NF-κB and IRF1 Induce Endogenous Retrovirus K Expression via Interferon-Stimulated Response Elements in Its 5' Long Terminal Repeat

Abstract

Thousands of endogenous retroviruses (ERV), viral fossils of ancient germ line infections, reside within the human genome. Evidence of ERV activity has been observed widely in both health and disease. While this is most often cited as a bystander effect of cell culture or disease states, it is unclear which signals control ERV transcription. Bioinformatic analysis suggests that the viral promoter of endogenous retrovirus K (ERVK) is responsive to inflammatory transcription factors. Here we show that one reason for ERVK upregulation in amyotrophic lateral sclerosis (ALS) is the presence of functional interferon-stimulated response elements (ISREs) in the viral promoter. Transcription factor overexpression assays revealed independent and synergistic upregulation of ERVK by interferon regulatory factor 1 (IRF1) and NF-κB isoforms. Tumor necrosis factor alpha (TNF-α) and LIGHT cytokine treatments of human astrocytes and neurons enhanced ERVK transcription and protein levels through IRF1 and NF-κB binding to the ISREs. We further show that in ALS brain tissue, neuronal ERVK reactivation is associated with the nuclear translocation of IRF1 and NF-κB isoforms p50 and p65. ERVK overexpression can cause motor neuron pathology in murine models. Our results implicate neuroinflammation as a key trigger of ERVK provirus reactivation in ALS. These molecular mechanisms may also extend to the pathobiology of other ERVK-associated inflammatory diseases, such as cancers, HIV infection, rheumatoid arthritis, and schizophrenia.

Importance: It has been well established that inflammatory signaling pathways in ALS converge at NF-κB to promote neuronal damage. Our findings suggest that inflammation-driven IRF1 and NF-κB activity promotes ERVK reactivation in neurons of the motor cortex in ALS. Thus, quenching ERVK activity through antiretroviral or immunomodulatory regimens may hinder virus-mediated neuropathology and improve the symptoms of ALS or other ERVK-associated diseases.

FIG 1

LIGHT, but not TNF-α, markedly enhances ERVK polyprotein and RT expression in astrocytes. SVGA cells were treated with various doses of TNF-α or LIGHT for 24 h. Western blotting and confocal microscopy were used to detect alterations in ERVK RT, IRF1, and NF-κB p65, p50, and p52 protein levels. (A) Western blot (cropped) showing that LIGHT strongly induced ERVK polyprotein (125 kDa) expression and cleavage to produce the small, 52/54-kDa RT without RNase H and the larger, 60-kDa RT with RNase H, concomitantly with upregulation of IRF1 and NF-κB protein levels. In comparison, TNF-α slightly enhanced ERVK polyprotein and RT subunit expression. β-Actin was used as the loading control (n = 3). Quantification of the 54-kDa band is depicted in green. (B) Representative confocal micrographs depicting LIGHT-mediated induction of ERVK RT expression. ERVK RT aggregates were deposited proximal to the nucleus and formed a perinuclear ring (n = 3). Quantification of the ERVK RT/DAPI staining ratio is presented in each merged micrograph.

FIG 2

TNF-α, but not LIGHT, markedly enhances ERVK polyprotein and RT expression in neurons. ReNcell CX cell-derived neurons were treated with various doses of TNF-α or LIGHT for 24 h. Western blotting and confocal microscopy were used to detect alterations in ERVK RT, IRF1, and NF-κB p65, p50, and p52 protein levels. (A) Western blot (cropped) showing that TNF-α strongly induced ERVK polyprotein (180 and 125 kDa) expression and cleavage to produce the small, 56/58-kDa RT without RNase H and the larger, 68-kDa RT with RNase H, concomitantly with upregulation of IRF1 and NF-κB protein levels. In comparison, LIGHT enhanced the expression of the RT subunit without RNase H but not RT with the RNase H domain, suggesting that optimal RT activity may occur during exposure of neurons to TNF-α. β-Actin was used as the loading control (n = 3). Quantification of the 56-kDa and 68-kDa bands is depicted in green. (B) Representative confocal micrographs depicting TNF-α-mediated induction of ERVK RT expression in neurons. The fluorescent Nissl stain was used to identify neurons (n = 3). Quantification of the ERVK RT/DAPI staining ratio is presented in each merged micrograph.

FIG 3

NF-κB and IRF1 interact with the ISREs in the ERVK 5′ LTR and synergize to enhance ERVK gene transcription. (A) In silico-predicted IRF1 and NF-κB binding sites, including two ISREs in the ERVK 5′ LTR consensus sequence. The data are from reference . (B) IRF1 and NF-κB p65 significantly enhance ERVK pol transcription in 293T cells. 293T cells were transfected with 1 μg of pCMVBL or pCMV2 empty vector (negative controls) or with plasmids encoding wild-type or phosphomimetic forms (5D, 4D, and 6D) of IRF and NF-κB for 48 h. The modulation of ERVK pol RNA levels was measured by using SYBR green detection and Q-PCR, and data were normalized to the negative control (**, P < 0.01; ****, P < 0.0001; n = 3). 18S rRNA was used as the endogenous control. Only IRF1 and NF-κB p65 significantly induced ERVK pol transcription. (C) IRF1 and NF-κB p65 and p50 synergize to significantly enhance ERVK pol transcription in astrocytes. SVGA cells were transfected with 1 μg of empty vector (negative control) or with plasmids encoding IRF1 and NF-κB isoforms, individually and in combinations, for 48 h. The modulation of ERVK pol RNA levels was measured by using SYBR green detection and Q-PCR, and data were normalized to the negative control (*, P < 0.05; ****, P < 0.0001; n = 3). 18S rRNA was used as the endogenous control. Although IRF1 or NF-κB p65 alone was sufficient to significantly induce ERVK pol transcription, IRF1 and NF-κB p65 and p50 synergized to further enhance ERVK pol RNA levels (up to 68-fold). (D) TNF-α and LIGHT markedly enhanced the binding of IRF1 and NF-κB p65 and p50 to both ISREs in the ERVK 5′ LTR, in a cell-type-dependent manner. Chromatin was extracted from SVGA cells and ReNcell CX cell-derived neurons treated with TNF-α (10 ng/ml) or LIGHT (10 ng/ml) for 8 h. Chromatin immunoprecipitation (ChIP) was performed with anti-human IRF1 or NF-κB p65, p50, or p52 antibody. Q-PCR was used to amplify immunoprecipitated ISRE sequences within the ERVK 5′ LTR, and products were detected using SYBR green detection. The fold enrichment of transcription factors at each ISRE was first normalized to the input control and then to the IgG negative control. All transcription factors were bound to the ISREs at basal levels. However, the binding of NF-κB p65 and p50 was significantly enhanced by LIGHT treatment, but not TNF-α treatment, in astrocytes (top panels) (n = 3; *, P < 0.05; ****, P < 0.0001). In contrast, the binding of NF-κB p65 and p50 was significantly enhanced by TNF-α treatment, but not LIGHT treatment, in neurons (bottom panels) (n = 2; *, P < 0.05; ***, P < 0.001).

FIG 4

Representative images of ERVK reverse transcriptase (RT) in cortical brain tissue. Representative images show ERVK RT expression (green) in prefrontal cortex autopsy tissues (Brodmann area 9; NIH NeuroBioBank) from an individual with cancer (neuronormal; individual 3371) and a patient with ALS (individual 5215). Five total tissues were examined for each clinical group. (Left) Mosaic tiling (magnification, ×10) reveals enhanced ERVK RT expression in deep cortical tissue (cortical layer V) and upper cortical tissue (cortical layer III) in an ALS specimen. (Right) Nissl-stained cells reveal ERVK RT staining mainly in large pyramidal neurons. Nuclear DAPI staining is shown in blue. Bars, 10 μm for middle and right panels.

FIG 5

The expression of ERVK RT is markedly enhanced in cortical neurons from patients with ALS and associates with increased levels and enhanced nuclear translocation of IRF1 and NF-κB. Representative confocal micrographs show ERVK RT, IRF1, and NF-κB p50 and p65 protein detection in Brodmann area 6 motor cortex tissues from an ALS patient (individual 5215) and a neuronormal control (individual 3371). ERVK RT expression was increased in the perinuclear region and in the axons of large pyramidal neurons in the ALS motor cortex. This occurred concomitantly with increased IRF1 (A), NF-κB p50 (B), and NF-κB p65 (C) nuclear translocation in cortical neurons. Five total tissues were examined for each clinical group.

FIG 6

Augmented ERVK RT levels in ALS cortical neurons correlate with enhanced nuclear localization of proinflammatory transcription factors. (A) The mean optical intensity (relative intensity) was used as a metric to quantify the level of total ERVK RT, as well as the level of nuclear IRF1, NF-κB p50, or NF-κB p65, in cortical neurons from neuronormal individuals (n = 86 cells [10 to 29 cells per case]) and patients with ALS (n = 105 cells [10 to 27 cells per case]). Clinical groups (n = 5 each) were compared using the nonparametric Mann-Whitney U test. The expression of total ERVK RT and nuclear translocation of each transcription factor increased significantly in ALS cortical neurons compared to the controls (***, P < 0.0001). (B) Pearson's correlation between the mean relative intensities of total ERVK RT and nuclear IRF1, NF-κB p50, or NF-κB p65 measured in neuronormal (NN) (red) and ALS (blue) cortical neurons. ERVK RT expression exhibited a moderate (p65 [0.6863]) to strong (IRF1 [0.8574] and p50 [0.8405]) correlation with the extent of nuclear localization for each transcription factor. (C) ROC curve analyses depicting the accuracy (area under the curve [AUC]) of augmented ERVK RT and proinflammatory transcription factor levels in discriminating between cortical neurons in controls and those in ALS tissues. IRF1 and NF-κB p50 levels were excellent predictors of ALS. In contrast, total ERVK RT and nuclear NF-κB p65 levels were less accurate at distinguishing between the normal and diseased states.

FIG 7

IRF1 and NF-κB drive ERVK reactivation via ISREs in the LTR. This figure summarizes how the proinflammatory cytokines TNF-α and LIGHT promote the cellular expression of ERVK. Following engagement of IRF1 and NF-κB isoforms p50 and p65 with the ERVK LTR, viral RNA and protein production is induced. Additional κB sites in the ERVK LTR may host noncanonical p50 homodimers, thus facilitating recruitment of additional transcriptional activators, such as Sp1/Sp3. Accumulation of ERVK reverse transcriptase in human neurons forms a cytoplasmic aggresome as well as nuclear foci.