Antiviral Interferon-Beta Signaling Induced by Designed Transcription Activator-Like Effectors (TALE)
Antiviral Interferon-Beta Signaling Induced by Designed Transcription Activator-Like Effectors (TALE)
Ivonne Renner 0
Nancy Funk 0
Rene Geissler 0
Susann Friedrich 0
Anika Penzel 0
Sven-Erik Behrens * 0
Matthias J. Schnell, Thomas Jefferson
University, United States of America
0 Institute of Biochemistry and Biotechnology, Section Microbial Biotechnology, Martin Luther University Halle- Wittenberg, Faculty of Life Sciences (NFI) , Kurt-Mothes-Str. 3, D-06120, Halle/Saale , Germany
Here we show that designed transcription activator-like effectors (TALEs) that bind to defined areas of the interferon beta promoter are capable to induce IFN-beta expression and signaling in human cells. Importantly, TALE-mediated IFN-beta signaling occurs independently of pathogen pattern recognition but effectively prohibits viral RNA replication as demonstrated with a hepatitis C virus replicon. TALEs were thus indicated to be valuable tools in various applications addressing, for example, virus-host interactions.
TALEs were originally characterized as virulence factors of plant pathogenic
bacteria that reprogram gene transcription of the host cells. TALEs contain a DNA
binding domain that is composed of similar tandem repeats of typically 34 amino
acids. For transcription activation, each repeat binds one base pair of the target
DNA, and a repeat-variable di-residue (RVD) specifies the bound base .
Thus, designer TALEs containing a defined order of repeats and a suitable
transcription activation domain can be constructed and applied to induce the
transcription of human genes .
An attractive target for transcription activation is the cytokine IFN-beta, which is
well characterized regarding its antiviral activity and also used during treatment of
multiple sclerosis [8, 9]. The IFN-beta promoter mainly consists of an enhancer that is
flanked by two nucleosomes, one masking the transcriptional TATA-box (Fig. 1A).
The transcription of the IFN-beta gene is stringently regulated. During cell
activation, which may be provoked by interactions of cellular pattern recognition
receptors (PRR) with pathogen associated molecular pattern (PAMP), signaling
cascades induce the assembly of transcription and nucleosome remodeling factors
(the so-called enhanceosome) at the enhancers positive regulatory domains
(PRD; Fig. 1A). This, in turn, enables the association of the TATA-binding
protein and Pol II-mediated transcription to initiate . Accordingly, in
non-activated cells (i.e., in the absence of the enhanceosome), the IFN-beta
promoter was considered to be accessible for the binding and
transcriptioninducing activity of TALEs.
In this report we show that TALEs directed to bind to certain sites of the
IFNbeta promoter induce an effective antiviral signaling cascade also in the absence of
an external stimulus.
Cloning of TALE genes
Using a repeat library and the Golden TALE technology  six single-repeat
modules were ligated into assembly vectors as a six-repeat array by cut-ligation
using BpiI. The procedure was essentially performed as described by Geissler et al.
. To assemble the complete TALE-coding sequence, three six-repeat arrays were
ligated with modules encoding a green fluorescent protein (GFP) tag, the N- and
C-terminus, and the herpes simplex virus VP16 transcription activation domain
(C-terminal 68 aa), respectively, into a pcDNA3 (Invitrogen) derivative using
BsaI. The amino acid sequence of the applied TALEs is given in Table S1 in File
Construction of reporter plasmid
The plasmid was based on pF12A RM Flexi (Promega) where the barnase gene was
replaced by a luciferase gene. To generate plasmids containing the TALE
recognition sites, pF12A RM (Luc) was amplified with primers containing target
boxes. The promoter and 59 untranslated region (675 bp) of the IFN-beta1
promoter were amplified by PCR and inserted into the luciferase reporter plasmid
In vitro transcription
The replicon-encoding plasmid pSGR-JFH1 was kindly provided by Dr. Wakita
(Tokyo Metropolitan Institute for Neuroscience) and modified as described in
. The plasmid was digested with XbaI and transcribed by run-off in vitro
transcription (standard protocol) with T7 RNA-polymerase (Stratagene) using the
protocol of Geissler et al. 2012 .
Cell culturing and transfection conditions
Huh7 cells  were cultured in DMEM (Invitrogen) supplemented with 10%
FCS (PAN-Biotech), 1% penicillin/streptomycin (Invitrogen), 0.1% d-Biotin and
0.1% hypoxanthin (Sigma) . Transfection of plasmids was performed with
70% confluent cells, 20 mg plasmid DNA/10 ml growth medium using Turbofect
(Fermentas) and the manufacturers instructions. The replicon RNA was
transfected using 300 ng (ca. 100 fmol) and the Bio-Rad Gene Pulser II (1 pulse
without controller at 0.2 kV and 950 mF).
The western-blot analysis was performed at 24 h p.t. of the TALE-expressing
plasmids. Ca. 26106 cells were centrifuged at 10006g, washed with phosphate
buffer saline and lysed by 1x lysis buffer (Promega). The protein amount was
determined by a standard Bradford assay (BioRad) and 20 mg of total cell protein
separated per lane by SDS-PAGE. Following the transfer to nitrocellulose
(Millipore), the reaction was carried out using standard conditions and the
following antibodies: anti-GFP (Invitrogen A-6455) 1:2000, anti-GAPDH (Santa
Cruz sc-47724) 1:15000; secondary antibodies (each at 1:5000 dilution)
antirabbit (Licor Cw 800 926-32213), anti-mouse (Licor Cw800 926-32212).
For qRT-PCR analysis, RNA was isolated from ca. 26106 cells with Trizol at the
indicated time points and the qRT-PCR performed using Revert Aid reverse
transcriptase (Thermo) and the PCR-MasterMix qRT (Roboklon GmbH,
Germany). For reverse transcription of total mRNA, an oligodT19 primer was
applied; for reverse transcription of the HCV replicon JFH, we applied the primer
JFH reverse (ACA TGA TCT GCA GAG AGA CCA G). The RT conditions were
1 h at 42C (amounts of applied RNA 500 ng); the PCR conditions were 94C,
15 sec; 60C, 25 sec; 72C 25 sec640 cycles. The HCV RNA levels were
normalized to GAPDH RNA as internal control. For further details, see Geissler et
al. . All applied DNA oligonucleotides (purchased from Eurofins, Germany)
are summarized in Table S2 in File S1.
Data evaluation and statistics
Data evaluation and statistics were done as described previously .
Results and Discussion
Following earlier work, which revealed the general option to induce IFN-beta
expression in human cells by a TALE , this study aimed at investigating if an
entire antiviral IFN-beta signaling cascade may be navigated by designed effectors.
For this purpose, we generated a set of six TALE-expressing constructs applying
the Golden TALE technology . The corresponding effectors were designed to
bind to different regions of the IFN-beta promoter (see Fig. 1A and Table 1).
After transient transfection of the expression plasmids, all six effectors were
comparably expressed in Huh7 cells (human hepatoma cells). This was
demonstrated by western-blot that detected the TALEs via a fused green
fluorescent protein (GFP) reporter (Fig. 1B). In a subsequent experiment, we
expressed the individual TALEs in Huh7 cells and measured the amount of
IFNbeta mRNA by qRT-PCR (Fig. 1C). This data revealed that with
promoterassociating TALEs, the transcription of the IFN-beta gene was up to 4-fold
increased in comparison to experiments with a non-related TALE. Interestingly,
most effective were TALEs1 and 6 that were binding to the sites of the promoter
that were indicated to be covered by nucleosomes (Fig. 1A and C). This suggests
that the effectors, besides attracting the Pol II transcription machinery, may also
facilitate nucleosomal remodeling.
Secreted IFNs function by binding to the IFN receptor (IFNAR) of neighboring
cells and by activating the canonical JAK/STAT pathway. This leads to the
formation of interferon-stimulated gene factor complexes (ISGF3) that drive the
transcription of interferon-stimulated genes (ISGs). ISGs encode antiviral proteins
like OAS (29-59-oligoadenylate synthetase), MX (GTPase), ADAR (adenosine
deaminase) and signaling proteins as the interferon regulatory factor IRF7 [16
18]. To understand next if IFN-beta expression correlated with ISG expression, we
performed qRT-PCR that measured the mRNA levels of IFN-beta side-by-side
with those of OAS1 and 2, MX1, IRF7 and ADAR1. This was done with two cell
types, Huh7 and Huh7.5. Huh7.5 differ from Huh7 such that RIG-I (retinoic
acid-inducible gene I), an important intracellular PRR  is defective in these
cells [20, 21]. Thus, with the pathogen hepatitis C virus (HCV) it is well
understood that PAMP as the tri-phosphate at the viral RNAs 59-end and an
HCV-specific RNA motif in the 39 untranslated region (39UTR) of the viral
genome are recognized by RIG-I and that RIG-I-induced signaling cascades lead
to the activation of transcription factors that regulate IFN gene expression [22
25]. To compare HCV- and TALE-induced IFN-beta signalling, both cell types
were transfected either with an HCV subgenomic RNA replicon  (Fig. 2A) or
with the TALE6-expressing plasmid. Interestingly, as shown in Fig. 2C, TALE6
induced the expression of IFN-beta and ISGs in both cell types, while replicating
HCV RNA did so only in Huh7 (Fig. 2B). Controls performed with a
nonreplicating HCV RNA or a non-specific TALE showed neither an induction of
IFN-beta nor of ISGs (Fig. 2B and C). This data highlights that TALE6 triggered
the entire IFN-beta signaling cascade. Most importantly, with the experiment
applying Huh7.5 cells, we confirmed that TALE-induced IFN-beta signaling
occurred independently of PAMP signaling.
In a final experiment, we addressed the question if the TALE6-mediated
induction of IFN-beta and ISGs was capable to interfere with viral RNA
replication. For this, Huh7 and Huh7.5 cells were transfected with the
TALE6encoding plasmid and, 24 h later, with the HCV replicon. As shown in Fig. 2D,
prior expression of TALE6 effectively inhibited viral RNA replication while
expression of a non-related TALE did not. A considerable inhibitory effect on viral
replication was also observed in cells when we expressed TALE6 in Huh7 cells
where HCV replication was already established (Fig. 2E).
The activation of IFN-beta gene expression via PRR-PAMP signaling is a
wellstudied model of how transcriptional output is regulated in the cell. Here we
demonstrate that TALEs, by operating directly on the transcriptional level, bypass
PAMP/RIG-I-mediated signaling. Moreover, TALE-triggered IFN-beta signaling
effectively prohibits HCV replication in hepatoma cells. Thus, in comparison to
the IFN-beta response that is induced by the HCV replicon in Huh-7 cells
(Fig. 2B), the TALE6-stimulated IFN-beta and ISG expression (Fig. 2C) leads to a
clearly detectable decrease in the level of viral RNA (Fig. 2D). We explained this
by the multiple ways of how viral factors, which are also encoded by the applied
replicon may inhibit IFN signaling . For example, the HCV NS3/4A protease
inhibits HCV PAMP-RIG-I signaling by proteolytic degradation of a RIG-I
signaling adapter .
Taken together, our data recommend IFN-beta inducing TALEs as potentially
valuable tools for future vaccination or treatment applications. Specifically
designed TALEs may also be helpful to unravel the function of yet insufficiently
characterized host factors participating in (antiviral) cell signaling or viral
File S1. Supporting tables. Table S1, Organization and amino acid sequences of
applied TALEs. Table S2, DNA oligonucleotides applied for qRT-PCR.
Conceived and designed the experiments: IR RG SF SEB. Performed the
experiments: IR NF RG SF AP. Analyzed the data: IR NF RG SF SEB. Contributed
to the writing of the manuscript: SEB.
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