Structural and functional characterization of Reston Ebola virus VP35 interferon inhibitory domain.
ABSTRACT Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-A crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 A, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for sequence variability, coupled with the multiple critical roles played by Ebola virus VP35 proteins, highlight the viability of VP35 as a potential target for therapeutic development.
- SourceAvailable from: Yiming Bao[Show abstract] [Hide abstract]
ABSTRACT: Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information's (NCBI's) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [ ( )/ / / / - ], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences.Viruses. 01/2014; 6(9):3663-82.
- [Show abstract] [Hide abstract]
ABSTRACT: Ebolavirus (EBOV) causes severe hemorrhagic fever with a mortality rate of up to 90%. EBOV is a member of the order Mononegavirales and, like other viruses in this taxonomic group, contains a negative-sense single-stranded (ss) RNA. The EBOV ssRNA encodes seven distinct proteins. One of them, the nucleoprotein (NP), is the most abundant viral protein in the infected cell and within the viral nucleocapsid. Like other EBOV proteins, NP is multifunctional. It is tightly associated with the viral genome and is essential for viral transcription, RNA replication, genome packaging and nucleocapsid assembly prior to membrane encapsulation. NP is unusual among the Mononegavirales in that it contains two distinct regions, or putative domains, the C-terminal of which shows no homology to any known proteins and is purported to be a hub for protein–protein interactions within the nucleocapsid. The atomic structure of NP remains unknown. Here, the boundaries of the N- and C-terminal domains of NP from Zaire EBOV are defined, it is shown that they can be expressed as highly stable recombinant proteins in Escherichia coli, and the atomic structure of the C-terminal domain (residues 641–739) derived from analysis of two distinct crystal forms at 1.98 and 1.75 Å resolution is described. The structure reveals a novel tertiary fold that is distantly reminiscent of the β-grasp architecture.Acta Crystallographica Section D Biological Crystallography 09/2014; 70(9). · 7.23 Impact Factor
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ABSTRACT: Ebolaviruses cause a severe hemorrhagic fever syndrome that is rapidly fatal to humans and nonhuman primates. Ebola protein interactions with host cellular proteins disrupt type I and type II interferon responses, RNAi antiviral responses, antigen presentation, T-cell-dependent B cell responses, humoral antibodies, and cell-mediated immunity. This multifaceted approach to evasion and suppression of innate and adaptive immune responses in their target hosts leads to the severe immune dysregulation and "cytokine storm" that is characteristic of fatal ebolavirus infection. Here, we highlight some of the processes by which Ebola interacts with its mammalian hosts to evade antiviral defenses.Cell. 10/2014; 159(3):477-486.
Structural and Functional Characterization of Reston
Ebola Virus VP35 Interferon Inhibitory Domain
Daisy W. Leung1, Reed S. Shabman2†, Mina Farahbakhsh1,3†,
Kathleen C. Prins2, Dominika M. Borek4, Tianjiao Wang1,
Elke Mühlberger5,6, Christopher F. Basler2and Gaya K. Amarasinghe1⁎
1Department of Biochemistry,
Biophysics, and Molecular
Biology, Iowa State University,
Ames, IA 50011, USA
2Department of Microbiology,
Mount Sinai School of
Medicine, New York,
NY 10029, USA
Program, Iowa State University,
Ames, IA 50011, USA
4Department of Biochemistry,
UT Southwestern Medical
Center at Dallas, 5323 Harry
Hines Boulevard, Dallas,
TX 75390, USA
5Department of Microbiology,
Boston University School of
Medicine, Boston, MA, USA
6National Emerging Infectious
Diseases Laboratories Institute,
72 East Concord Street, Boston,
MA 02118, USA
Received 22 December 2009;
received in revised form
10 April 2010;
accepted 12 April 2010
24 April 2010
Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and
nonhuman primates. Among the filoviruses characterized thus far, Reston
Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to
humans despite the fact that REBOV can cause lethal disease in nonhuman
primates. Previous studies also suggest that REBOV is less effective at
inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or
Marburg virus. Virally encoded VP35 protein is critical for immune
suppression, but an understanding of the relative contributions of VP35
proteins from REBOV and other filoviruses is currently lacking. In order to
address this question, we characterized the REBOV VP35 interferon
inhibitory domain (IID) using structural, biochemical, and virological
studies. These studies reveal differences in double-stranded RNA binding
and interferon inhibition between the two species. These observed
differences are likely due to increased stability and loss of flexibility in
REBOV VP35 IID, as demonstrated by thermal shift stability assays.
Consistent with thisfinding, the 1.71-Å crystal structure of REBOV VP35 IID
reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall
backbone r.m.s.d. of 0.64 Å, but contains an additional helical element at the
linker between the two subdomains of VP35 IID. Mutations near the linker,
including swapping sequences between REBOV and ZEBOV, reveal that the
linker sequence has limited tolerance for variability. Together with the
previously solved ligand-free and double-stranded-RNA-bound forms of
ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID
reinforce the importance of VP35 in immune suppression. Functional
differences observed between REBOV and ZEBOV VP35 proteins may
contribute to observed differences in pathogenicity, but these are unlikely to
be the major determinant. However, the high level of similarity in structure
and the low tolerance for sequence variability, coupled with the multiple
critical roles played by Ebola virus VP35 proteins, highlight the viability of
VP35 as a potential target for therapeutic development.
© 2010 Elsevier Ltd. All rights reserved.
Edited by M. F. SummersKeywords: viral protein structure; Ebola VP35; immune evasion
Filoviruses, including Ebola virus (EBOV) and
Marburg virus (MARV), are enveloped negative-
sense RNA viruses associated with zoonotic infec-
tions in humans.1–4Only a single species of MARV
(Lake Victoria) has been identified thus far; how-
ever, currently, five known species of EBOV have
been reported: Zaire, Sudan, Ivory Coast, Bundibu-
gyo, and Reston.1–4The ability to cause lethal
disease in humans and nonhuman primates—
*Corresponding author. E-mail address:
† R.S.S. and M.F. contributed equally to this work.
Abbreviations used: REBOV, Reston Ebola virus;
ZEBOV, Zaire Ebola virus; IID, interferon inhibitory
domain; EBOV, Ebola virus; MARV, Marburg virus; IFN,
interferon; dsRNA, double-stranded RNA; PDB, Protein
doi:10.1016/j.jmb.2010.04.022 J. Mol. Biol. (2010) 399, 347– 357
Available online at www.sciencedirect.com
0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.
primarily the ability of Zaire Ebola virus (ZEBOV)—
has resulted in outbreaks, with human fatality rates
near 90%.4In contrast, Reston Ebola virus (REBOV)
is unique among filoviruses in that is has never
caused lethal disease in a human. Only a few
documented cases of human infection with REBOV
have been documented. Moreover, these infections
were not associated with illness or death, suggest-
ing that REBOV may be attenuated and likely
avirulent in humans. The recent discovery of the
presence of REBOV in samples collected from
domestic swine in the Philippines5–7suggests that
REBOV has a greater potential to enter the human
food chain than previously recognized. Comparison
of global cellular transcriptional responses to
ZEBOV, REBOV, and MARV suggests that
REBOV is less efficient than other EBOVs in
inhibiting host cell interferon (IFN) responses,8
although interpretation of these cellular responses
is complicated by the fact that REBOV replicates
more slowly in cell culture than ZEBOV.9However,
the molecular basis for species variation among
EBOVs is currently lacking. Therefore, studies
directed at examining structural and functional
comparisons may shed light on this highly lethal
family of negative-stranded RNA viruses and
suggest novel antiviral strategies against EBOVs.
Among the seven structural proteins encoded by
the negative single-stranded RNA genome of EBOV,
VP24 and VP35 display immune antagonism. In
particular, VP24 functions through inhibition of the
JAK/STAT pathway, while VP35 functions to
inhibit IFN-α/β signaling.10–12Additionally, VP35
is critical for RNA-dependent protein kinase (PKR)
inhibition,13,14viral replication,15,16and suppression
of RNA silencing.17As shown in Fig. 1a, VP35
contains an N-terminal coiled-coil domain that is
important for oligomerization22,23and a C-terminal
interferon inhibitory domain (IID) that binds dou-
ble-stranded RNA (dsRNA).15,24–26The coiled-coil
domain is required for several VP35-mediated
functions, including viral replication and nucleo-
capsid formation,22,23,27–30as well as immune
suppression, as EBOV VP35 IID is unable to
suppress IFN induction to the same level as the
full-length protein.23,24However, we and others
have shown that VP35 binds dsRNA through the C-
terminal IID, which alone is sufficient for IFN
inhibition.15,24–26Our recent crystal structure of
ZEBOV VP35 IID revealed a cluster of conserved
basic residues that are important for dsRNA
binding.18From this first structure of a VP35 IID
and associated solution NMR experiments, we
demonstrated that VP35 IID consists of two sub-
domains that form a single independently folded
unit. The α-helical subdomain forms a four-helix
bundle, whereas the β-sheet subdomain forms a
four-stranded β-sheet with a short helix. Examina-
tion of the structure identified two distinguishable
charged patches: first basic patch and central basic
patch. Interestingly, this region was previously
identified as a critical element for IFN inhibition,
and many residues within these charged patches
display a high level of sequence similarity among
members of the filoviral family (Fig. 1a).25Mutation
of R312 in ZEBOV VP35 IID (corresponding to R301
displays attenuated IFN inhibition. Several studies
have extensively characterized mutations from the
ZEBOV central basic patch and demonstrated the
functional importance of residues within this
patch.15,16,25,26Consistent with these reports, recent
ZEBOV viruses show that growth rates of R312A
and K319A/R322A mutation were greatly attenuat-
ed compared to viruses with wild-type VP35.34
Moreover, the K319A/R322A mutant virus was
avirulent in a lethal guinea pig model.33A high
level of sequence similarity, including the presence
of identical residues at key sites, within the central
basic patch suggests that VP35 proteins in all EBOVs
may function in a similar manner.9,24
Previous studies have shown that nearly all
components from the REBOV and ZEBOV nucle-
ocapsid (viral polymerase L, NP, VP30, and VP35)
are interchangeable in a minigenome assay that
mimics viral replication.9Only the combination of
ZEBOV VP35 and REBOV viral polymerase L
displayed diminished replication function.9There-
fore, despite significant differences in their viru-
lence in humans, limited information on functional
comparisons between REBOV and ZEBOV gene
products is currently available.35Our studies des-
cribed below address these questions in the
context of critical VP35 proteins through combined
biochemical, structural, and virological studies by
comparing the VP35 IID proteins from REBOV
and ZEBOV. Biochemical and cell biological
studies show that VP35 IID proteins from
REBOV and ZEBOV function similarly but display
different structural stabilities. Consistent with
these findings, our crystal structure of REBOV
VP35 IID, solved to 1.71 Å resolution, reveals
similar folds, with a single difference at the
interface of the α-helical and β-sheet subdomains.
While this difference is likely responsible for the
higher structural stability observed for REBOV
VP35 IID, structural and functional studies show
that only sequence swapping between REBOV and
ZEBOV near the linker is tolerated, as additional
mutations result in loss of structure and function.
While the observed differences in structure,
dsRNA binding, and IFN inhibition likely result
from loss of conformational plasticity, our results
suggest that VP35 proteins from REBOV and
ZEBOV probably function in a similar manner.
Therefore, while VP35 may contribute to observed
pathogenic differences, it is unlikely to be the main
determinant. Given the structural and functional
similarities, coupled with a low tolerance for
mutations at the subdomain interface, our results
suggest that VP35 IID is a viable candidate for
antiviral development, and the availability of a
high-resolution crystal structure from our study
should facilitate these efforts.
Reston Ebola VP35 Interferon Inhibitory Domain
Fig. 1 (legend on next page)
Reston Ebola VP35 Interferon Inhibitory Domain
Sequence analysis of REBOV VP35 IID
One of the hallmarks of EBOV infections is the
inhibition of host immune responses that impair
innate and adaptive immunity. In vivo studies
comparing REBOV and ZEBOV infections show
marked differences in immune suppression capac-
ities. Although REBOV and ZEBOV show very
different disease outcomes in humans, examination
of the corresponding VP35 IID sequences reveals
that only 14 residues are different between the two
proteins within the IID region (Fig. 1a). Since VP35
is an important EBOV-encoded virulence factor
responsible for immune suppression and evasion,
we examined the functional properties of REBOV
VP35 IID. Protein samples were prepared by
modifying previously published protocols for
ZEBOV VP35 IID.36Given the high level of
sequence homology, we were able to identify
constructs that were well behaved based on
hydrodynamic characterization, including size-ex-
clusion chromatography and dynamic light scatter-
ing (data not shown). Based on these studies, we
identified a REBOV VP35 IID construct containing
residues 204–329, corresponding to ZEBOV VP35
IID residues 215–340.
REBOV VP35 IID displays slightly diminished
dsRNA binding and IFN-β inhibition
We recently determined the dsRNA-bound struc-
ture of ZEBOV VP35 IID, which revealed that the
VP35 IID protein uses multiple strategies to engage
in protein–protein and protein–RNA interactions in
order to facilitate viral infection and replication.26
We also demonstrated that dsRNA binding is
important for VP35-mediated antagonism of host
immune responses that originate from cytosolic
RIG-I like receptors. As an initial test of REBOV
VP35 characterization and comparison, we assessed
the ability of REBOV VP35 IID to bind 8 bp of
dsRNA by isothermal titration calorimetry (Fig. 1b).
Both VP35 IIDs bound 8 bp of dsRNA with a
stoichiometry of 1:4 (dsRNA/VP35 IID ratio).
However, comparison of binding constants revealed
that REBOV VP35 IID displayed approximately
2-fold to 3-fold lower dsRNA binding for the same
dsRNA when compared to ZEBOV VP35 IID.
Moreover, the relative heats released upon binding
were ∼25% lower for REBOV VP35 IID titration
than for ZEBOV VP35 IID titration, indicating that
dsRNA interactions with REBOV and ZEBOV VP35
IIDs may not be identical despite the highly similar
primary sequence, particularly near the central basic
We next tested the inhibition of Sendai-virus-
mediated activation of the IFN-β promoter by
REBOV and ZEBOV VP35 proteins. As shown in
Fig. 1c, both REBOV and ZEBOV VP35 proteins can
inhibit IFN-β promoter activation, but REBOV VP35
inhibits this activation to levels lower than those
observed for ZEBOV VP35. Examination of
corresponding Western blot analyses indicates that
the expression levels of REBOV and ZEBOV VP35
proteins are similar, suggesting that the differences
Fig. 1. REBOV VP35 is not as potent an IFN antagonist as ZEBOV VP35. (a) Domain organization of REBOV VP35
based on previous biochemical and structural studies.18VP35 sequences for the IID region from REBOV (REBOV residues
204–329; accession number AAN04449.1) and ZEBOV (ZEBOV residues 215–340; accession number AF086833) were
aligned using CLUSTAL W, version 1.81.19Identical residues (black) and similar residues (gray) are highlighted. (b)
Representative isothermal titration calorimetry data showing raw heat released (top) and integrated heats per injection
(bottom) for REBOV VP35 IID (left) and ZEBOV VP35 IID (right) binding to 8 bp of dsRNA corresponding to the
palindromic sequence 5′-CGCAUGCG-3′. A vector containing the coding region for REBOV and ZEBOV18was used as
IID constructs were grown and overexpressed as maltose-binding protein fusion proteins in BL21(DE3) cells (Novagen) in
either LB or minimal medium. Protein expression in E. coli was induced at an optical density at 600 nm of 0.8 with 0.5 mM
IPTG and grown overnight at 18 °C.Cells were harvested, resuspended in lysis buffer [25 mMNa-phosphate (pH 7.0), 1 M
NaCl, 20 mM imidazole, and 5 mM β-mercaptoethanol], lysed using an EmulsiFlex-C5 homogenizer (Avestin), and
clarified by centrifugation at 30,000g at 4 °C for 30 min. The supernatant was purified by affinity and ion-exchange
chromatographies. Fusion tags were removed prior to final purification by size-exclusion chromatography. The purity of
the samples was assessed by SDS-PAGE. The average dissociation constants are 1200 nM and 500 nM for REBOV and
(EV), increasing amounts of hemagglutinin-tagged VP35 expression plasmid, IFN-β promoter firefly luciferase reporter
plasmid,andaconstitutivelyexpressedRenillaluciferasereporter plasmid.At18hposttransfection, thecellswereinfected
with Sendai virus (SeV). At 24 h postinfection, the cells were harvested and lysates were prepared. Reporter gene assays
were performed using a dual-luciferase reporter assay (Promega). Firefly luciferase activity was normalized to Renilla
luciferase activity, and the results were expressed as percent induction relative to positive control. Error bars represent the
ThermoFluor assays of REBOV and ZEBOV VP35 IID proteins reveals differences in Tmvalues for mutant proteins. The
melting temperature (Tm) of VP35 IID variants was measured by following established ThermoFluor assay protocols.20,21
λex=470–505 nm and λem=540–700 nm. Data were acquired using a temperature gradient from 30 °C to 90 °C in
increments of 0.5 °C. Samples contained 20 μM VP35 IID protein, 1× SYPRO Orange (Invitrogen), 10 mM Hepes (pH 7),
150 mM NaCl, and 2 mM tris(2-carboxyethyl)phosphine. Fluorescence increases due to the association of SYPRO Orange
with exposed hydrophobic residues as theprotein unfolds with increasing temperature. Fluorescence data were analyzed,
and the derivative of the curve represents the melting temperature.
Reston Ebola VP35 Interferon Inhibitory Domain
observed in dsRNA binding and IFN inhibition may
result from the reduced ability of REBOV VP35 to
sequester dsRNA, leading to higher levels of IFN
REBOV VP35 IID is more stable than ZEBOV
To further examine the physical basis for the
difference in REBOV and ZEBOV VP35 functions,
we conducted ThermoFluor assays20,21with REBOV
and ZEBOV VP35 IID proteins. Using a temperature
gradient from 30 °C to 90 °C, we monitored the
changes in fluorescence upon SYPRO Orange bind-
derivative of fluorescence change, shown in Fig. 1d,
revealed that the average melting temperature (Tm)
for REBOV VP35 IID is 63.4±0.3 °C, while ZEBOV
VP35 IID displayed a Tmvalue of 57.0±0.1 °C. Given
that the reported values are an average of at least 15
independent measurements, our results suggest that
REBOV VP35 IID is thermally more stable than
ZEBOV VP35 IID by at least 6 °C. However, the
source of the difference in thermal stability is not
immediately evident, as these two proteins only
differ at 14 residues in the region encoded for VP35
IID (Fig. 1a).
Crystal structure of REBOV VP35 IID
In an effort to structurally characterize the REBOV
VP35 IID protein, we determined the crystal struc-
ture of REBOV VP35 IID containing residues 204–
329. Initial crystallization conditions were obtained
from a commercial crystal screen (Hampton Re-
search), and these conditions were subsequently
optimized to obtain single crystals that were suitable
data collection statistics are shown in Table 1. Data
reduction and indexing revealed that the REBOV
VP35 IID crystals belong to space group P41, with
unit cell dimensions of a=51.55 Å, b=55.55 Å,
c=50.87 Å, α=90°, β=90°, and γ=90°. The asym-
metric unit contained one molecule of REBOV VP35
IID, and the Matthews coefficient was calculated at
2.55 with a solvent content of 52%.
The structure of REBOV VP35 IID was determined
by molecular replacement using our previously
determined structure of ZEBOV VP35 IID as a
search model [Protein Data Bank (PDB) code
3FKE]18(Fig. 2a). REBOV VP35 IID shows remark-
able similarity to ZEBOV VP35 IID (backbone r.m.s.
d. value of 0.64 Å) (Fig. 2b). Overall, the REBOV
VP35 IID structure retained the α-helical subdomain
and the β-sheet subdomain present in the ZEBOV
VP35 IID structure, which aligned with r.m.s.d.
values of 0.3 Å and 0.5 Å for the α-helical and β-
sheet subdomains, respectively.18These results
suggest that other filoviral VP35 IID proteins are
likely to contain a similar fold and, more impor-
tantly, that the VP35 IID fold is substantially
different from the canonical double stranded RNA
binding domain fold of αβββα. Comparisons using
the DALI server21reveal that the α-helical subdo-
main has a topology similar to those of many
functionally unrelated proteins (DALI z-score N2).
However, like the ZEBOV IID structure, we were
unable to identify other known structurally or
functionally related proteins, confirming the unique-
ness of the REBOV VP35 IID fold.
Previous studies have shown that mutation of
individual residues within the central basic patch
results in loss of dsRNA binding and concomitant
loss of IFN inhibition. Initial solution NMR analysis
revealed that mutant VP35 proteins retain their
overall fold. In contrast, recent crystal structures
Table 1. Data collection and refinement statistics
a, b, c (Å)
α, β, γ (°)
Multiplicity of observation
51.55, 55.55, 50.87
90, 90, 90
Number of reflections
Number of atoms
Bond lengths (Å)
Bond angles (°)
Each data set was collected using one crystal.
Crystals were grown at 25 °C by hanging-drop vapor-diffusion
method with 20 mg/mL protein solutions diluted with 100 mM
sodium acetate (pH 3.0), 14% Tacsimate, and 16% (wt/vol)
polyethylene glycol 3350. Crystals grew within 2–4 days to
dimensions of 10 μm×20 μm×200 μm. Crystals were soaked in a
reservoir solution containing a final glycerol concentration of 25%
(wt/vol) and frozen in nitrogen stream. Data were collected from
a single crystal at SBC beamline 19ID of the Advanced Photon
Source at 100 K. A complete data set of 180°, with 1° oscillations,
was collected at a wavelength of 0.979 Å. Data were processed
using HKL2000,37and intensities were converted into structure
factors using the CCP4 program38TRUNCATE. Phases were
determined using molecular replacement with the native wild-
type structure of ZEBOV VP35 IID (PDB code 3FKE36) using
either MOLREP39or PHASER.40Refinement was performed with
REFMAC5 interspaced with manual rebuilding with Coot.41
Water molecules were initially added using ARP/wARP42,43if a
peak greater than 3.0σ was present on Fourier maps with
coefficients (Fobs−Fcalc)eiαcalcand later manually inspected with
Coot.44The model was further refined using REFMAC5 with
MLKF residual function, bulk solvent scaling, and individual
isotropic B-factors. TLS parameters were determined with
TLMSD.45The quality of the refined model was validated with
MOLPROBITY.46The final refinement statistics for all structures
are shown above.
aValues in parentheses are for the highest-resolution shell.
Reston Ebola VP35 Interferon Inhibitory Domain
Fig. 2. Crystal structure of REBOV IID. (a) Ribbon representation of the 1.71-Å REBOV VP35 IID (PDB code 3L2A)
contains six α-helices, including the same four α-helices in the α-helical subdomain and four β-strands in the β-sheet
subdomain of ZEBOV VP35 IID.18The linker between the two subdomains of REBOV VP35 IID contains a short α-helix
(α4b). (b) Alignment between REBOV (blue) and ZEBOV (green; PDB code 3FKE) VP35 IID structures. The α4b helix
observed in the REBOV VP35 IID structure is highlighted by a box. (c) Comparison of electrostatic surface representations
in REBOV VP35 IID (left) and ZEBOV VP35 IID (right) (scale of −10 kT/e to +10 kT/e) highlighting the central basic patch
residues. Structure figures were prepared using PyMOL.47
Reston Ebola VP35 Interferon Inhibitory Domain
Fig. 3. Structural comparison of REBOV and ZEBOV VP35 IID proteins. (a) Structural alignment of the α-helical
subdomains of REBOV (blue; PDB code 3L2A) and ZEBOV (green; PDB code 3FKE) shows the structural and orientation
differences between the β-sheet subdomains relative to the α-helical subdomains. (b) Same alignment as in (a) rotated
180° on the y-axis (c). Comparison of critical residues in VP35 IID that form the dsRNA end-cap structure, which is
important for dsRNA blunt-end recognition. Evaluation of interactions between the REBOV VP35 IID residue K328 side-
chain and the oxygen atoms (OXT1 and OXT2) of terminal residue I329 reveals important differences between REBOV
VP35 IID and ZEBOV VP35 IID that may affect the properties of the dsRNA end cap. REBOV VP35 IID residue K328 is
located ∼5 Å away from I329 OXT1, whereas ZEBOV VP35 IID residue K339 Nɛis within 4 Å of OXT1 and OXT2 of the
terminal I340 residue (two black dotted lines for ZEBOV VP35 IID versus only one black dotted line for REBOV VP35 IID).
Structure figures were prepared using PyMOL.47
Reston Ebola VP35 Interferon Inhibitory Domain
of three ZEBOV VP35 IID central basic patch
mutants—R312A (PDB code 3L27), K319A/R322A
(PDB code 3L29), and K339A (PDB code 3L28)—
showed that substantial changes to the electrostatic
surface are evident as a result of mutating charged
residues. However, in these mutant structures, only
subtle changes to the overall protein conformation
are observed. Comparison of the electrostatic
surface of the REBOV and ZEBOV IID structures,
shown in Fig. 2c, confirms our predictions that
residues previously known to be important for
dsRNA binding in ZEBOV VP35 IID form a
similarly charged surface in REBOV VP35 IID.
Structural comparison of Reston versus Zaire
Despite the remarkable similarities at the global
structure level, we observe one notable difference
between the REBOV VP35 IID structure and the
ZEBOV VP35 IID structure at the subdomain
interface: In the REBOV VP35 IID structure, the
linker between the α-helical subdomain and the
β-sheet subdomain forms an additional short five-
residue α-helix in the REBOV VP35 IID structure
(Fig. 2a, α4b). This helix is absent in the ZEBOV
VP35 IID structure and is replaced by a loop. We
have previously demonstrated that the subdomain
interface, formed by highly conserved residues, is
critical for the folding and stability of ZEBOV
VP35 IID.18It is likely then that the presence of the
α4b helix provides additional stabilization of the
REBOV VP35 IID structure near the subdomain
interface and contributes to the increased thermal
stability observed in ThermoFluor assays. As a
consequence of this helix, the relative orientation
between the α-helical subdomain and the β-sheet
subdomain is rotated by 1.0–4.0° when the
structures of the dsRNA/VP35 complex (PDB
codes 3L25 and 3L26), as well mutant structures,
are compared by aligning the α-helical subdomain
(or the β-sheet subdomain) (Fig. 3a and b).
Moreover, we observed that ZEBOV VP35 IID
was able to bind both dsRNA backbone and the
blunt dsRNA ends forming an “end cap,” and this
interaction also depends on the ability of the
penultimate lysine residue (Lys328 in REBOV or
Lys339 in ZEBOV) to coordinate the terminal
carboxylate group. As shown in Fig. 3c, charge
neutralization of the terminal carboxylate is less
efficient in REBOV VP35 IID due to the relative
orientation (Fig. 3c). Thus, the helical nature of the
linker in REBOV VP35 IID likely limits the
conformational flexibility required for multiple
interactions between VP35 IID and dsRNA, such
as those observed in the ZEBOV IID/dsRNA
In order to further assess the differences between
REBOV and ZEBOV VP35 IID linker regions and to
identify structural characteristics that may be func-
tionally relevant, we generated a series of mutations
for REBOV and ZEBOV VP35 IIDs near the linker
to a flexible linker(Table 2, linker1), swapping of the
linker region between ZEBOV and REBOV (Table 2,
linker 2), and insertion of a flexible linker (Table 2,
linker 3). As shown in Fig. 4, swapping the linker
sequence between REBOV and ZEBOV IIDs did not
affect the overall thermal stability (Fig. 4a), structure
(Fig. 4b, left), or function (Fig. 4c). However,
substitution of the linker sequences (linker 1) or
Ser residues resulted in highly destabilized VP35 IID
when expressed in Escherichia coli under conditions
we were able to purify the linker 3 proteins, they
show diminished stability [Fig. 4a, compare black
(wild type) with blue (linker 3)] and a large number
(Fig. 4b, right). Consistent with these observations,
expression of both linker 1 and linker 3 in the context
diminished IFN inhibitory activity in the Sendai
virus infection assay (Fig. 4c). In contrast, even at
plasmid concentrations lower than those used in
Fig. 4d, linker 2 constructs show wild-type behavior.
Together, the results described above highlight
the importance of the linker sequence connecting
the two subdomains in VP35 IID to activity and
demonstrate that EBOV VP35 sequences have a very
low sequence tolerance for variability at the sub-
The critical role played by VP35 in immune
suppression and its importance to viral replication
and EBOV pathogenesis are well established. We
have recently provided additional experimental
evidence that links the ability of ZEBOV VP35 to
binddsRNA toitsabilityto antagonizehostimmune
responses.26Consistent with these observations,
recent reports show that antiviral pathways initiated
by cytosolic RIG-I-like helicases are important for
EBOV infections,50–56as preactivation of RIG-I alone
can reduce EBOV infections up to ∼1000-fold.57
Table 2. Mutant ZEBOV and REBOV linker construct sequences
REBOV linker 1
REBOV linker 2
REBOV linker 3
ZEBOV linker 1
ZEBOV linker 2
ZEBOV linker 3
Reston Ebola VP35 Interferon Inhibitory Domain
Therefore, inhibition of RIG-I-like receptor function,
which detects viral nucleic acids such as dsRNA
during infection and replication, is likely important
for EBOV propagation. Comparison of EBOV VP35
IID structures with those of other viruses suggests
that VP35 is significantly different from other viral
and cellular proteins, but these properties are
highly conserved among filoviral VP35 proteins.
Therefore, the availability of multiple structures of
Ebola VP35 IID proteins, including the REBOV
VP35 IID structure described here, coupled with
the high level of sequence similarity among VP35
IIDs from different filoviruses and the low toler-
ance for sequence variation, provides unique
information that will facilitate new opportunities
for therapeutic and diagnostic drug design.
Coordinates and structure factors for the REBOV
VP35 IID protein have been deposited in the PDB
under PDB code 3L2A. (While this manuscript was in
preparation, a structure of REBOV VP35 IID, free and
bound to 18 bp of dsRNA, appeared online. Based on
published images, the two structures appear
similar.58However, we were unable to compare the
structures, as the coordinates for these structures had
not been released at the time of initial submission.)
Fig. 4. REBOV VP35 IID is more stable than ZEBOV VP35 IID. (a) The first derivative of normalized fluorescence
emission data for the ThermoFluor assays of REBOV VP35 IID proteins reveals differences in Tmvalues for REBOV
proteins with different linkers between the α-helical subdomain and the β-sheet subdomain. A vector containing the
coding region for REBOV was used as template to generate PCR products containing alanine mutations at V279, P280,
and I283 (REBOV VP35 linker 2) or a 10-residue stretch of glycine and serine residues between P281 and P282
(REBOV VP35 linker 3). Experiments were carried out as described in the legend to Fig. 1d. The Tmvalues are 62.9 °C,
62.4 °C, and 53.4 °C for wild-type REBOV VP35 IID (black), REBOV VP35 IID linker 2 (red), and REBOV VP35 IID
linker 3 (blue), respectively. (b)1H/15N heteronuclear single quantum coherence spectra of REBOV VP35 IID wild-
type (black) overlaid with REBOV VP35 linker 2 (red; left) or REBOV VP35 linker 3 (blue; right). Samples were
generated using previously described methods.18,26,36Data were acquired on a Bruker Avance II spectrometer
operating at 700.13 MHz for1H at 25 °C. Data were processed with NMRPipe/NMRDraw48and NMRView.49(c and
d) IFN inhibition function of REBOV and ZEBOV VP35 constructs was tested as described above using varying VP35
vector concentrations, as indicated in the figure. Representative Western blot analyses and antibodies are indicated
below each graph.
Reston Ebola VP35 Interferon Inhibitory Domain
We thank the ISU Biotechnology Facilities and
Dr. J. Hoy for providing access to instrumentation
and support. We also thank P. Ramanan, J.
Binning, L. Tantral, D. Peterson, and C. Brown
for laboratory assistance, and Drs. S. Ginell, N.
Duke, F. Rotella, M. Cuff, and J. Lazarz at APS
Sector 19. Use of Argonne National Laboratory
Structural Biology Center beamlines at the Ad-
vanced Photon Source was supported by the US
Department of Energy under contract number DE-
AC02-06CH11357. This work was supported, in
part, by National Institutes of Health grants
(1F32AI084324 to D.W.L., 1F32AI084453 to R.S.S.,
R01AI059536 to C.F.B., and R01AI081914 to G.K.A.),
by the German Research Foundation (SFB 535 to
Virgin(PI) to G.K.A.), and by the Roy J. Carver
Charitable Trust (09-3271 to G.K.A.).
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