loaded. Lanes denoted m0, m4, or m24 represent 0, 4, or 24 h after mock infection, respectively. A representative blot of three independent experiments is shown. The sizes of molecular weight markers (in kilodaltons) are shown to the left of the blot.

FIG 10

Schematic of VP16 CRM sequences important for GR- and Slug-mediated transactivation. (A) DNA sequence of the GRE consensus, consensus 1/2 GREs, and two 1/2 GREs in VP16 CRM sequences. Nucleotides above the consensus 1/2 GRE indicate changes that still result in GR binding and transactivation (lowercase denotes less likelihood for the nucleotide to be bound by GR monomer), and “N” refers to any base. (B) Shown is the 55-bp region within VP16 CRM sequences that contain both 1/2 GREs, the E-box, and NF-κB consensus binding sites. Predicted interactions, in part defined by ChIP studies in Fig. 7, that occur with the denoted transcription factors and VP16 sequences are shown. For details, see the text. This figure was drawn using BioRender.