The 3′ terminal region of Zika virus RNA contains a conserved G-quadruplex and is unfolded by human DDX17

Abstract

Zika virus (ZIKV) infection remains a worldwide concern, and currently no effective treatments or vaccines are available. Novel therapeutics are an avenue of interest that could probe viral RNA-human protein communication to stop viral replication. One specific RNA structure, G-quadruplexes (G4s), possess various roles in viruses and all domains of life, including transcription and translation regulation and genome stability, and serves as nucleation points for RNA liquid-liquid phase separation. Previous G4 studies on ZIKV using a quadruplex forming G-rich sequences Mapper located a potential G-quadruplex sequence in the 3′ terminal region (TR) and was validated structurally using a 25-mer oligo. It is currently unknown if this structure is conserved and maintained in a large ZIKV RNA transcript and its specific roles in viral replication. Using bioinformatic analysis and biochemical assays, we demonstrate that the ZIKV 3′ TR G4 is conserved across all ZIKV isolates and maintains its structure in a 3′ TR full-length transcript. We further established the G4 formation using pyridostatin and the BG4 G4-recognizing antibody binding assays. Our study also demonstrates that the human DEAD-box helicases, DDX3X132-607 and DDX17135-555, bind to the 3′ TR and that DDX17135-555 unfolds the G4 present in the 3′ TR. These findings provide a path forward in potential therapeutic targeting of DDX3X or DDX17’s binding to the 3′ TR G4 region for novel treatments against ZIKV.

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OPEN ACCESS | Article
The 3terminal region of Zika virus RNA contains a
conserved G-quadruplex and is unfolded by human
DDX17
Dannielle L. Gemmilla, Corey R. Nelsona, Maulik D. Badmaliaa, Higor S. Pereira a,LiamKerr a, Michael
T. Wolfinger b,c,d, and Trushar R. Patel a,e,f
aAlberta RNA Research and Training Institute & Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge,
AB T1K 3M4, Canada; bBioinformatics and Computational Biology, Faculty of Computer Science, University of Vienna, Währinger
Strasse 29, 1090, Vienna, Austria; cDepartment of Theoretical Chemistry, University of Vienna, Währinger Strasse 17, 1090, Vienna,
Austria; dRNA Forecast e.U., 1140 Vienna, Austria; eDepartment of Microbiology, Immunology and Infectious Disease, Cumming
School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada; fLi Ka Shing Institute of Virology and Discovery Lab,
University of Alberta, Edmonton, AB T6G 2E1, Canada
Corresponding author: Trushar R. Patel (email: trushar.patel@uleth.ca)
Abstract
Zika virus (ZIKV) infection remains a worldwide concern, and currently no eective treatments or vaccines are available.
Novel therapeutics are an avenue of interest that could probe viral RNA-human protein communication to stop viral repli-
cation. One specific RNA structure, G-quadruplexes (G4s), possess various roles in viruses and all domains of life, including
transcription and translation regulation and genome stability, and serves as nucleation points for RNA liquid-liquid phase sep-
aration. Previous G4 studies on ZIKV using a quadruplex forming G-rich sequences Mapper located a potential G-quadruplex
sequenceinthe3
terminal region (TR) and was validated structurally using a 25-mer oligo. It is currently unknown if this
structure is conserved and maintained in a large ZIKV RNA transcript and its specific roles in viral replication. Using bioin-
formatic analysis and biochemical assays, we demonstrate that the ZIKV 3TR G4 is conserved across all ZIKV isolates and
maintains its structure in a 3TR full-length transcript. We further established the G4 formation using pyridostatin and the
BG4 G4-recognizing antibody binding assays. Our study also demonstrates that the human DEAD-box helicases, DDX3X132-607
and DDX17135-555, bind to the 3TR and that DDX17135-555 unfolds the G4 present in the 3TR. These findings provide a path
forward in potential therapeutic targeting of DDX3X or DDX17’s binding to the 3TR G4 region for novel treatments against
ZIKV.
Key words: Zika virus, 3terminal region, G-quadruplex, BG4 antibody, DDX17, helicase assay
Introduction
Zika virus (ZIKV) is an endemic, neurovirulent arbovirus
whose primary vectors are Aedes aegypti and Aedes albopictus
mosquitoes. Its primary transmission mode is the horizon-
tal transfer of mosquito-infectious saliva during blood feed-
ing (Musso and Gubler 2016). Currently, the Centers for Dis-
ease Control and Prevention has designated 86 countries as
ZIKV risk areas (Prevention 2022). ZIKV is popularly known
for the 2015/2016 epidemic in Brazil; during that time, it
was revealed that the virus can be vertically transmitted
from an infected pregnant woman to their fetuses, which
can lead to congenital disabilities such as microcephaly in
utero (Tang 2016). ZIKV can also cause debilitating symptoms
in adults, including Guillain-Barré Syndrome, fever, rash,
headaches, and joint pain, and can also be sexually trans-
mitted (Barzon et al. 2016;Mansuy et al. 2016;Tang 2016).
Currently, there are no treatments or vaccines available
(USA Food and Drug Administration 2022). ZIKV possesses
a positive-sense, non-segmented RNA genome of ∼10.4 kb,
which is composed of a 5terminal region (TR) (∼108 nt),
three structural genes——capsid (C), membrane (prM), and
envelope (E)——and seven nonstructural genes——NS1, NS2A,
NS2B, NS3, NS4A, NS4B, and NS5, followed by a long, 3TR
(∼432 nt). The TRs play vital roles in viral genome cyclization,
which regulates viral transcription and translation, and they
alter host mRNA turnover, suppresses interferon response,
and modulate the host-cell environment (Donald et al. 2016;
Chavali et al. 2017;Sirohi and Kuhn 2017;Sanford et al.
2019;Schneider and Wolfinger 2019;Michalski et al. 2019;
Wolfinger 2021).
An area of interest for antiviral targeting is G-quadruplexes
(G4s)——noncanonical structures identified in all domains of
life and almost all viral groups in the Baltimore classifica-
tion (Ruggiero and Richter 2020). Viral G4s are known to play
96 Biochem. Cell Biol. 102: 96–105 (2024) | dx.doi.org/10.1139/bcb-2023-0036

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Fig. 1. Schematic of a G-quadruplex and RNA transcripts under study. A schematic of a G4 in parallel conformation with mono-
valent cations coordinated in the core of the stacked guanines is shown in (A). Transcripts that were used in the experiments,
with the potential G-quadruplex sequence at the ends of the 3terminal region (TR) (green) and 3SL (blue) are shown in (B).
The G4 mutant is shown in red, along with the G4 mutation sequence shown below compared to the wild-type sequence.
critical roles throughout regulatory processes in their life
cycles, such as transcriptional and translational activities,
as well as alternative splicing (Patel et al. 2000;Métifiot et
al. 2014;Murat et al. 2014;Meinke et al. 2016). G4 struc-
tures consist of at least two consecutive guanine tetrads in
close proximity Hoogsteen base pairing with each other in
bothDNAandRNA(Wang and Patel 1993;Burge et al. 2006;
Bhattacharyya et al. 2016), altogether stabilized by a mono-
valent cation (preferably K+) at the coordinating site (Fig.
1A). DNA G4s can adopt parallel, antiparallel, and hybrid con-
formations, whereas RNA G4s exclusively adopt propeller-
type parallel topology due to the 2-OH preventing syn-
conformation torsion angles (Fay et al. 2017)(Fig. 1A).
It has been shown that many human proteins, including
DExD-box helicases, interact with G4s (Linder et al. 1989;
Brázda et al. 2014;Zhang et al. 2021). The DExD-box family
proteins are involved in a wide range of functions, including
embryonic development, cell proliferation, hematopoiesis,
metabolism, immune response, cancer pathogenesis, inflam-
mation, autoimmune diseases, and influence of viral replica-
tion (Meier-Stephenson et al. 2018;Andrisani et al. 2022). We
have previously demonstrated that the DEAD-box helicases,
DDX3X132-607 or DDX17135-555, interact and unfold the inter-
genic region (IGR) and 5noncoding region of the Rift Val-
ley fever virus and the Japanese encephalitis and Zika virus
5TRs, respectively (Nelson et al. 2020;Nelson et al. 2021).
DDX3X has been reported as a critical factor required for
dengue and hepatitis C viral replication and as an inhibitory
factor for West Nile virus replication (Ariumi et al. 2007;Brai
et al. 2019). It has also been shown to interact with RNA
G4s. Conversely, it is unclear if DDX17 directly binds to G4s,
since it is involved in G4 binding with other proteins, but
no direct interaction has been demonstrated (Fortuna et al.
2021;Dardenne et al. 2014). A previous study on the identi-
fication of the ZIKV genomic RNA structure of both African
and Asian/American lineages using selective 2-hydroxyl acy-
lation analyzed by primer extension identified a conserved,
canonical stem-loop structure within the 3TR (classified as
the 3stem-loop [SL]) (Li et al. 2018). Interestingly, a study by
Fleming et al. (2016) found potential G-quadruplex sequences
within ZIKV using the quadruplex-forming G-rich sequences
Mapper, and they identified and characterized a G4 contained
in the 3SL, albeit using a 25-mer oligo short-range interac-
tion approach (Kikin et al. 2006;Fleming et al. 2016). From
the perspective of long-range RNA interactions, it is still un-
clear if a G4 is maintained in the 3SL. Additionally, a previ-
ous study indicated that conformational switching could oc-
cur between G4 and canonical RNA structures, as in the case
of this study, a hairpin (Bugaut et al. 2012). These events are
based on the proximity of specific mono and divalent cations,
which are altogether generated by RNA liquid–liquid phase
separation (X. Liu et al. 2021). From these recent discover-
ies, the 3SL could exist as canonical and noncanonical states
based on ion proximity.
Because of the lack of treatment options for ZIKV, investi-
gation of alternative targets within the viral structure or life
cycle is being explored, and G4s is one of them (Majee et al.
2021). The G4 binding molecules, Braco-19, TMPyP4, Berber-
ine, NiL, 360 A, and pyridostatin (PDS), have all been shown to
interact with ZIKV G4s in vitro and cell culturing assays and
showed that these molecules altogether reduce viral replica-
tion and protein production to varying degrees (Majee et al.
2021;Zou et al. 2021). It remains unclear if the 3TR G4 ex-
ists in a larger RNA structure containing long-range interac-
tions, as previous studies focus on a short-range approach us-
ing short oligos. Moreover, deciphering the various human
proteins that interact with this G4 could provide insight into
its function during viral replication.
In this study, we investigated the presence of a G4 in
the full-length ZIKV 3TR. For this purpose, we first used
bioinformatics analyses to demonstrate that the 3SL G4
sequence is present in multiple ZIKV isolates. By imple-
menting a long-range interaction approach with long RNA
transcripts of the 3TR (432 nt) and 3SL (104 nt) (Fig. 1B),
we were able to identify the presence of a G4 in the 3TR
transcript using the BG4 antibody, TMPyP4, and PDS, which
all specifically bind to G4s. Subsequently, we elucidated that
human DDX3X132-607 and DDX17135-555 interact with the 3TR
and that DDX17135-555 unwinds the G4 present in the 3SL in
an ATP-dependent manner.

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Materials and methods
Bioinformatics analysis of conserved G4s in the
3TR
ZIKV viral genome data were downloaded from NCBI Gen-
Bank on 14 January 2021, comprising 1028 ZIKV isolates.
Filtering for isolates that cover the complete G4-containing
3SL regions was performed with an Infernal covariance
model (Nawrocki and Eddy 2013) constructed from the 3
SL seed alignment of the Spondweni group featured in the
viRNA GitHub repository (https://github.com/mtw/viRNA), re-
sulting in 113 ZIKV isolates. Filtering these for a nonredun-
dant set of 3SL regions resulted in a total of 14 representa-
tive ZIKV isolates. The ZIKV isolates from Haiti used in this
study (KU509998.3) are identical to strain MN577544.1, fea-
tured in our nonredundant set, throughout the entire 3SL
region. A structural multiple sequence alignment of the 3SL
region of 14 representative ZIKV isolates was computed with
LocARNA (Will et al. 2007) and visualized with Jalview (Clamp
et al. 2004).
RNA transcript expression of 3TR, 3SL, and
G4 mutant
The ZIKV 3TR (10375–10807) (432 nt) sequence was derived
from the ZIKV isolate from Haiti (GenBank: KU509998.3)
(Lednicky et al. 2016). This isolate also has a nearly identi-
cal sequence to the Asian/American lineage, one of the pre-
dominant lineages across the globe (Beaver et al. 2018). We
designed three plasmid constructs from this sequence con-
taining the full 3TR, the 3SL, and a 3TR with the G4
sequence mutated (G4 mutant). The G4 mutant construct
was designed wherein five nucleotide mutations were made
(5-GGCGGCCGGUGUGGGG-3→5-GACGACCGAUGUGAGA-
3)(Fig. 1B) in the hypothesized G4 sequence to disrupt the G4
formation. All constructs were made using pUC57-Kan plas-
mids with a T7 promoter upstream of the desired transcript,
with an XbaI cut site at the end of the transcript to linearize
the plasmid such that T7 polymerase dissociates during in
vitro transcription (IVT) reactions. The 3TR and 3SL plas-
mid constructs were commercially synthesized by Integrated
DNA Technologies, and BioBasic synthesized the G4 mutant.
The synthesized plasmids were transformed into compe-
tent Escherichia coli NEB5ɑcells (New England Biolabs), and
the cells were further propagated to replicate the plasmid
DNA for IVTs. The plasmid DNA was extracted, linearized us-
ing XbaI restriction digestion, and subsequently used for IVT
reaction. The T7 polymerase-based (expressed and purified in-
house) IVT reaction was performed by mixing transcription
buer (200 mM Tris-Cl [pH 7.5], 75 mM MgCl2, 10 mM sper-
madine, and 50 mM NaCl), 100 mM DTT, 25 mM nucleoside
triphosphates, 100 mM guanosine monophosphate, 0.5 U/μL
IPPase, 10 μM T7 polymerase, Ribolock, and the digested plas-
mid and performed for 3 h at 37 ◦C. This was followed im-
mediately by purification via size-exclusion chromatography
(SEC) using a Cytiva Superdex 200 Increase 10/300 GL for the
ZIKV 3SL and a Sephacryl S-400 HR for the 3TR and G4 mu-
tant RNA (Fig. S1A/B). The columns were pre-equilibrated in
G4 buer (10 mM HEPES [pH 7.5] and 100 mM KCl). For purity
analysis, the peak fractions were analyzed on a 2% agarose gel
(Fig. S1C). The purified RNA was then ethanol precipitated at
−80 ◦C in preparation for RNA labeling for subsequent exper-
iments.
Expression and purification of BG4 antibody, as
well as DDX17135-555 and DDX3132-607 proteins
The plasmid coding for the BG4 antibody was obtained
from Addgene (pSANG10-3 F-BG4) and transformed into E. coli
BL21 DE3 for protein expression. The transformed cells were
grown at 37 ◦C until the optical density reached 0.6 at 600 nm.
Subsequently, cells were induced with 1 mM isopropylthio-β-
galactoside and were grown for 16 h at 16 ◦C, followed by cell
harvesting through centrifugation. The harvested cells were
resuspended in lysis buer (50 mM Tris-Cl [pH 7.5], 500 mM
NaCl, 0.2% tween, 5% glycerol, 5 mM β-mercaptoethanol, and
10 mM imidazole), supplemented with 0.1 mg/mL lysozyme,
200 units of DNase I (Thermofisher), 2 mM PMSF, as well as
protease inhibitors (pepstatin, aprotinin, and AEBSF) from
Biobasic. The cell suspension was sonicated and clarified us-
ing centrifugation (30 000×g); the clarified lysate was loaded
onto a 1 mL Histrap high-performance column from Cytiva
mounted on an Äkta Start system and purified using an elu-
tion gradient from 10 to 150 mM imidazole supplemented to
the lysis buer. The fractions were purified via SEC using a
Superdex 75 increase 10/300 GL (Cytiva) pre-equilibrated with
1X PBS mounted on an Äkta Pure system (Fig. S2). The frac-
tions were analyzed on a 12% SDS-PAGE gel, pooled, and con-
centrated to 14 μM and stored at −20 ◦C until further use.
DDX3X132-607, DDX17135-555, and DDX17 mutants are alto-
gether truncated versions of the protein (Fig. S3A), and each
was expressed according to protocols described previously us-
ing an E. coli expression system similar to the BG4 antibody
(Nelson et al. 2020,2021) (Fig. S3B). DDX17 mutant was also
purified using a pET28a cloned vector with glycine mutations
on amino acids which coordinate ATP hydrolysis; the Q motif,
which interacts with the adenine of ATP; and motifs 1 and 2
in the ATP binding domain, which interact with the triphos-
phate (Fig. S3A,C) (Ali 2021a;Ali 2021b;Tanner et al. 2003).
The mutant assays were performed to prevent ATP hydroly-
sis, and consequently, helicase activity.
Alexa Fluor 488 labeling of the RNA
Each purified RNA transcript was labeled at the 5
end with Alex fluor 488. Note that 1.25 mg of 1-ethyl-
3- (3-dimethylaminopropyl) carbodiimide hydrochloride was
mixed with 8 μL of concentrated RNA in water and 5 μLAlexa
Fluor 488 in 0.2 M KCl, and 20 μL of 0.1 M imidazole (pH 6). All
reactions were incubated at room temperature for 18 h. The
RNA was then diluted in 460 μL of G4 buer (10 mM HEPES
[pH 7.5] and 100 mM KCl) and loaded onto a Cytiva Superdex
200 Increase 10/300 GL to purify the labeled RNA from the
unlabeled fluorophore. Each RNA sample was then collected
and heat-cooled at 95 ◦C for 5 min, followed by room tem-
perature incubation for 15 min. The RNA was tested for fluo-
rescence counts on the NanoTemper Technologies Monolith
NT.115 microscale thermophoresis (MST) device to ensure op-
timal labeling for downstream experiments, similar to previ-

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ous experiments that used Alex Fluor 488 for labeling their
nucleic acids (Yoo et al. 2004;Johnson et al. 2006;Kynast et al.
2004). The labeled RNA was then vitrified in liquid nitrogen
and stored at −80 ◦C until downstream experimentation.
Interaction studies using microscale
thermophoresis
A Monolith NT.115 MST from NanoTemper Technologies
was used to evaluate the anity of RNA constructs with their
ligands. The ZIKV 3TR, G4 mutant, and 3SL RNA transcripts
were evaluated at 80 nM at 100% excitation at medium MST
power for the BG4 binding anity and PDS binding check
runs. For the MST binding studies, the samples were seri-
ally diluted two-fold in the MST capillaries with concentra-
tions ranging from 6.8 μM–0.8nMforBG4,7μM – 14.6 nM
for DDX3X132-607,and19μM – 0.58 nM for DDX17135-555.All
MST data were collected in triplicate at room temperature us-
ing the Nano-Blue filter and medium IR-laser power for BG4,
while high power was used for DDX3X132-607 or DDX17135-555.
The dissociation constants were calculated using the Mono-
lith NT.115 analysis software by plotting the ligand % bound,
and for the PDS runs, the average response amplitude was
recorded in triplicate, and the Monolith NT.115 analysis soft-
ware provided the signal-to-noise ratio values.
Electrophoretic mobility shift assays (EMSAs) were per-
formed to support the MST binding data (Fig. S4) with the
ZIKV 3TR mixed with two-fold serial diluted concentrations
of DDX3X132-607 or DDX17135-555. Furthermore, the oligonu-
cleotides used in the MST helicase assay were also mixed with
DDX3X132-607 or DDX17135-555 to ensure that the helicase assay
results on the MST were a result of the 3SL being unwound,
not the oligo binding to the protein. The labeled ZIKV RNA
was scanned on Cy2, whereas the oligo was scanned on Cy5.
Unwinding assays of the ZIKV 3TR G4
To assess the ability of DDX3X132-607 and DDX17135-555
to unwind the noncanonical structured area in the
3SL, we used MST and a complementary RNA (5-
AGUUUCCACCACGCUGGCCGCCA-3) that would only base
pair to the G4 portion of the target RNA, if it was unfolded
by DDX3X132-607 or DDX17135-555, as performed previously
(Nelson et al. 2020,2021). When fluorescence and (or) mi-
gratory dierences occur between the BSA control and the
helicase, this provides evidence that the helicase unfolded
the noncanonical region, as the oligo base pairing causes a
change in fluorescence, shape, charge, and (or) hydration
shell of the target RNA (Huang and Zhang 2021). The reaction
was performed using 1 μMZIKV3
SL RNA sample, 4.25 μM
ATP, 40 nM Cy-5 DNA Oligo, and 20 μM of either DDX3X132-607
or DDX17135-555. BSA was selected as a negative control. The
reaction was incubated for 15 min prior to the measure-
ments. The experiments were performed eight times in total
over two binding check runs on the Nanotemper Monolith
NT.115. At 50% excitation and medium MST power, the
signal-to-noise ratio was assessed for each run to determine
successful unwinding activity, where, again, a signal-to-noise
ratio of ≥5 indicates that binding is occurring.
A spectrofluorimetric unwinding assay was also performed
using a Quanta Master 60 fluorescence spectrometer (Pho-
ton Technology International). The reactions were performed
using a mixture of 2 μM protein, 50 nM RNA, and 3.5 μM
ThT——a well-characterized G4 binding biosensor (Xu et al.
2016). The mixture was incubated for 5 min to allow ThT to
bind to the G4 on the RNA, followed by a fluorescence mea-
surement taken at 425 nm excitation and emission collected
from 450–510 nm with a step size of 1 nm increments. Fol-
lowing initial data collection, 60 μM of ATP was titrated into
the mixture, followed by a 5 min incubation to allow the he-
licase to unfold the G4, preventing ThT from binding, as the
G4s is unfolded, and the same fluorescence parameters were
used to collect the emission. The change in fluorescence was
then plotted (n=3), and a t-test was performed to check for
statistical significance. DDX17135-555, DDX3X132-607, and a con-
trol, BSA, were used to validate the MST results of unwinding.
Results
The G4 sequence contained in the 3SL of the 3
TR is conserved across the globe in ZIKV isolates
To obtain a comprehensive view of the sequence diversity
within the 3TR of all known ZIKV isolates, we collected all
complete ZIKV genomes from NCBI GenBank (Clark et al.
2016) and extracted the genomic regions that fold into the 3
SL element. Filtering for nonredundant sequences in the 3SL
region resulted in 14 representative ZIKV isolates, comprising
both African and Asian/American ZIKV lineages. A structural
multiple sequence alignment is shown in Fig. 2, highlighting
strong primary sequence conservation of the region of inter-
est that could potentially form a G4 structure.
ZIKV 3TR contains a G4 structure
We performed MST binding checks with PDS, and bind-
ing anity studies with the BG4 antibody, altogether with
the 3TR of ZIKV to investigate if the 3TR contains a G4
(Fig. 1A). PDS is a small molecule that selectively binds to the
top quartet of a G4 and stabilizes the noncanonical structure
(Rodriguez et al. 2012;Zhang et al. 2014;Moruno-Manchon et
al. 2017;Hou et al. 2022). PDS showed a significant response
amplitude in the 3TR and 3SL compared to the G4 mutant
in samples treated with PDS versus no PDS (Fig. 3A). These
results suggest that any potential for the G4 formation in the
G4 mutant is significantly impeded or nonexistent, as PDS is
below the signal-to-noise (SNR) threshold, and the response
amplitude is significantly lower than the 3TR and 3SL. The
Nanotemper analysis software states that a binding event ev-
idently occurs when the SNR value is ≥5 when comparing
treated versus untreated samples. Previously, it was demon-
strated that a ∼20 nt oligo from the 3SL of ZIKV contains a
G4 structure using PDS (Hou et al. 2022) indicating that PDS
is a reliable molecule in the study/identification of G4s.
We further investigated the presence of a G4 using an an-
tibody (BG4) that specifically targets the G4 structure. The
BG4 antibody is a well-established, reliable antibody that has
been widely used to identify G4s of both DNA and RNA in
vivo and in vitro (Bi et al. 2013,2014a,2014b;Byrd et al.

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Fig. 2. The 3stem-loop (SL) G4 sequence is conserved across 1028 Zika virus (ZIKV) isolates. Fourteen nonredundant ZIKV
3SL sequences from African and Asian/American lineages were aligned using the LocARNA software (Will et al. 2007). The
consensus sequence and its accompanying dot-bracket notation are also included, noting that the software predicts a canonical
structure within the dot-bracket notation.
Fig. 3. AG4existsinthe3
stem-loop (SL) of the 3terminal region (TR). The change in normalized fluorescence data with
RNA alone compared to RNA +pyridostatin (PDS) (n=3) is shown in (A). Binding anities of BG4 and TMPyP4 with Zika virus
(ZIKV) 3SL (blue), 3TR (green), and 3G4 mutant (red) are shown in (B and C). The determined Kdfor each transcript for BG4
were as follows: 3SL =0.0106 ±0.0023 μM, 3TR =1.27 ±0.087 μM, 3G4 mutant =11.11 ±1.71 μM, and for TMPyP4: 3
SL =0.0471 ±0.0079 μM, 3TR =0.0339 ±0.0117 μM, 3G4 mutant =no binding (n=3).
2016;Mao et al. 2018;David et al. 2019;Canesin et al. 2020;
Javadekar et al. 2020;Xu et al. 2020;Kom ˚
urková et al. 2021;
Varshney et al. 2021). The MST experiments using the BG4
antibody suggested that BG4 interacts with the 3SL at the
nanomolar range, 0.0106 ±0.0023 μM(Fig. 3B, blue line),
whereas the binding anity of the 3TR was determined to
be 1.27 ±0.08 μM(Fig. 3B, green line). The binding studies
of BG4 with G4 mutant suggested a Kdof 11.11 ±1.71 μM
(Fig. 3B, red line), which is 10-fold weaker compared to the
wild-type sequence. This change in the Kdvalue suggests that
the mutant sequence cannot form a G4. A potential reason
for the kinetic dierences between the 3SL and 3TR could
be due to steric/structural hindrance specific to the ZIKV 3
TR that is not present in the 3SL, as previous literature
shows that kinetics can dier depending on access to the
binding site (Dupuis et al. 2014). These data were further
supported by performing binding anity MST studies with
TMPyP4 (Fig. 3C), which is also a well-characterized G4 bind-
ing/unfolding small molecule (Morris et al. 2012;Zamiri et
al. 2014;Ji et al. 2020). The anity determined for each RNA
with TMPyP4 was as follows: 3SL =0.0471 ±0.0079 μM, 3
TR =0.0339 ±0.0117 μM, and 3G4 mutant =no binding.
DDX3X132-607 and DDX17135-555 interact with the
ZIKV 3TR
The interaction studies between the ZIKV 3TR and the
human helicases, DDX3X132-607 and DDX17135-555, were per-

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Biochem. Cell Biol. 102: 96–105 (2024) | dx.doi.org/10.1139/bcb-2023-0036 101
Fig. 4. DDX3X132-607 and DDX17135-555 bind to the 3TR. Mi-
croscale thermophoresis (MST) traces demonstrate the inter-
action of the 3terminal region (TR) of Zika virus (ZIKV) with
DDX3X132-607 (green) and DDX17135-555 (blue). RNA concentra-
tion was a constant 50 nM, while the dilution series started
from 19 μMforDDX17
135-555 and 15 μMforDDX3X
132-607
(n=3). Measurements were performed using 20% excita-
tion and high MST power. The Kdwas determined to be
1.16 ±0.02 μMforDDX17
135-555 and 3.70 ±0.10 μMfor
DDX3X132-607.
formed using MST. Figure 4 shows the binding anities of
DDX17135-555 and DDX3X132-607 with the ZIKV 3TR, with a
Kdof 1.16 ±0.02 μM and 3.70 ±0.10 μM, respectively. Addi-
tionally, these interactions were confirmed through a native-
PAGE and agarose EMSA where a shift is observed in con-
centrations measured around the Kdfor both DDX3X132-607
and DDX17135-555 (Fig. S4). These anities align with previous
studies on the ZIKV 5TR, which suggested that DDX3X132-607
binds with an anity at 7.05 μM(Nelson et al. 2021). It is cur-
rently unclear what role(s) these helicases play in viral repli-
cation, as it could be detrimental or advantageous to the viral
life cycle (Meier-Stephenson et al. 2018;Hernández-Díaz et al.
2021).
DDX17135-555 unwinds the 3TR G4 using the 3
SL
Once we established that both helicases interact with ZIKV
3TR, we asked if DDX17135-555 and DDX3X132-607 can un-
fold the G4 in the 3SL, as there is currently no evidence
demonstrating that DDX3X or DDX17 unfolds G4s. The he-
licase assays were performed using the 3SL to investigate if
DDX3X132-607 and DDX17135-555 unwind the G4 present in 3
SL. MST helicase assay utilizes a fluorescently labeled oligo
that was designed to be complementary to a portion of
the target RNA that was predicted to be double-stranded,
as previously described (Nelson et al. 2021). If the pres-
ence of the oligo results in a change in the migration of
the fluorescently labeled molecules, we can infer that the
RNA was unwound, permitting the binding to occur. The
MST experiments indicated that only DDX17135-555 caused
a shift in the fluorescent migration compared to the BSA
control and DDX17 mutant (Fig. 5A). An unpaired t-test was
performed for DDX3X132-607, DDX17135-555, and DDX17 mu-
tant, and the p-value at 95% confidence indicated a value of
0.029 for DDX3X132-607, 0.001 for DDX17135-555, and no statis-
tical significance for DDX17 mutant. Collectively, these ex-
periments suggest that the Cy5-DNA oligo binds to the un-
wound RNA in the presence of DDX17135-555. The SNR ratio for
DDX17135-555 was above 5, which confirms that the assay was
successful.
We also performed a spectrofluorimetric helicase assay by
replacing the complimentary oligo with ThT, a G4-binding
fluorescence sensor that stacks G-tetrads stabilizing K+ions.
This system allows for the collection of the ThT fluores-
cence emission when it recognizes a properly folded G4 com-
pared to unwound molecules in the presence of helicases
and ATP. A fluorescence decrease was observed upon the ad-
dition of ATP into DDX17135-555 and RNA sample, whereas
for BSA, DDX3X132-607 and DDX17 mutant showed no statis-
tically significant change (Fig. 5B). These results suggest that
DDX17135-555 unwound the G4, specifically on the 3SL pre-
venting ThT from binding/fluorescing, whereas DDX3X132-607
and DDX17 mutant are unable to unwind the same RNA.
DDX3X132-607 and DDX17 mutant also failed to produce a sig-
nificant change in the fluorescent migration of the oligo in
the MST assays, which is seen in the SNR ratio, which was
below 5. These combined results demonstrate a previously
unreported function of DDX17135-555: unwinding of a G4. The
MST studies also demonstrate that while DDX17135-555 can un-
fold the 3SL G4, DDX3X132-607 cannot.
Discussion
Our work demonstrates the presence of G4 in ZIKV 3TR
and establishes that human helicase DDX17135-555 can un-
wind the G4 structure. These results unveil a previously un-
reported function of DDX17. Further work is required to elu-
cidate the function(s) of the 3TR G4 in the ZIKV life cy-
cle. DDX3X has been extensively studied for its ability to in-
teract with various RNA structures, including G4 (Herdy et
al. 2018). Consistent with this finding, our previous study
demonstrated the capacity of DDX3X132-607 unwinding sec-
ondary structure within the ZIKV and Japanese Encephalitis
virus 5TRs (Nelson et al. 2021). We hypothesize that the ob-
served inability to unwind G4 structures might be attributed
to a structural feature within the substrate rather than the
absence of a functional domain within the helicase. How-
ever, it is still unclear whether DDX3X can unwind G4s (H.
Liu et al. 2021;Caterino and Paeschke 2022). Previous litera-
ture porposes hypotheses into the role of G4s in the 3TR of
the viral genome, such as serving as a “capped” degradation-
resistant region comparable to G overhangs in telomeres, eva-
sion of the host intracellular immune response, assisting in
viral transcription initiation, or act as another element in
host protein “sponging,” as this structure is contained in sub-
flaviviral RNA (Moon et al. 2015;Lista et al. 2017;Reznichenko
et al. 2019;Michalski et al. 2019;Bryan 2020). Moreover, pro-
tein sponging could also be assisted with a G4 serving as a
nucleation point for RNA phase separation, potentially giv-
ing the virus more control of ion proximity and structural

Canadian Science Publishing
102 Biochem.Cell Biol. 102: 96–105 (2024) | dx.doi.org/10.1139/bcb-2023-0036
Fig. 5. DDX17135-555 unfolds the 3SL G4. (A) and (B) are the helicase assay experiments performed using microscale ther-
mophoresis (A) and fluorescence spectroscopy (B). Experiments in (A) contain the complementary-labeled oligonucleotide that
base pairs to the G4 sequence if unfolded by the helicase protein in the presence of ATP with the relative fluorescence repre-
sented (n=4). During this binding check, it is essential to run a BSA control alongside each individual sample being tested,
as BSA is the reference point for each individual run. The significance of these runs is the signal-to-noise (SNR) in the fluores-
cence dierence. The fluorescence spectroscopy in (B) contains the G4 binding biosensor, ThT with the relative fluorescence
dierences measured between the presence and absence of ATP in the reaction mixture (n=3). The decrease in fluorescence
in (B) indicates that the G4 is unfolding in the 3SL; hence, ThT is unable to bind. Similar to the microscale thermophoresis
(MST) analysis in A, the fluorescence in (B) need not be normalized, as a comparison of the highest fluorescence peaks within
each measurement is collected. When comparing the fluorescence, it was performed in pairs, each with a BSA control for the
individual proteins, rather than a comparison of the dierent proteins against each other. In (A), statistically significant shifts
in each assay indicate that the oligo is bound to the 3SL. A t-test was performed for all assays to signify the statistical relevance
of our data. The asterisks indicate the p-value (ns =p>0.05, ∗p≤0.05, ∗∗p≤0.01, ∗∗∗ p≤0.001, ∗∗∗∗p≤0.0001).
switching between a G4 and stem-loop (Bugaut et al. 2012;
X. Liu et al. 2021). This would potentially provide ZIKV an
extension of the number of human proteins that can be re-
cruited to the terminal regions as well, thus providing a more
streamlined replication. Furthermore, it has also been shown
that the 12 regions of the 3TR contain N6-methyladenosines
(m6A) enrichment, and eight out of the 12 m6A regions were
on the 3SL G4. Previous literature showed that in the case
of ZIKV, when m6A was suppressed, it increased viral repli-
cation, suggesting that m6A could be a mechanism the cell
uses to slow viral replication (Fleming et al. 2019). Overall,
further investigation into the role of G4 from ZIKV 3TR is
required.
ZIKV remains a significant public health concern globally,
and due to concerns of potential antibody-dependent en-
hancement caused by antibodies generated from flaviviral
vaccines or infection, novel treatment avenues to stop vi-
ral replication are urgently required. To this end, our study
demonstrated that ZIKV contains a G4 within the 3SL of the
3TR that is maintained across multiple ZIKV isolates and is
unfolded by human DDX17135-555. This discovery allows for
further investigation to exploit this structured region by pro-
viding potential treatments using G4 binding molecules or
other molecules that disrupt human DDX17–3SL interaction.
Further studies of the function of the 3SL G4 during viral
replication are essential, but the foundation of the identifi-
cation of the G4 and two RNA-binding proteins allows for a
benchmark to start exploring this region as a target to at-
tempt to stop ZIKV replication, providing a novel treatment
for ZIKV infection.
Acknowledgement
This project was funded by an NSERC Discovery Grant to
TRP (RPGIN 2017–04003 and RPGIN 2022–03391). DLG’s and
CRN stipends were supported by NSERC Discovery Grant to
TRP and Alberta Innovates, respectively. Infrastructure sup-
port was provided by the Canadian Foundation for Innova-
tion grant to TRP (CFI 37115). MDB’s and HSP’s salaries were
supported by a Canada Research Chair stipend and Alberta
Innovates grants to TRP. TRP acknowledges the Canada Re-
search Chair program.
Article information
History dates
Received: 14 February 2023
Accepted: 19 September 2023
Accepted manuscript online: 29 September 2023
Version of record online: 23 October 2023
Copyright
© 2023 The Author(s). This work is licensed under a Creative
Commons Attribution 4.0 International License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduc-
tion in any medium, provided the original author(s) and
source are credited.
Data availability
All data have been presented in this article.

Canadian Science Publishing
Biochem. Cell Biol. 102: 96–105 (2024) | dx.doi.org/10.1139/bcb-2023-0036 103
Author information
Author ORCIDs
Higor S. Pereira https://orcid.org/0000-0003-4761-8361
Liam Kerr https://orcid.org/0000-0002-5459-4639
Michael T. Wolfinger https://orcid.org/0000-0003-0925-5205
Trushar R. Patel https://orcid.org/0000-0003-0627-2923
Author notes
Present address for Michael T. Wolfinger is Bioinformat-
ics Group, Department of Computer Science, University
of Freiburg, Georges-Köhler-Allee 106, 79110 Freiburg, Ger-
many.
Dannielle L. Gemmill and Corey R. Nelson contributed
equally to this work.
Trushar R. Patel served as an Editorial Board Member at the
time of manuscript review and acceptance; peer review and
editorial decisions regarding this manuscript were handled
by another Editorial Board Member.
Author contributions
Conceptualization: TRP
Data curation: DLG, CRN, HSP, LK
Formal analysis: DLG, CRN, MDB, HSP, MTW
Funding acquisition: TRP
Investigation: MDB, TRP
Methodology: DLG, CRN, HSP, MTW
Project administration: TRP
Software: MTW
Supervision: TRP
Writing – original draft: DLG, CRN, MDB
Writing – review & editing: DLG, HSP, MTW
Competing interests
The authors declare no competing interests.
Supplementary material
Supplementary data are available with the article at https:
//doi.org/10.1139/bcb-2023-0036.
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