Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Feb 20:8:240.
doi: 10.3389/fmicb.2017.00240. eCollection 2017.

HELZ2 Is an IFN Effector Mediating Suppression of Dengue Virus

Dahlene N Fusco  1 , Henry Pratt  2 , Stephen Kandilas  3 , Scarlett Se Yun Cheon  4 , Wenyu Lin  2 , D Alex Cronkite  2 , Megha Basavappa  2 , Kate L Jeffrey  2 , Anthony Anselmo  5 , Ruslan Sadreyev  5 , Clarence Yapp  6 , Xu Shi  7 , John F O'Sullivan  8 , Robert E Gerszten  9 , Takuya Tomaru  10 , Satoshi Yoshino  10 , Tetsurou Satoh  10 , Raymond T Chung  2
Affiliations

HELZ2 Is an IFN Effector Mediating Suppression of Dengue Virus

Dahlene N Fusco et al. Front Microbiol. .

Abstract

Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral response is mediated by HELZ2 transcriptional upregulation, enrichment of HELZ2 protein levels in the nucleus, and activation of a transcriptional program that appears to modulate intracellular lipid state. IEGs identified in this study may serve as both (1) potential targets for host directed antiviral design, downstream of the common flaviviral subversion point, as well as (2) possible biomarkers, whose variation, natural, or iatrogenic, could affect host response to viral infections.

Keywords: genes that are required for IFN-mediated suppression of virus; interferon; interferon effector gene (IEG).

PubMed Disclaimer

Figures

Figure 1
Figure 1
Targeted Dengue Interferon Effector Screen Identifies 56 DENV-HCV IEGs, including nuclear receptor pathway factors. (A) IFN signals through IFNAR, JAK1, TYK2, STAT1/2 to activate transcription of IFN stimulated genes, including HELZ2. HELZ2 is a nuclear receptor co-activator and a helicase. (B) A targeted RNAi screen was performed to identify host factors required for interferon's antiviral activity against DENV, or DENV IFN effector genes (DENV IEGs). HeLa cells were reverse transfected with 4 unique siRNAs for each of 120 HCV IEGs, followed by 48 h incubation, treatment with IFN, then infection with DENV2, followed by 48 h incubation then staining, imaging, and quantitative analysis. (C) Raw Images from DENV IEG screen. HeLa cells were transfected with indicated siRNA, processed as in (B), then stained with anti-DENV antibody (green) or Hoechst nuclear stain (blue) and imaged for quantitative analysis. Cell count and percent infection are indicated on 10x images. (D) Rank plot of mean fold rescue for all screened siRNA duplexes. Fold rescue above 1.5 was considered positive. Y axis indicates mean fold rescue, X axis indicates rank of rescue. (E) Bioinformatic analysis of DENV IEGs identified two nuclear receptor pathways, the aryl hydrocarbon receptor pathway and the PPARα/RXR pathway.
Figure 2
Figure 2
DENV IEG Screen Hits Confirmation. (A) HeLa Cell Out-of-Screen DENV IEG Confirmation. RNAi mediated knockdown of select DENV IEG screen hits confirmed rescue of DENV from IFN (HeLa Cells, n = 3–5). (B) IEG Phenotype Evaluation in a more clinically relevant cell type: hepatocytes. Huh7.5.1 cells were transfected with indicated siRNA followed by treatment with IFN, then infection with DENV, and staining, quantitative imaging. (C) Stable HELZ2 knockdown cells exhibit IFN resistance. Huh7.5.1 cells stably transduced with vector control (APM), shRNA targeting HELZ2 long and short isoforms (85 s) or long isoform only (91) were evaluated for IFN resistance. HELZ2 knockdown cells exhibit IFN resistance compared to vector transduced controls. For (A–C), cells were treated with IFN then infected with DENV, followed by staining for DENV and nucleus, then quantitative microscopy for percent infection. Stars indicate significant differences by two-tailed t-tests, unequal variance. (D) Knockdown shRNA target schematic.
Figure 3
Figure 3
Validation of HELZ2 knockdown in HeLa cells at the mRNA and protein levels. HeLa cells were transfected with four unique siRNAs targeting HELZ2 (h1-4), or non-targeting control (nt), followed by 48 h incubation, then IFN treatment for 24 h, then protein or RNA harvest. Quantification of HELZ2 protein, normalized to actin, from a representative western blot is shown for HELZ2 long isoform (A), short isoform (B), with western blot (C). Non transfected (null) sample is indicated by n. qRT PCR was used to validate HELZ2 mRNA knockdown (D). *p < 0.05 compared to control.
Figure 4
Figure 4
HELZ2 long (beta, v1) and short (alpha, v2) isoforms are both IFN responsive. (A,B) HELZ2 alpha, beta promoter activation. Huh751 or HeLa cells were transiently transfected with plasmids containing HELZ2v1 (beta, long isoform) or HELZv2 (alpha, short isoform) promoter with luminescence reporter. Cells were treated with medium, IFN (dose indicated) or virus for 24 h, followed by development and luminescence read. Virus used was JFH1 HCV in Huh751 s and NGC2 DENV in HeLas. (A) IFN led to greater HELZ2 v1/beta than v2/alpha promoter activation in Huh7.5.1 human hepatocytes. (B) HeLa IFN response is attenuated compared to Huh7.5.1 cells. Virus does not affect promoter response. (C) Promoter reporter schematic. The top purple lines indicate two unique HELZ2 promoters, with different transcription start sites for the two HELZ2 isoforms identified based on genomic sequence information from the UCSC genome browser. The lower blue schema represent the two unique luciferase promoter reporters generated for each of the HELZ2 isoform promoters. (D) HELZ2 isoform specific mRNA expression. Response of HELZ2 mRNA to IFN time course was measured using isoform-specific primers and qRT PCR. (E–H) HELZ2 long (v1/alpha) and short (v2/beta) isoform proteins are upregulated by IFN but not the PPARα agonist fenofibrate. (E) Huh7.5.1 cellular compartments were fractionated followed by western blot (wb) for compartment specific HELZ2 expression analyses after 24 h stimulation with medium (M), IFN 1,600 IU/ml (I), or 10 um fenofibrate (F). Experiment was performed thrice and a representative WB is shown (E). HELZ2 long isoform was detected between 420 and 247 kDa bands at highest levels during IFN treatment, with IFN induction in both nuclear and non-nuclear compartments. HELZ2 short isoform protein was also enhanced by IFN, most significantly in the nuclear compartment. (F,G) Representative WB results from e. were quantified for each compartment (x axis), with y axis indicating fold increase above background, which was set to 1. mem, membrane; nuc, nucleus; chr, chromatin; cyt, cytoplasm; ske, cytoskeleton. (H,I) Pooled quantitative results for three compartment—specific HELZ2 WBs, including (E) are shown for HELZ2 long (H) and short (I) isoforms. Raw data for each pooled experiment is presented in Datasheets S2 and S5.
Figure 5
Figure 5
PPARα does not appear to be the HELZ2-IFN Nuclear Receptor. (A) HeLa cells were transfected with four unique siRNAs targeting PPARα, without any significant rescue of DENV from IFN as measured by percent infected cells, using IF as in Figure 1. (B) Huh7.5.1 cells were transfected with four unique siRNAs targeting PPARα, and two of four siRNAs led to significant rescue of DENV from IFN. Knockdown of two other nuclear-receptor family genes, MAP2K4 (map) and SLC27A2 (s) led to rescue of DENV from IFN for four of four unique siRNA duplexes. *p < 0.05 compared to control.
Figure 6
Figure 6
HELZ2 CHiP seq and Metabolomics identify HELZ2 as a lipid regulator. (A) CHiP seq to identify DNA bound by HELZ2 during IFN stimulation in hepatocytes. Chromatin immunoprecipitation was performed using anti-HELZ2 antibody on Huh7.5.1 human hepatocyte nuclear extracts following 24 h treatment with IFN. Sequencing of DNA pulled down by anti-HELZ2 revealed enrichment for LMF1 transcription start site region sequences in the presence, but not absence, of IFN. LMF1 encodes lipase maturation factor 1, a protein involved in the maturation and transport of lipoprotein lipase through the secretory pathway. The plot indicates the chromasome location of enriched sequences detected by chromatin IP, harvested from cells with (top) and without (bottom) IFN treatment, on the x axis, and coverage density, or quantity of DNA detected per nucleotide, on the y axis. The vertical line indicates the location of the TSS for LMF1, and the annotated Sox8 TSS is within 1 kb. (B) Metabolomics to determine the lipid profile of HELZ2 knockdown cells. Huh7.5.1 hepatocytes stably transduced with lentivirus containing vector control (AP), shRNA targeting HELZ2 long and short isoforms (85) or shRNA targeting HELZ2 long isoform only (91) were treated with medium (M) or IFN (I) followed by intracellular lipid extraction and analysis using mass spectrometry. HELZ2 knockdown cells were found to contain a highly significant decrease in the triglyceride TAG 54:6 (number of carbons in the lipid acyl chain: number of double bonds in the lipid acyl chain), compared to vector controls, both in the presence and absence of IFN treatment. In the box, the central line is the median, the lower margin of the box is the first quartile, the upper margin of the box is the third quartile. The upper whisker extends from the hinge to the highest value that is within 1.5*IQR of the hinge, and the lower whisker extends from the hinge to the lowest value that is within 1.5*IQR of the hinge.
Figure 7
Figure 7
AHR is an IFN Effector Gene and Interacts with HELZ2. RNAi-mediated knockdown of AHR with four unique siRNAs leads to weak but consistent rescue of DENV from IFN in HeLa cells (A) and Huh7.5.1 hepatocytes (B). (C) Knockdown of AHR by the four unique siRNAs from (A) was validated at the protein level using western blot (C) with quantification (D) and at the mRNA level using qRT PCR (E). (F) Co-immunoprecipitation of AHR with HELZ2 (H) and AHR (A) antibodies and isotype control (I) was performed in Huh7.5.1 cells treated with IFN 1,600 IU/ml × 24 h or mock, followed by immunoblot for AHR. Load controls (Ld) were run in parallel, and immunoblotted for AHR and actin (G). Co-immunoprecipitation was repeated in the setting of mock (M), IFN 1,600 IUml (I), or the AHR agonist FICZ 0.9 um (F) × 24 h, using HELZ2 and AHR antibodies for IP, followed by HELZ2 and AHR immunoblot. Load controls were stained for HELZ2, AHR, and actin. Ladder is indicated by L. *p < 0.05 compared to control.
Figure 8
Figure 8
Strong but not weak AHR agonist exhibits antiviral effect against DENV NGC2 in Huh7.5.1 cells. Huh7.5.1 cells were plated then, 24 h later, treated with AHR agonist suspended in DMSO vs. DMSO alone, at indicated concentration, with or without submaximal dose IFN (25 IU/ml), for 24 h. Cells were then infected with DENV NGC2 at MOI of 1, followed by 48 h incubation, then fixation, permeabilization, and staining for DENV, followed by quantitative microscopy for cell counts and percent infected cells. Fold infection, vs. mock-treated cells, is shown. For the WEAK AGONIST (ITE), 0 um concentration, the % infected cells for three unique experiments were 18, 29, 40. For the STRONG AGONIST (FICZ), 0 um concentration, the % infected cells for three unique experiments were 20, 30, 32. Treatment groups are: (A) Weak agonist (ITE), no IFN. (B) Weak agonist (ITE), submax IFN. (C) Strong agonist (FICZ), no IFN. (D) Strong agonist (FICZ), submaximal IFN. *p < 0.05 compared to control.
Figure 9
Figure 9
Primary bone marrow derived macrophages of HELZ2 knockout mice display enhanced DENV infectivity compared to wild type controls. Age and sex matched HELZ2 knockout and WT mice were euthanized followed by isolation of primary bone marrow derived macrophages. Cells were plated in 96 well black clear bottomed plates then, the next day, treated with IFN or mock, then, the next day, infected with DENV NGC2, followed by 48 h incubation. Cells were then fixed, permeabilized, stained, imaged, and analyzed for percent DENV infection. Graphs depict pooled data from three separate experiments which include a total of 7 WT mice and 6 K/O mice. (A) Indicates DENV fold infection. (B) Indicates cell count per field. *p < 0.05 compared to control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Acosta E. G., Bartenschlager R. (2016). The quest for host targets to combat dengue virus infections. Curr. Opin. Virol. 20, 47–54. 10.1016/j.coviro.2016.09.003 - DOI - PubMed
    1. Adams O., Besken K., Oberdörfer C., MacKenzie C. R., Rüssing D., Däubener W. (2004). Inhibition of human herpes simplex virus type 2 by interferon gamma and tumor necrosis factor alpha is mediated by indoleamine 2,3-dioxygenase. Microbes Infect. 6, 806–812. 10.1016/j.micinf.2004.04.007 - DOI - PubMed
    1. Ahn Y. H., Yang Y., Gibbons D. L., Creighton C. J., Yang F., Wistuba I. I., et al. . (2011). Map2k4 functions as a tumor suppressor in lung adenocarcinoma and inhibits tumor cell invasion by decreasing peroxisome proliferator-activated receptor gamma2 expression. Mol. Cell. Biol. 31, 4270–4285. 10.1128/MCB.05562-11 - DOI - PMC - PubMed
    1. Arnold R., Neumann M., Konig W. (2007). Peroxisome proliferator-activated receptor-gamma agonists inhibit respiratory syncytial virus-induced expression of intercellular adhesion molecule-1 in human lung epithelial cells. Immunology 121, 71–81. 10.1111/j.1365-2567.2006.02539.x - DOI - PMC - PubMed
    1. Aye K. S., Charngkaew K., Win N., Wai K. Z., Moe K., Punyadee N., et al. . (2014). Pathologic highlights of dengue hemorrhagic fever in 13 autopsy cases from Myanmar. Hum. Pathol. 45, 1221–1233. 10.1016/j.humpath.2014.01.022 - DOI - PubMed
-