Seesuay et al.

Emerging Microbes & Infections (2018)7:41

DOI 10.1038/s41426-018-0031-3 Emerging Microbes & Infections

ARTICLE Open Access

Human transbodies that interfere with the

functions of Ebola virus VP35 protein in
genome replication and transcription and
innate immune antagonism
Watee Seesuay1, Surasak Jittavisutthikul1, Nawannaporn Sae-lim1, Nitat Sookrung1,2, Yuwaporn Sakolvaree1 and
Wanpen Chaicumpa1

Abstract
Small molecular inhibitors and passive immunization against Ebola virus disease (EVD) have been tested in animal
models, including rodents and non-human primates, as well as in clinical trials. Nevertheless, there is currently no Food
and Drug Administration (FDA)-approved therapy, and alternative strategies must be pursued. The aim of this study
was to produce cell-penetrable human single-chain antibodies (transbodies) that are able to interfere with the
activities of interferon inhibitory domain (IID) of the VP35 protein, a multifunctional virulence factor of Ebola virus
(EBOV). We speculated that effective VP35-IID-specific transbodies could inspire further studies to identify an
alternative to conventional antibody therapies. Phage display technology was used to generate Escherichia coli-derived
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human single-chain antibodies (HuscFvs) that bind to IID. HuscFvs were linked to nona-arginine (R9) to make them cell
penetrable. Transbodies of transformed E. coli clones 13 and 3, which were predicted to interact with first basic patch
residues (R9-HuscFv13), central basic patch, and end-cap residues (R9-HuscFv3), effectively inhibited EBOV
minigenome activity. Transbodies of E. coli clones 3 and 8 antagonized VP35-mediated interferon suppression in VP35-
transduced cells. We postulate that these transbodies formed an interface contact with the IID central basic patch,
end-cap, and/or residues that are important for IID multimeric formation for dsRNA binding. These transbodies should
be evaluated further in vitro using authentic EBOV and in vivo in animal models of EVD before their therapeutic/
prophylactic effectiveness is clinically evaluated.

Introduction viral replication complex2. VP35 interacts with NP for

VP35 protein is a multifunctional virulence factor for viral assembly3 and inhibits innate interferon (IFNα/β)
Ebola virus (EBOV) replication1. One of the functions of production to facilitate host immune evasion4–7. The
this protein is its polymerase co-factor activity that VP35 latter function is mediated by blocking the signaling
protein and other EBOV proteins, such as NP (nucleo- pathways of RIG-1 and MDA5, either through seques-
protein), VP30 (transcription factor), and L (RNA- tration of viral dsRNA8,9 or through direct contact with
dependent RNA polymerase) and viral RNA, form the IKKε and TBK-1, which impairs the kinase interaction
with downstream IRF-3/IRF-7 and IPS-110 and leads to an
absence of several integral antiviral factors that are nor-
Correspondence: Wanpen Chaicumpa (wanpen.cha@mahidol.ac.th) mally generated via the autocrine and paracrine actions of
1
Center of Research Excellence on Therapeutic Proteins and Antibody IFNs, including PKR and 2΄5΄-OAS11.
Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital,
The EBOV VP35 comprises 340 residues that contain
Mahidol University, Bangkok 10700, Thailand
2
Department of Research and Development, Faculty of Medicine Siriraj the N-terminal oligomerization domain (NOD) (residues
Hospital, Mahidol University, Bangkok 10700, Thailand

© The Author(s) 2018

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Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 2 of 15

1–220) and C-terminal portion that has IFN antagonistic It is typically difficult for hydrophilic and large mole-
activity (termed the IFN-inhibitory domain, IID)12–14. cules, such as conventional four-chain antibodies, to
VP35 forms multimers via the coiled-coil motif within penetrate the mammalian plasma membrane41. Thus, the
NOD, which facilitates the interferon-antagonist activity antibodies are unable to access intracellular targets.
of IID15. Residues 20–48 bind NP and regulate NP Recently, cell-penetrating peptides (CPPs) have been
assembly on viral RNA to facilitate RNA synthesis16,17. shown to deliver their molecularly linked biologically
Residues 71–75 interact with host dynein LC8 to enhance active molecules into living cells42. Typically, CPPs are
viral RNA synthesis18. The IID consists of two sub- positively charged, which facilitates electrostatic interac-
domains: an N-terminal α-helical subdomain (residues tions with negatively charged cell-surface constituents.
221–283) that contains four alpha helices (α1–α4) and a Nona-arginine (R9) is an example of a CPP that effectively
C-terminal β-sheet subdomain (residues 294–340) that delivers its cargo into the cytoplasm42,43. Given that VP35
comprises four antiparallel strands (β1–β4), a small α5, is associated with several pivotal activities in the EBOV
and a type II polyproline helix13. Within the IID, there are infectious cycle1, the aim of this study was to generate
two conserved basic patches and additional border basic cell-penetrable human scFvs (R9-HuscFvs) or transbodies
residues that play different but cooperative roles in EBOV that can effectively interfere with VP35-IID functions. We
replication19. The first basic patch (K222, R225, K248, and speculate that effective VP35-IID-specific R9-HuscFvs
K251) is located in the N-terminal helical subdomain and could inspire further analyses to identify an alternative to
is important for VP35 polymerase co-factor function as conventional antibody therapies.
well as NP binding for the formation of nucleocapsid19.
The second or central basic patch (R305, K309, R312, Results
K339, R322, and K319) binds to dsRNA to inhibit the IFN Recombinant VP35 and IID
signaling pathway1,13. Conserved F239 and I340 form a Schematic diagrams of bacterially produced recombi-
hydrophobic pocket called an “end-cap” that binds the nant full-length EBOV VP35 (bVP35FL) and the
blunt ends of dsRNA20. Several other basic residues C-terminal IID of VP35 (bVP35IID) are shown in Fig. 1a.
(K282, R283, R298, and R300) located at the IID periphery SDS-PAGE-separated patterns of the purified recombi-
contribute to VP35 polymerase co-factor function20. nant proteins are shown in Fig. 1b.
H240, which is located near the first basic patch, is critical
for VP35 activities19. VP35-bound transbodies
There is currently no effective FDA approved agent or Phage clones that bound to bVP35FL were selected
protocol for the treatment of Ebola virus disease (EVD). from a HuscFv phage display library44 by bio-panning
Thus, EBOV-infected patients can only be provided sup- using 1 μg of bVP35FL as antigen. E. coli HB2151 infected
portive care21. Several agents and treatment regimens with recombinant bVP35FL-bound phages were screened
have been tested in both animal models of EVD and in for huscfv sequences by PCR. Lysates of 17 and 34 huscfv-
naturally infected humans22–29. Whether any are effective positive E. coli clones bound and did not bind to
in naturally infected humans could not be determined bVP35FL, respectively (Fig. 1c). Supplementary Figure S1
because the outbreak was dwindling, numbers of treated provides details of the binding of individual clones to
cases were relatively small, and other treatments were also VP35 (test antigen) and BSA (control antigen).
administered. Passive immunization using EVD- DNA coding for bVP35FL-bound HuscFvs of the 17
convalescent serum and KZ52 monoclonal antibody has clones (No's. 3, 6, 7, 8, 10, 13, 15, 21, 23, 24, 25, 28, 29, 31,
had limited success30–33, while newer polyclonal approa- 33, 36, and 38) was classified into seven different types
ches (such as ZMapp, MB-003 and other antibody cock- based on the deduced amino acid sequences: type 1 (clones
tails) are able to reverse advanced EVD in non-human 3 and 33); type 2 (clones 6, 7, 8, 10, 31, 36 and 38); type 3
primates (NHPs) and/or effectively prevent morbidity and (clones 13 and 21); type 4 (clone 15); type 5 (clone 23); type
mortality in NHPs when administered as a post-exposure 6 (clones 24 and 29); and, type 7 (clones 25 and 28). Clones
prophylactic34–38. It should be noted that the antibodies 3, 8, 13, 15, 23, 24, and 28 were selected as the repre-
used in the passive immunization were mostly in an intact sentatives of individual types for further experiments.
four-chain format that either inhibited cellular entry of HuscFvs of E. coli clones 3, 8, 13, 15, 23, 24, and 28 were
the virus (antibody to GP1)36,38, neutralized secreted linked molecularly to R9, which is a CPP. Figure 2a shows a
glycoprotein (sGP) for mitigation of pathogenicity39, schematic diagram of the cell-penetrable HuscFv construct.
inhibited the release of endosomal RNP into the cyto- Recombinant R9-HuscFvs were expressed as inclusion
plasm (antibody to GP2)32 or caused antibody-dependent bodies (IBs) by the transformed E. coli. After IB purification
cell-mediated cytotoxicity40. The antibodies lacked the and subsequent protein refolding, the purity of individual
ability to interfere with the activities of intracytoplasmic R9-HuscFvs (∼34 kDa) was checked by SDS-PAGE and
proteins of the replicating virus. CBB staining (Fig. 2b). The refolded R9-HuscFvs were
Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 3 of 15

tested for binding to bVP35FL and bVP35IID by indirect

ELISA. All R9-HuscFvs retained their binding activity to
bVP35FL; however, only the R9-HuscFvs of clones 3, 8, 13,
and 24 bound to bVP35IID (ELISA signals for bVP35IID
were more than three times the OD405nm of that of the BSA
control) (Fig. 2c). The EC50 values of R9-HuscFv3, 8, 13,
and 24 bound to bVP35IID were 1.6, 1.12, 1.41, and 2.06
μM, respectively (Supplementary Figure S2). The R9-
HuscFvs of these four clones were further tested for their
cell entry ability. To achieve this goal, HepG2 cells were
incubated with R9-HuscFvs from individual E. coli clones.
Intracellular antibodies were probed with Chromeo 488-
labeled anti-Strep tag II antibody and evaluated by confocal
microscopy. The R9-HuscFvs of all clones were found to be
cell penetrable, and they were located predominantly in the
cytoplasm. Figure 2d depicts the intracellular localization
of the R9-HuscFv3 as a representative model.

Presumptive residues of VP35-IID that interact with

HuscFvs
The orientations of the complexes formed between
VP35-IID and modeled HuscFvs are shown in Fig. 3a. The
predicted presumptive residues on the contact interface of
VP35-IID and individual HuscFvs are presented in Fig. 3b–e
and Supplementary Table S1. According to the docking, the
presumptive binding sites of HuscFv3 were at the spatially
juxtaposed IID central basic patch interface (R305, K309,
R312, R322, and K339), border basic residues (K282 and
R300), and end-cap residues (F239 and I340). HuscFv8 was
predicted to bind to the border basic residues opposite the
IID first basic patch (K282 and R283), central basic patch
(R322 and K339), and end-cap residue (I340). HuscFv13
interacted with the first basic patch interface (K222, R225,
K248, and K251) as well as with H240. HuscFv24 formed a
predictive interface with the IID helical subdomain and
K222, R225, K248, and K251 of the first basic patch. Based
on the important residues of IID that were predicted to
form contact interfaces with HuscFvs (Supplementary
Fig. 1 Productions of recombinant VP35 proteins and selection Table S1), these antibodies were further tested to evaluate
of VP35-bound HuscFvs. a Schematic representations of constructs their ability to regulate VP35-IID activities.
of bacterially produced recombinant full-length EBOV VP35 (bVP35FL)
and the C-terminal interferon inhibitory domain of VP35 (bVP35IID). b Binding of R9-HuscFvs to VP35 produced from mammalian
Recombinant bVP35FL and bVP35IID proteins purified from
transformed E. coli clones. M, pre-stained protein ladder; lane 1,
cells (mVP35)
purified bVP35FL; and, lane 2, purified bVP35IID. Numbers at the left Figure 4a shows the DNA construct encoding VP35
represent the protein molecular masses in kDa. c bVP358FL-bound with the N-terminal Flag-tag. The mVP35 found in the
HuscFv clones, as determined by indirect ELISA using purified transfected COS-7 cell lysate is shown in Fig. 4b. Ten
bVP35FL as antigen. The bound group was selected from the OD405nm micrograms of cell lysate containing mVP35 was mixed
signal above mean + 3SD of the background binding control (lysate of
original E. coli HB2151; HB). Statistical significance was determined
with 3 μg of purified R9-HuscFvs, and the individual
using one-way ANOVA and Tukey’s post hoc test. Supplementary mixtures were added to the wells of a 96-well plate con-
Figure S1 provides details of the binding of individual clones to taining immobilized anti-Flag antibody. After incubation
bVP35FL (test antigen) and BSA (control antigen) and washing, Strep-Tactin®-HRP conjugate was added to
detect the R9-HuscFv-mVP35 complexes (Fig. 4c). The
Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 4 of 15

Fig. 2 Antigen binding and cell entry ability of purified R9-HuscFvs. a Schematic representation of the construct for preparing cell-penetrable
HuscFvs (R9-HuscFvs). b SDS-PAGE and CBB-stained R9-HuscFvs purified and refolded from transformed E. coli clones 3, 8, 13, 15, 23, 24, and 28. R9-
HuscFvs had a molecular mass of ~34 kDa under reducing condition. c Binding activities of R9-HuscFvs to bVP35FL and bVP35IID compared to BSA
(control antigen), as demonstrated by indirect ELISA. Positive binding to the tested antigens yielded an OD405nm signal three times higher than to
that of the control antigen. Supplementary Figure S2 shows the EC50 value derived from selected bVP35IID-bound R9-HuscFvs. d Intracellular
localization of R9-HuscFv was revealed by confocal immunofluorescence microscopy. HepG2 cells were incubated with R9-HuscFv3 (representative of
the R9-HuscFvs) for 3 h, and then the cells were fixed, permeabilized, and stained. Cell border, white line; R9-HuscFv, green; nuclei, blue

lower panel of Fig. 4d illustrates the input proteins in the cells were co-transfected with a minigenome consisting of
co-immunoprecipitation assay. The ELISA results shown a plasmid cocktail, i.e., EBOV-Gaussia luciferase (Gluc)
in upper panel of Fig. 4d demonstrate that all R9-HuscFvs +NP−IRES−VP35+VP30+L. R9-HuscFv3, R9-HuscFv8,
bound to VP35 in the transfected COS-7 cell lysate. R9-HuscFv13, R9-HuscFv24, irrelevant R9-HuscFv (Irr),
or medium alone (M, for positive minigenome activity
Effect of transbodies on hepatic cells control) that was then added to the cells. The negative
HepG2 cells were mixed with 100 μL of complete minigenome activity controls were COS-7 cells co-
medium containing 25 μg/mL of individual VP35-bound- transfected with Gluc+NP−IRES−VP35+VP30 (−L)
transbodies, control (irrelevant) transbody (Irr), or buffer and Gluc+L+NP+VP30 (−VP35). The preparations were
(B) for 24 h. Cells with added digitonin (D) served as a maintained at 37 °C in a 5% CO2 incubator for 36 h. The
maximal cytotoxic control. Cell death was measured using activities of the minigenome, as indicated by Gluc inten-
the luminescent assay. The numbers of viable cells for all sities in cell culture wells, were monitored. As shown in
treatments were then calculated and found to be more Fig. 5b, the tested R9-HuscFvs caused a significant
than 90% in all cases. The viability of transbody-treated decrease in minigenome activity when compared to the
cells did not differ from that of cells with only buffer added positive minigenome activity (M) and Irr (p < 0.05). The
(100% viable cells) (p > 0.05), indicating that the transbo- most effective transbody was R9-HuscFv13 [90% inhibi-
dies were biocompatible with human hepatic cells (Fig. 4e). tion, which was not significantly different from the
minigenome activity without the VP35 construct (NP-
Efficacy of the transbodies in the suppression of EBOV IRES-VP35)]. R9-HuscFv3, R9-HuscFv24, and R9-
minigenome activity HuscFv8 inhibited VP35 genome activity by ∼70, ∼50,
The plasmids used in the assay for determining the and ∼40%, respectively. The irrelevant transbody (Irr) also
efficacy of transbodies for inhibiting EBOV minigenome inhibited EBOV minigenome activity by ∼10% when
activity are shown in Fig. 5a. In this experiment, COS-7 compared to M (p < 0.05).
Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 5 of 15

Fig. 3 (See legend on next page.)

Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 6 of 15

(see figure on previous page)

Fig. 3 Computerized-generated structures of VP35-IID-HuscFv complexes and presumptive VP35-IID epitopes. a Overall structures of EBOV
VP35-IID (PDB ID: 3FKE) (cyan) after complexing with HuscFvs (blue white) of E. coli clones 3, 8, 13, and 24 derived from molecular docking. Computer-
generated animated images of VP35-IID that formed direct interface contact with antibodies are colored in orange, while the interfaces that fell
within 5 Å thresholds of the van der Waals radii of the HuscFvs are colored in gray. b–e Contact interfaces between VP35-IID and HuscFv3, HuscFv8,
HuscFv13, and HuscFv24. Residues of the IID that make contact with their respective HuscFvs are shown as sticks. For details, see Supplementary
Table S1

Transbodies mediated restoration of host innate gene (Fig. 6a). At 24 h post-transduction, the spent culture
expression in VP35-transduced cells medium in each well was replaced with fresh medium
Figure 6a shows a schematic diagram of the VP35 containing VP35-specific R9-HuscFvs. Controls included
expression cassette used for HepG2 transduction. The untreated cells, cells stimulated with poly(I:C), VP35-
conceptual diagram regarding how EBOV VP35 mediates transduced cells without any treatment, VP35-transduced
inhibition of the innate interferon signaling pathway and cells stimulated with poly(I:C), and VP35-transduced cells
the expected VP35 antagonistic activity of the transbodies treated with irrelevant R9-HuscFv. The treated cells were
is illustrated in Fig. 6b. The ability of extra-chromosomal incubated for 12 h, and the expression levels of IFNB1 and
transgene expression to trigger the interferon response EIF2AK2 were determined. R9-HuscFv3 and R9-HuscFv8
circuit45 was investigated. Expression levels of IFNB1 and mediated the upregulation of both innate immune
EIF2AK2 (encode IFN-β and PKR, respectively), which are response genes compared with VP35-transduced cells in
the integral antiviral genes in the interferon signaling medium alone (p < 0.0001) (Fig. 6g, h). VP35-transduced
pathway in HepG2 cells transduced with the VP35 gene cells treated with R9-HuscFv13 and cells transduced with
cassette, was determined at 12-h and 36-h post-trans- added poly(I:C) showed an equivalent and modest upre-
duction. Expression of the IFNB1 and EIF2AK2 genes was gulation of IFNB1 activity when compared with VP35-
assessed by quantitative reverse transcription-polymerase transduced cells alone (p < 0.05) (Fig. 6g). Irrelevant R9-
chain reaction (qRT-PCR). Controls were normal cells, HuscFv did not cause any change in IFNB1 expression.
cells transfected with poly(I:C), and cells transduced with Regarding EIF1AK2 (Fig. 6h), R9-HuscFv3 and R9-
VP35H240E gene cassette and empty vector. At 12 h, cells HuscFv8 upregulated gene expression by ∼7-fold, which
transduced with VP35 gene and poly(I:C) showed com- was comparable to the observed gene expression in poly(I:
parable upregulation of IFNB1 (∼400-fold) compared with C)-treated VP35-transduced cells. The effectiveness of
normal cells (Fig. 6c). At 36 h, the expression level of R9-HuscFv13 on innate gene expression did not differ
IFNB1 in cells with the VP35 transgene returned to the from that of irrelevant R9-HuscFv and untreated VP35-
level in normal cells, while the expression level of IFNB1 transduced cells.
in cells treated with poly(I:C) was sustained at 36 h. In
contrast, the EIF2AK2 expression patterns were similar to Discussion
those observed in cells transduced with empty vector (Fig. An effective therapeutic agent and protocol for EVD are
6d). Cells stimulated with poly(I:C) showed upregulated needed. Passive immunization and previously tested
EIF2AK2 at both time points. It is noteworthy that while immunotherapy could not interfere with the replication of
the VP35 construct could induce IFNB1 expression, the virus that has gained entry into host cells. The strategy
VP35H240E construct could not. At the protein level, it being proposed in this study is to use small antibody
has been previously shown that VP35H240E has lost its fragments that can penetrate the cell and gain access to
co-polymerase and IFN antagonistic activities19. pivotal proteins of the replicating virus. The VP35 protein
The effects of R9-HuscFvs on the innate immune was selected not only to inhibit viral replication, transla-
response genes of HepG2 cells were investigated. VP35- tion, and RNP assembly but also to restore host immunity
specific R9-HuscFvs and irrelevant R9-HuscFv were and reduce immunopathophysiology caused by
added to cells in culture wells. After 12 h, the expression EBOV46,47.
levels of IFNB1 and EIF2AK2 were determined. R9- HuscFvs to VP35 were derived from human immu-
HuscFvs did not cause a significant upregulation of IFNB1 noglobulin genes, and therefore they should not be
and EIF2AK2 compared with the untreated control cells immunogenic in humans. Each molecule of the HuscFv
(p > 0.05) (Fig. 6e, f). contains six complementarity-determining regions
To investigate the effect of VP35-specific R9-HuscFvs (CDRs), and each CDR comprises several residues that
on the interferon antagonistic activity of VP35, HepG2 can cooperate in capturing the target, which in this study
cells maintained in complete medium were transduced consisted of several highly conserved critical residues
with lentivector carrying the EBOV VP35 gene cassette pivotal for the function of VP35-IID12. Two recent
Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 7 of 15

Fig. 4 Interactions of the transbodies with VP35 and their

biocompatibility. a Schematic representation of the VP35 construct
for production in mammalian cells (mVP35). b mVP35 produced from
COS-7 cells (∼40 kDa; arrow). COS-7 cells were transiently transfected
with the VP35 construct. At 48-h post-transfection, the cells were
lysed, and the presence of mVP35 in the clarified lysate was
determined by Western blotting using anti-Flag antibody. The lysate
of cells transfected with empty vector was used as a negative control.
c Diagram of co-immunoprecipitation and ELISA for detecting the
interaction between mVP35 and R9-HuscFvs. COS-7 cell lysate
containing mVP35 was mixed with R9-HuscFvs, and individual
mixtures were added to wells containing immobilized anti-Flag
antibody. Captured complexes were detected using Strep-Tactin®-HRP
conjugate and HRP substrate. d Upper, the OD405nm of ELISA for
demonstrating the interaction between mVP35 and R9-HuscFvs
compared with mVP35 mixed with irrelevant R9-HuscFv (Irr) and the
background binding control (Ctrl; mVP35 without R9-HuscFv). Lower,
the input reactants of co-immunoprecipitation revealed by Western
blotting are shown. e The biocompatibility of R9-HuscFvs with human
hepatic cells shown as the percent viability of HepG2 cells after
incubation with R9-HuscFvs for 24 h. The protease activity of dead
cells was measured after addition of luminogenic substrate to wells
containing treated cells, and the percent cell viability was calculated.
Statistical significance was determined using one-way ANOVA and
Tukey’s post hoc test

studies, one employing VHH as cross-linkers of highly

conserved regions of filoviral nucleoprotein to inhibit viral
replication48 and the other evaluating the anti-MARV
VHH epitopes as highly conserved49, antecede that tar-
geting vital conserved regions of intracellular viral anti-
gens may be less prone to selection of escape mutants.
Escape mutants of GP were identified in EBOV-infected
Rhesus monkeys treated with antibody cocktails (MB-
003)50. Thus, a novel approach for antiviral therapeutics
should be directed towards the conserved regions of the
viral proteins, which are exemplified by residues in the
first, border, and central basic patches of VP35 in this
study (mutations at those residues render the protein
inactive or functionally impaired19,20). Given that the
function of the VP35 N-terminal domain is still elusive in
contrast to the well-known structure and activities of the
C-terminal domain (IID), we selected only the HuscFvs
(clones 3, 8, 13, and 24) that recognized VP35-IID for
further functional assays. HuscFvs were converted into a
cell-penetrable format by linking them to a CPP, R9. R9-
HuscFvs readily entered the cells and did not cause
cytotoxicity to mammalian cells, which indicated their
biocompatibility.
Basic residues of the IID first basic patch (R225, K248,
and K251) interacted with the viral nucleoprotein (NP) for
RNP assembly; this interaction is also essential for viral
polymerase activity19. R225A, K248A, and K251A show
markedly reduced NP-binding activity and a complete
absence of polymerase co-factor activity19. The other
basic amino acids located at the border of the IID (K282,
Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 8 of 15

R283, R298, and R300) are also important for

VP35 polymerase co-factor function. Alanine mutants of
these residues have diminished polymerase co-factor
activity, although they retain their NP-binding capa-
city19. The polymerase co-factor activity was determined
by the basic charge of the amino acids and not their
identity19. A computerized simulation indicated that R9-
HuscFvs of all clones formed contacts with residues that
are important for VP35 polymerase co-factor function
and/or interactions with NP, either the border basic
residue(s) and/or the first basic patch residues. Therefore,
they were tested for their ability to inhibit EBOV mini-
genome activity.
In this study, the EBOV minigenome activity assay was
used to determine the antagonistic role of transbodies
toward VP35-IID during EBOV transcription and replica-
tion. The system described by Jasenosky et al.51 consisting
of the RNA polymerase I-driven minigenome and bicis-
tronic NP and VP35 was used, which is more robust than
the conventional minigenome system51,52. R9-HuscFv13,
which was predicted to dock on K222, R225, K248, and
K251 of the first basic patch and on the H240 end-cap, was
the most effective transbody for inhibiting minigenome
activity, which is consistent with a previous finding showing
that alanine mutation of R225, K248, and K251 not only
leads to a loss of polymerase co-factor function but also a
reduced NP interaction19. The H240E mutant was found to
lack both IFNβ-antagonistic and minigenome activities19,
which indicated the important dual role of this amino acid
in IID. The K222A mutation, however, retained activity
comparable to that of wild type19. R9-HuscFv3, which was
predicted to form contacts with K282 and R300 on the IID
border, was the second most effective transbody for inhi-
biting EBOV minigenome activity, consistent with a pre-
vious observation that individual mutations of these two
residues to alanine result in diminution of polymerase co-
Fig. 5 Transbody-mediated inhibition of EBOV minigenome factor activity19. Presumptive R9-HuscFv24-mediated con-
activity. a Schematic diagrams of the plasmids used in the RNA tact with K222, K225, K248, and K251 was similar to that of
polymerase I-driven EBOV minigenome system. EBOV-like reporter HuscFv13; however, this transbody was not as effective as
(Gluc) RNA was transcribed under the regulation of human Pol-I
R9-HuscFv13 for reducing EBOV minigenome activity,
promoter and the Sal box transcription termination element. The viral
protein expression cassettes were under the regulation of CMV I.E. potentially due to a lack of H240 functional inhibition or
enhancer/promoter and the SV40 late poly(A) signal element. b different orientations of the antibody–IID interactions, as
Percent EBOV minigenome activity of COS-7 cells after treatment with shown by molecular docking. The least effective
R9-HuscFvs. COS-7 cells transfected with EBOV minigenome were R9-HuscFv8 was predicted to bind to the border basic
treated with R9-HuscFv3, R9-HuscFv8, R9-HuscFv13, R9-HuscFv24, and
residues K282 and R283. Although mutations of residues in
irrelevant R9-HuscFv (Irr). Minigenome activity was measured by
detecting Gluc bioluminescent intensity. Controls included (M) COS-7 the border basic patch have been shown to interfere with
cells co-transfected with the minigenome without antibody polymerase co-factor function19, R9-HuscFv8-inhibited
treatment, (−L) COS-7 cells co-transfected with EBOV minigenome minigenome activity by only ~40%, which indicated that
without L plasmid, and (−VP35) COS-7 cells co-transfected with the these residues might be less important for genome activity.
minigenome without VP35 plasmid. Differences in percent
The mutations that led to discontinued co-polymerase
minigenome activity were compared using one-way ANOVA and
Tukey’s post hoc test activity observed previously19 may have been due to struc-
tural alterations of the protein.
Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 9 of 15

Fig. 6 (See legend on next page.)

Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 10 of 15

(see figure on previous page)

Fig. 6 INF-β gene response in VP35-transduced cells after treatment with transbodies. a Schematic diagram of the EBOV VP35 expression
cassette (pLVX-VP35) for HepG2 cell transduction. b Conceptual diagram illustrating how the transbodies rescued the IFN-β gene response of host
cells from VP35 interferon antagonistic activity. After transduction with lentivector carrying EBOV VP35 gene cassette, transcription of the EBOV-VP35
episome was initiated (cyan solid arrows). The ability of VP35 mRNA to activate MDA5 signaling cascade was antagonized after VP35 production.
Cytoplasmic VP35 bioactivity could be blocked by the VP35-targeting transbody (gray solid arrow), which resulted in IFNB1 expression (orange solid
arrows) by triggering transgene mRNA. As a consequence, the produced IFN-β induced interferon-stimulated genes (e.g., EIF2AK2 (magenta solid
arrow)). Dotted black arrows indicate signaling cascades. c, d Fold change of IFNB1 (c) and EIF2AK2 (d) mRNAs in cells after EBOV VP35 transduction
for 12 and 36 h compared to untreated control cells. Cells transfected with poly(I:C) served as positive stimulation, and cells transduced with EBOV
VP35H240E and empty vector were transduction controls. Significant differences were determined by two-way ANOVA and the Šidák t test. e, f
Expression of IFNB1 (e) and EIF2AK2 (f) mRNAs of untreated control HepG2 cells after incubation with transbodies for 12 h in comparison to the
control cells in culture medium alone. g, h Fold change of IFNB1 (g) and EIF2AK2 (h) mRNAs in VP35-transduced cells after exposure to transbodies for
12 h compared with VP35-transduced cells. Controls included untreated cells with and without poly(I:C) transfection, VP35-transduced cells with and
without poly(I:C) transfection, and VP35-transduced cells treated with irrelevant transbody (Irr). Statistically significant differences were determined
using one-way ANOVA and Dunnett’s post hoc test

VP35-IID in multimeric form binds efficiently to dif- via obstruction or suppression of these critical IID resi-
ferent viral RNA ligands, which results in inhibition of dues. R9-HuscFv8 showed comparable effectiveness to
innate IFN signaling20. Important amino acids involved in R9-HuscFv3 relative to host innate immunity restoration,
the IID intermolecular interaction are located in the which could be due to binding of the transbody to R322
central basic patch, including R312 and R322 of the first and K339 of the central basic patch, which is important
monomer, which form hydrogen bonds to residues of the for the interaction and recognition of dsRNA53. In addi-
second monomer (G270, D271, and E269 and Q262 and tion, R9-HuscFv8 was predicted to bind to I340 of the end
E269, respectively)20. The interaction of IID with dsRNA cap and to S272, C275, I278, and Q279, which should
was mediated by P233, T237, F239, S272, N274, C275, disrupt dsRNA binding. Moreover, relative to that pre-
I278, A306, S310, and I340 of the first and third monomer diction, this transbody also bound to residues (E269,
via van der Waals and water forces, while R312 and R322 Q270, and D271), which form multimers during dsRNA
of the second and fourth IID molecules interacted with binding of VP35. From computerized intermolecular
the phosphodiester backbone of the dsRNA20. In this docking analysis, R9-HuscFv13 docked on F235 for end-
study, computerized simulations suggested that HuscFv3, cap recognition and on H240 near the first basic patch. A
HuscFv8, and HuscFv13 formed an interface contact via previous study found that IFN antagonistic activity was
van der Waals forces, hydrophobic forces, π effect, and/or not affected by the VP35-F235A mutant, while H240E
hydrogen interactions with several residues of the IID caused increased IFN-β promoter activity19,20. However,
(Supplementary Table S1). The end-capped dsRNA of R9-HuscFv13 only modestly rescued IFNB1, which did
these residues directly contacted the dsRNA and/or not result in a significant upregulation of the PKR gene
formed a multimeric complex during dsRNA binding20. compared with the upregulation observed in VP35-
Therefore, the R9-HuscFvs of the three clones were tested transduced cells. H240 may not be directly involved in
for their ability to inhibit VP35-IID IFN antagonistic interferon antagonistic activity. The structural instability
activity and to restore innate immune response gene of or configuration change in the H240E mutant may
expression in HepG2 cells that had been transduced with render the IID unable to retain its functions. The
a lentivector carrying the EBOV-VP35 gene. VP35- observed lowest level of effectiveness of R9-HuscFv13 for
transduced cells treated with R9-HuscFv3 and R9- restoring the innate immune response might be because
HuscFv8 showed an upregulation of IFNB1 and this transbody did not bind to the critical residues
EIF2AK2 compared to the expression of IFNB1 and involved in dsRNA contact, such as those in the central
EIF2AK2 in VP35-transduction control and irrelevant R9- basic patch or the border basic residues.
HuscFv-treated VP35-transduced cells. The predicted IID In conclusion, VP35-IID-specific transbodies are able to
residues specific by HuscFv3 included the end-cap motif inhibit protein functions associated with the EBOV
(F235, F239, and I340), other residues that have been replication cycle, including polymerase co-factor activity
known to contact dsRNA (P233, T237, Q274, C275, I278, and host IFN-antagonism. Further experiments are nee-
A306, and S310), residues of the central basic patch that ded to elucidate the actual interaction between the anti-
are important for interferon inhibitory activity (R305, bodies and VP35-IID. These transbodies should be further
K309, R312, R322, and K339), and D271 making up the tested using authentic EBOV before they can be con-
IID multimeric contact20. Therefore, it is plausible that sidered a promising alternative to existing treatment
host innate immunity rescued by R9-HuscFv3 occurred approaches.
Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 11 of 15

Materials and methods soluble E. coli fractions to bVP35FL was tested by indirect
Cell cultures ELISA (Supplementary Method S2). Phagemid DNA from
COS-7 cells (African green monkey kidney cell line), E. coli clones that produced rVP35FL-bound HuscFvs
HepG2 cells (human hepatoma cell line), and Lenti-X 293 were subjected to nucleotide sequencing, and then the
T cells were grown in complete medium (Dulbecco’s CDRs and immunoglobulin framework regions of all
Modified Eagle’s Medium) (Gibco, Thermo Fisher Sci- sequences were identified using the integrative database of
entific, Waltham, MA, USA) supplemented with 10% germ-line variable genes (VBASE2, http://www.vbase2.
heat-inactivated fetal bovine serum (FBS) (HyClone, GE org)55.
Healthcare Life Sciences, Marlborough, MA, USA), 100
units/mL penicillin and 100 μg/mL streptomycin, and 2 Production of R9-HuscFvs (transbodies) to VP35
mM L-alanine-L-glutamine dipeptide (Gibco) at 37 °C in a An optimized DNA construct of R9-HuscFv-Strep tag II
5% CO2 atmosphere. with an additional stop codon was synthesized and cloned
into pET24a+ (GenScript). The DNA was introduced into
Production of bacterially produced recombinant full- E. coli BL21 (DE3). For production of R9-HuscFvs, the
length EBOV VP35 and the C-terminal interferon inhibitory transformed E. coli BL21 (DE3) clones were cultured in
domain of VP35 LB broth containing 30 μg/mL kanamycin at 37 °C with
The VP35 consensus sequence was obtained by multiple aeration overnight. An aliquot of 25 μL of the overnight
sequence alignments of Zaire EBOV VP35 from the uni- culture were inoculated into 250 mL of fresh auto-
versal protein resource54. Optimized DNA coding for induction KPM medium containing 50 μg/mL of kana-
VP35 (accession MF801599) was synthesized (GenScript, mycin. The cultures were maintained at 30 °C with
Picataway, NJ, USA) and used as a template for preparing shaking at 250 rpm overnight. Bacterial cells were har-
bVP35FL and VP35-IID (residues A221-I340; bVP35IID) vested by centrifugation (4000 × g at 4 °C for 20 min). The
by PCR. DNA fragments were cloned into pLATE52 antibodies were purified as described in Supplementary
(Thermo Fisher Scientific) using the LIC technique. The Method S1 before use in subsequent experiments. The
respective recombinant plasmids were transformed into E. purified and refolded recombinant R9-HuscFvs were
coli BL21 (DE3). Appropriate colonies of transformed E. verified by SDS-PAGE and Coomassie Brilliant Blue G-
coli were grown in LB broth containing 100 μg/mL 250 (CBB) staining. Their binding activity and half max-
ampicillin (LB-A) and 1 mM IPTG (Thermo Fisher Sci- imal effective concentrations (EC50) were determined
entific). IBs containing bVP35FL and bVP35IID were (Supplementary Method S2).
purified, and both proteins were refolded (Supplementary
Method S1). Confocal immunofluorescence microscopy
HepG2 cells (6 × 104 cells) were placed in an 8-chamber
Phage bio-panning cell imaging cover-glass (Eppendorf, Hamburg, Germany)
Phage clones that bound to bVP35FL were selected and kept at 37 °C in a 5% CO2 incubator overnight. The
from a HuscFv phage display library44 by bio-panning. culture medium was removed, and the cells were
One microgram of bVP35FL in 100 μL of 0.2 M sodium replenished with 0.5 mL fresh complete medium con-
carbonate-bicarbonate buffer, pH 9.4 was coated into a taining 25 µg/mL of individual R9-HuscFvs and then
well of an EIA/RIA strip (Corning, NY, USA). After further incubated for 3 h. The cells were gently rinsed
incubation, the antigen-coated well was blocked with with Dulbecco’s PBS (DPBS) (Gibco), fixed with a 1:2
protein-free blocking buffer, followed by addition and dilution of IC fixation buffer (eBioscience, Thermo Fisher
incubation of the phage library with the immobilized Scientific) in DPBS at 25 °C for 1 h, washed with PBS,
antigen. After removing antigen-unbound phages by permeabilized using 0.1% v/v Triton X-100 in PBS at 37 °
washing, log-phase E. coli HB2151 were added, and phage C for 30 min, and blocked with blocking solution (1% w/v
infection was permitted for 10 min. Bacteria were spread BSA, 22.52 mg/mL glycine, and 0.1% v/v Tween-20 in
onto LB-A agar plates and incubated at 37 °C overnight. PBS) at 25 °C for 1 h. After blocking, the cells were washed
Phagemid-transformed E. coli colonies that appeared on with PBS and stained with StrepMAB ImmoChromeo
the plates were screened for HuscFv genes (huscfvs) by 488-conjugated antibody (IBA Life Sciences GmbH) and
direct colony PCR44. The huscfv-positive E. coli clones 1 µg/mL Hoechst 33342 (Biotium, Fremont, CA, USA) in
were grown in 2YT broth containing 100 μg/mL ampi- blocking solution at 4 °C overnight. After staining, the
cillin (2YT-A) and 2% w/v glucose at 37 °C for 3 h. The cells were washed with PBS, mounted with 50% w/v gly-
bacterial pellet was suspended in 2YT-A medium con- cerol in PBS, and observed under a confocal microscope
taining 0.2 mM IPTG and incubated at 30 °C with shaking (Carl Zeiss Laser Scanning System LSM 510, Carl Zeiss
for 5 h. Cells were harvested from individual cultures, Microscopy GmbH). Images were processed using the
suspended in PBS and sonicated. Binding of HuscFvs in Zeiss LSM Image Browser (version 3.2.0.115).
Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 12 of 15

Computerized simulation Quick Start Bradford Protein Assay (Bio-Rad Labora-

The crystal structure of the VP35-IID (PDB ID: 3FKE) tories, Hercules, CA, USA). The presence of the Flag tag-
was obtained from the Protein Data Bank (RCSB PDB). fusion protein was verified by Western blot analysis using
Amino acid sequences of HuscFvs were subjected monoclonal anti-Flag M2 (Sigma-Aldrich, St. Louis, MO,
to homology modeling by iterative threading USA), goat anti-mouse immunoglobulin-alkaline phos-
assembly refinement (I-TASSER)56. To improve the local phatase (AP) conjugate (SouthernBiotech, Birmingham,
geometric and physical quality of the predicted 3D AL, USA), and KPL BCIP/NBT substrate (SeraCare Life
structure, I-TASSER predicted models were refined Sciences). For co-immunoprecipitation, cell lysates con-
using high-resolution protein structure refinement taining 10 µg of protein and 3 µg of individual R9-HuscFvs
and fragment-guided molecular dynamics simulation. were added to the mixture in 1 mL of
The VP35-IID structure and the modeled HuscFvs binding buffer (IBS containing 0.1% v/v Tween-20 and
were subjected to antibody-protein docking (ClusPro)57. 0.01% w/v skim milk). A total of 200 μL of that mixture
The largest cluster size that showed all of the HuscFv- was added to an ANTI-FLAG® M2-coated 96-well plate
CDRs within the 5 Å distance of the VP35-IID (Sigma-Aldrich), which was pre-blocked with Pierce
was selected. For interaction analysis, the antibody- Protein-Free Blocking Buffer (Thermo Fisher Scientific).
protein complexes in the NAMD simulated environ- The plate was kept at room temperature for 2 h on a
ment were adopted58. Pymol software (version 1.3r1 rotating platform. After washing with wash buffer (IBS
edu) (Schrödinger, New York, NY, USA) was used to containing 0.1% v/v Tween-20), all wells were blocked
visualize the modeled complex. Residues of VP35-IID with 240 μL of 1:1,000 biotin-blocking buffer (IBA) and
with side chains that fell within the threshold of 5 Å of kept at room temperature for 10 min. After removal
HuscFv radii (van der Waals radii of interacting atoms of the blocking buffer, 200 μL of 1:4,000 Strep-Tactin®-
and water) were defined as making interface contact with HRP conjugate (IBA) was added, kept for an additional 1
the antibody. h, and washed, and 200 µL of KPL ABTS substrate (Ser-
aCare Life Sciences) was added. The OD405nm of the
Co-immunoprecipitation assay content of each well was spectrometrically determined.
The NheI-Flag tag-MCS-Myc tag-NotI DNA fragment The remaining portion of each mixture was subjected to
(Integrated DNA Technologies, Coralville, IA, USA) Western blot analysis. Flag-tagged VP35 was detected
was used to replace the original multiple-cloning sites of using mouse monoclonal anti-Flag M2 (Sigma-Aldrich)
the pCI-neo vector (Promega, Fitchburg, WI, USA). The and goat anti-mouse immunoglobulin-AP conjugate
recombinant vector was designated pCI-Flag/Myc. (SouthernBiotech). Strep tag II-tagged R9-HuscFvs were
Codon-optimized DNA coding for EBOV VP35 (acces- detected using Strep-Tactin®-AP conjugate (IBA Life Sci-
sion no. MF801600) for mammalian expression was ences, GmbH). BCIP/NBT substrate was used for color
used as a template for preparing a Flag-VP35-expressing development.
construct. EBOV VP35-coding DNA was amplified and
cloned into pCI-Flag/Myc via the EcoRI and NotI Biocompatibility of the transbodies
restriction sites. The recombinant plasmid and the Culture medium (100 μL) containing 1 × 104 HepG2
empty vector were propagated separately in E. coli JM110, cells was placed in the wells of a 96-well white polystyrene
and they were isolated using a Presto™ Endotoxin Free microplate (Corning) and kept at 37 °C in a 5% CO2
Mini Plasmid Kit (Geneaid Biotech, New Taipai City, incubator overnight. The culture medium was then
Taiwan). COS-7 cells were placed in wells of a 6-well cell replaced with 100 μL of fresh complete medium con-
culture plate (Corning) (5 × 105 cells/well) and incubated taining 25 µg/mL of transbodies (from titration) or storage
overnight. Five micrograms of each plasmid preparation buffer. Controls included cells in medium alone (minimal
was transfected separately into cells using Xfect Single cytotoxicity/maximal cell viability), cells with added
Shots (Midi) Transfection Reagent (Takara Bio, Shiga, digitonin (maximal cytotoxicity), and complete medium
Japan). At 48-h post-transfection, the cells were rinsed without cells (luminescence background control). After
twice with DPBS and lysed with 1.5 mL M-PER Mam- leaving the microplate at 37 °C in a 5% CO2 atmosphere
malian Protein Extraction Reagent (Thermo Fisher Sci- for 24 h, cytotoxicity was measured using a CytoTox-Glo
entific) supplemented with a 1:200 dilution of protease Cytotoxicity Assay (Promega, Madison, WI, USA). Briefly,
inhibitor cocktail (set-III/EDTA-free) (Calbiochem, assay reagent (50 μL) was added to each well, mixed with
Merck KGaA, Darmstadt, Germany) and 25 U/mL Ben- the medium containing cells, and the mixture was kept at
zonase® Nuclease (Novagen), and incubated at room room temperature for 15 min before the luminescent
temperature for 10 min with shaking. The preparation signal was recorded with a Synergy™ H1 Hybrid Multi-
was centrifuged (15,000 × g, 4 °C, 5 min), and the protein Mode Microplate Reader (BioTek Instruments, Winooski,
content of the clear lysate was determined using the VT, USA).
Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 13 of 15

Construction of the EBOV minigenome reporter system signal of each well was recorded using a Synergy™ H1
Plasmids for expressing Zaire EBOV NP, VP35, VP30, Hybrid Multi-Mode Microplate Reader (BioTek
and L were designed as previously described53 with Instruments).
modifications. Optimized DNA coding for EBOV NP
(accession MF801600), NP-IRES-VP35 (accession Generation of lentivector carrying EBOV VP35
MF801600), VP30 (accession MF801601), and L (acces- Optimized DNA sequence coding for full-length VP35
sion MF801602) was synthesized and cloned into the pCI- of Zaire EBOV (accession MF801600) and mutant VP35
neo mammalian expression vector (with CMV promoter) (H240E; codon CAC to GAG) were synthesized (Gen-
(GenScript). The RNA polymerase I-driven transcription Script) and subcloned into the pLVX-Puro vector back-
cassette for the synthesis of EBOV-like RNA (i.e., EBOV bone via the XhoI and BamHI restriction sites. The
leader-Gluc-EBOV trailer (accession MF801603)) was recombinant constructs (pLVX-VP35 and pLVX-
generated in-house by splice overlapped extension PCR VP35H240E) were introduced into E. coli DH5α, and
(SOE-PCR) using gBlocks® Gene Fragments (Integrated replicated plasmids were recovered using an EndoFree
DNA Technologies) as a template. The cassette was Plasmid Maxi Kit (Qiagen). To generate the VSV-G
cloned anti-directionally between the Pol I promoter and pseudotyped lentivector, lentiviral packaging was
the terminator (Sal box) of pPol I (accession MF882921) prepared by co-transfecting Lenti-X 293 -T cells with
using an In-Fusion HD Cloning Plus Kit (Takara Bio). The pLVX-VP35, pLVX-VP35H240E or empty vector in a
resulting construct, designated pPol-I-EBOV-Gluc, was Lenti-X™ HTX Packaging System (Integrase
introduced into E. coli JM110 and recovered using an Deficient; Takara Bio). Lenti-X 293 -T cells (5 × 106 cells)
EndoFree Plasmid Maxi Kit (Qiagen, Hilden, Germany). in 10 mL of tetracycline-free complete medium were
Plasmid cocktails used in the minigenome assay and in placed in a 100-mm cell culture dish (Eppendorf) and kept
transbody-mediated inhibition of EBOV gene transcrip- overnight in a 37 °C CO2 incubator. DNA-Xfect Solution
tion and replication were prepared in endotoxin-free (Takara Bio) was added to the cells and incubated further
water, as follows: 5 µg/mL pCI-NP-IRES-VP35 or pCI-NP, for 4 h. After discarding the transfection medium, the cells
3 µg/mL pCI-VP30, 30 μg/mL pCI-L, and 2.5 μg/mL pPol- were replenished with fresh medium containing
I-EBOV-Gluc. tetracycline-free FBS. Viral protein expression and pack-
ing were allowed for 48 h in a 37 °C CO2 incubator. The
Inhibition of EBOV minigenome transcription by presence of lentivectors in culture supernatants was
transbodies checked using Lenti-X™ GoStix™ (Takara Bio), con-
Trypsinized COS-7 cells (8 × 105 cells in 900 μL com- centrated using a Lenti-X™ Concentrator (Takara Bio),
plete medium) were transfected by adding them to 100 μL and measured using a Lenti-X™ qRT-PCR Titration
of Xfect™ Single Shots (Midi) Transfection Reagent Kit (Takara Bio). The viral vector stock (adjusted to
(Takara Bio) containing 4.05 μg of minigenome plasmid 108–109 copies/mL) was kept at −80 °C in single-use
cocktail. The transfection mixture was added to cells in a aliquots until use.
sterile microcentrifuge tube and mixed gently. Immedi-
ately thereafter, 2 × 104 cells in a 25-μL volume were Effect of VP35 transgene on the innate immune response
dispensed into the individual wells of a 96-well cell culture of hepatic cells
cluster (Corning). Complete medium (100 μL) containing HepG2 cells (3 × 105 per well) in a 12-well cell culture
25 µg/mL of R9-HuscFvs or antibody storage buffer was cluster (Corning) were transduced with lentivector car-
added to the transfected cells and incubated at 37 °C in a rying the VP35 gene or an empty cassette at an MOI of 1.0
5% CO2 incubator for 36 h. The Gaussia luciferase (Gluc) by spinoculation at 30 °C for 1 h. The cells were washed
activities were measured using a Pierce Gaussia Lucifer- with DPBS, replenished with 1 mL fresh complete med-
ase Glow Assay Kit (Thermo Fisher Scientific) according ium, and then incubated for 12 or 36 h. Control cells were
to manufacturer’s instructions. Briefly, the culture med- replenished with fresh complete medium or medium
ium was collected, and the cells were added to 100 µL of containing 1 μg/mL poly(I:C)-HMW/LyoVec complexes
1× lysis buffer with continuous shaking (400 rpm) at (InvivoGen, San Diego, CA, USA). After the indicated
room temperature for 30 min. To obtain total luciferase incubation times, the culture medium was removed from
activity expressed by the minigenome system, each cell each well; the cells were lysed with 0.4 mL of TRIzol®
lysate (10 µL) and cell culture medium (equal volume) Reagent (Ambion, Thermo Fisher Scientific). Total RNA
were mixed and added to the appropriate well of a was extracted from the TRizol aqueous phase using the
96-well white polystyrene microplate (Corning). Total RNA Mini Kit (Geneaid Biotech). The RNA con-
A bioluminescent reaction was generated by adding 50 μL centration was measured (NanoDrop 2000 Spectro-
of co-elenterazine solution to individual wells and photometer, Thermo Fisher Scientific) and kept at −80 °C
kept at room temperature for 5 min. The luminescent until use.
Seesuay et al. Emerging Microbes & Infections (2018)7:41 Page 14 of 15

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Acknowledgements
18. Luthra, P., Jordan, D. S., Leung, D. W., Amarasinghe, G. K. & Basler, C. F. Ebola
This study was supported by a grant from the Faculty of Medicine Siriraj
virus VP35 interaction with dynein LC8 regulates viral RNA synthesis. J. Virol.
Hospital, Mahidol University, Bangkok, Thailand (R015834001), and an NSTDA
89, 5148–5153 (2015).
Chair Professor Grant funded by the Crown Property Bureau of Thailand
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(P-1450624). N.S.R. was supported by a TRF-RSA scholarship (RSA5780025).
required for its viral polymerase cofactor function. J. Virol. 84, 10581–10591
(2010).
Authors’ contributions 20. Leung, D. W. et al. Structural basis for dsRNA recognition and interferon
W.C.C. conceived the project. W.C.C., W.S.S., N.S.R. and Y.S.K. designed the antagonism by Ebola VP35. Nat. Struct. Mol. Biol. 17, 165–172 (2010).
experiments. W.S.S., S.J.V., and N.S.L. performed the experiments. W.C.C. and 21. Clark, D. V., Jahrling, P. B. & Lawler, J. V. Clinical management of Filovirus-
WSS interpreted the data, prepared the figures, and wrote the manuscript. infected patients. Viruses 4, 1668–1686 (2012).
22. Geisbert, T. W. et al. Treatment of Ebola virus infection with a recombinant
inhibitor of factor VIIa/tissue factor: a study in rhesus monkeys. Lancet 362,
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