Alteration of a Second Putative Fusion Peptide of Structural Glycoprotein E2 of Classical Swine Fever Virus Alters Virus Replication and Virulence in Swine

10  Download (0)

Full text

(1)

Alteration of a Second Putative Fusion Peptide of Structural

Glycoprotein E2 of Classical Swine Fever Virus Alters Virus

Replication and Virulence in Swine

L. G. Holinka,aE. Largo,bD. P. Gladue,aV. O’Donnell,a,cG. R. Risatti,cJ. L. Nieva,bM. V. Borcaa ARS, USDA, Plum Island Animal Disease Center, Greenport, New York, USAa

; Biophysics Unit (CSIC, UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, Spainb; Department of Pathobiology and Veterinary Science, CANR, University of Connecticut, Storrs, Connecticut, USAc

ABSTRACT

E2, the major envelope glycoprotein of classical swine fever virus (CSFV), is involved in several critical virus functions, including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on a Wimley-White interfacial hydrophobicity distribution predicted the involvement of a loop (residues 864 to 881) stabilized by a disulfide bond (869CKWGGNWTCV878, named FPII) in establishing interactions with the host cell membrane. This loop further contains an872GG873dipeptide, as well as two aromatic residues (871W and875W) accessible to solvent. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within FPII may affect replication of BICvin vitroand virus virulence in swine. Recombinant CSFVs containing mutations in different residues of FPII were constructed. A particular construct, harboring amino acid substitutions W871T, W875D, and V878T (FPII.2), demonstrated a significantly decreased ability to replicate in a swine cell line (SK6) and swine macrophage pri-mary cell cultures. Interestingly, mutated virus FPII.2 was completely attenuated in pigs. Also, animals infected with FPII.2 virus were protected against virulent challenge with Brescia virus at 21 days postvaccination. Supporting a role for the E2 the loop from residues 864 to 881 in membrane fusion, only synthetic peptides that were based on the native E2 functional sequence were competent for insertion into model membranes and perturbation of their integrity, and this functionality was lost in synthetic peptides harboring amino acid substitutions W871T, W875D, and V878T in FPII.2.

IMPORTANCE

This report describes the identification and characterization of a putative fusion peptide (FP) in the major structural protein E2 of classical swine fever virus (CSFV). The FP identification was performed by functional structural analysis of E2. We character-ized the functional significance of this FP by using artificial membranes. Replacement of critical amino acid residues within the FP radically alters how it interacts with the artificial membranes. When we introduced the same mutations into the viral se-quence, there was a reduction in replication in cell cultures, and when we infected domestic swine, the natural host of CSFV host, we observed that the virus was now completely attenuated in swine. In addition, the virus mutant that was attenuatedin vivo

efficiently protected pigs against wild-type virus. These results provide the proof of principle to support as a strategy for vaccine development the discovery and manipulation of FPs.

C

lassical swine fever (CSF) is a highly contagious disease of swine caused by CSF virus (CSFV), a small enveloped virus with a positive-sense, single-strand RNA genome (1). The approx-imately 12.5-kb CSFV genome contains a single open reading frame that encodes a polyprotein composed of 3,898 amino acids that ultimately yields up to 12 final cleavage products (NH2-Npro

-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) through co- and posttranslational processing of the polyprotein by cellular and viral proteases (2).

Structural components of the virion include the capsid (C) protein and glycoproteins: Erns, E1, and E2. Erns, a secreted pro-tein, is loosely associated with the viral envelope (3–5) and does not have a hydrophobic transmembrane anchor domain. Erns

does, however, possess a C-terminal charged amphipathic seg-ment that can mediate translocation of Ernsacross bilayer mem-branes (6). E1 and E2 are transmembrane proteins with an N-ter-minal ectodomain and a C-terN-ter-minal hydrophobic anchor (5). E2 is considered essential for CSFV replication, as virus mutants con-taining partial or complete deletions of the E2 gene are nonviable

(7). E2 has been implicated, along with Erns(8) and E1 (9), in viral

adsorption to host cells (10,11). Modifications introduced into this glycoprotein appear to have an important effect on CSFV virulence (12–16).

Using proteomic computational analysis, E2 has been charac-terized as a truncated class II fusion protein harboring an internal fusion peptide (IFP) that is located after secondary structural fold-ing at distal locations from the transmembrane anchor (17). We

Received2 August 2016 Accepted29 August 2016

Accepted manuscript posted online7 September 2016

CitationHolinka LG, Largo E, Gladue DP, O’Donnell V, Risatti GR, Nieva JL, Borca MV. 2016. Alteration of a second putative fusion peptide of structural glycoprotein E2 of classical swine fever virus alters virus replication and virulence in swine. J Virol 90:10299 –10308.doi:10.1128/JVI.01530-16.

Editor:S. Perlman, University of Iowa

Address correspondence to M. V. Borca, manuel.borca@ars.usda.gov. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

on November 7, 2019 by guest

http://jvi.asm.org/

(2)

have functionally characterized an FP similar to one that was orig-inally predicted to occur in hepatitis C virus (HCV) (17) located between amino acid residues 818 and 828 of CSFV E2. Reverse genetics utilizing a full-length infectious clone of the highly viru-lent strain Brescia (BICv) was used to demonstrate that specific amino acid substitutions resulted in replication-deficient viruses. In addition, we demonstrated a correlation between the func-tional effects induced by the amino acid substitutions and the capacity of synthetic FPs for insertion into membranes and breaching the permeability barrier (18).

We present here the identification and the functional charac-terization of a putative second fusion peptide (FPII) in the prox-imity of the previously described one (18). It is shown that FPII (869CKWGGNWTCV878) critically contributes to interactions with artificial membranes. Reverse genetics demonstrated that combined specific amino acid substitutions W871T, W875D, and V878T within the FPII region can affect virus replication in cell cultures and severely decrease virus virulence in swine. In addi-tion, studies performed with synthetic vesicles using synthetic peptides demonstrated that the region869CKWGGNWTCV878 ac-tually possesses FP activity and that amino acid substitutions W871T, W875D, and V878T severely affect that property.

MATERIALS AND METHODS

Viruses and cells.Swine kidney cells (SK6) (19), free of bovine viral diar-rhea virus (BVDV), were cultured in Dulbecco’s minimal essential me-dium (DMEM) (Gibco, Grand Island, NY) with 10% fetal calf serum (FCS) (Atlas Biologicals, Fort Collins, CO). CSFV Brescia strain was prop-agated in SK6 cells and was used for the construction of an infectious cDNA clone (12). Growth kinetics was assessed using primary swine mac-rophage cell cultures prepared as previously described (20). Titration of CSFV from clinical samples was performed using SK6 cells in 96-well plates (Costar, Cambridge, MA). After 4 days in culture, viral infectivity was assessed using an immunoperoxidase assay utilizing the CSFV mono-clonal antibody (MAb) WH303 (21) and the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Titers were calculated according to a method that has been previously described (22) and expressed as 50% tissue culture infective dose (TCID50) per milliliter. As performed, test sensitivity wasⱖ1.8 log10TCID50s/ml.

Construction of CSFV mutants.A full-length infectious clone (IC) of the virulent Brescia strain (pBIC) (12) was used as a template to obtain all cDNA IC constructs described in this report. Constructs containing mu-tations in the FP area were obtained using the QuikChange XL site-di-rected mutagenesis kit (Stratagene, San Diego, CA) per the manufactur-er’s instructions using full-length pBIC as the template and the primers described inTable 1. The product was then digested with Dpn1, leaving only the newly amplified plasmid, transformed into XL10-Gold ultracom-petent cells, and grown on terrific broth agar plates with ampicillin (Teknova, Hollister, CA). Positive colonies were selected for by sequence analysis of the E2 gene and grown for plasmid purification using a Maxiprep kit (Qiagen Sciences, Hilden, Germany). Each of the IC

con-structs was completely sequenced to verify that only site-directed mu-tagenesis-induced changes were present.

In vitrorescue of CSFV Brescia and FP mutants.Full-length genomic clones were linearized withSrfI and in vitrotranscribed using the T7 Megascript system (Ambion, Austin, TX) (12). RNA was precipitated with LiCl and transfected into SK6 cells by electroporation at 500 V, 720 ⍀, and 100 W with a BTX 630 electroporator (BTX, San Diego, CA). Cells were seeded in 12-well plates and incubated for 4 days at 37°C and 5% CO2. Virus was detected by immunoperoxidase staining as described above, and stocks of rescued viruses were stored at⫺70°C.

DNA sequencing and analysis.Full-length clones andin vitro-rescued viruses were completely sequenced with CSFV-specific primers by the dideoxynucleotide chain termination method (23). Viruses recovered from infected animals were sequenced in the region of the genome that contained the desired mutations. Sequencing reactions were prepared with a dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). Reaction products were sequenced on a Prism 3730xl auto-mated DNA sequencer (Applied Biosystems). Sequence data were assem-bled using Sequencher 4.7 software (Genes Codes Corporation, Ann Arbor, MI). The final DNA consensus sequence represented, on average, a 3- or 4-fold redundancy at each base position.

Animal infections.Animal experiments were performed under bio-safety level 3 conditions in the animal facilities at the Plum Island Animal Disease Center (PIADC) following a protocol approved by the Institu-tional Animal Care and Use Committee. Virulence of FP mutant viruses relative to BICv was initially assessed in 10- to 12-week-old, 40-lb com-mercial-breed pigs inoculated intranasally (i.n.) with 105TCID

50s of each virus. Pigs were randomly allocated into 7 groups of 4 animals each and were inoculated with an FP virus mutant or BICv. Clinical signs (anorexia, depression, purple skin discoloration, staggering gait, diarrhea, and cough) and changes in body temperature were recorded daily throughout the experiment (24). Blood was collected at the desired times postinfec-tion from the anterior vena cava into EDTA-containing tubes (Vacu-tainer) for quantification of viremia by virus titration as described above.

To assess the protective effect of FPII.2 virus, 105TCID

50s were inoc-ulated i.n., and at 3 and 21 days postinfection (dpi), FPII.2-infected ani-mals were (i.n.) challenged with 105TCID50s of highly virulent parental BICv. Clinical signs (as described above) and changes in body tempera-ture were recorded daily throughout the experiment.

Peptide-based assays.Peptides EDLFYCKWGGNWTCVKGE, EDLF YCKTGGNDTCTKGE, and EDLFYCKWGGDDTCVKGE, represent-ing, respectively, the CSFV E2 FPII (spFPII.wt) and its derived mu-tants FPII.2 (spFPII.2) and FPII.5 (spFPII.5), were commercially synthesized (Thermo Scientific, Waltham, MA). The purified peptides were dissolved in dimethyl sulfoxide (DMSO; spectroscopy grade) and their concentrations determined by the bicinchoninic acid microassay (Pierce, Rockford, IL). Small, diluted aliquots (typically 20␮l; 1 mg/ml) were stored frozen and were thawed only once upon use.N-(Lissamine rhodamine B sulfonyl) phosphatidylethanolamine (PE-Rho), 1-palmi-toyl-2-oleoylphosphatidylglycerol (POPG), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) were purchased from Avanti Polar Lipids (Birmingham, AL). The 8-aminonaphthalene-1,3,6-trisulfonic acid so-dium salt (ANTS) andp-xylenebis(pyridinium)bromide (DPX) were

ob-TABLE 1Nucleotide sequences of primers used for the production of FPII recombinant virusesa

Mutant name Forward primer sequence(s)

FPII.1 5=-ATTCTACTGTAAATGGGGGGGCAATGATACATGTACGAAAGGTGAACCAGTGACCTACACG-3=

FPII.2 5=-TGGAAAATGAAGATCTATTCTACTGTAAAACGGGGGGCAATGATACATGTACGAAAGGTGAA-3=

FPII.3 5=-CTACTGTAAATGGGGGGACAATTGGACATGTGTGA-3=

FPII.4 5=-ACTGTAAATGGGGGGGCGATTGGACATGTGTGAAA-3=

FPII.5 5=-CTATTCTACTGTAAATGGGGGGGCGATGATACATGTGTGAAAGGTGAACCAGTG-3=

FPII.6 5=-TGGAAAATGAAGATCTATTCTACAGTAAATGGGGGGGCAATT-3=and 5=-GGGGGGCAATTGGACAAGTGTGAAAGGTGAACC-3=

aOnly forward primers are presented; bold indicates nucleotide substitution.

on November 7, 2019 by guest

http://jvi.asm.org/

(3)

tained from Molecular Probes (Junction City, OR). Dodecylphosphocho-line was from Anatrace (Maumee, OH).

Circular dichroism (CD), lipid monolayer insertion, bilayer insertion, and vesicle permeability measurements were carried out as described in our previous paper (18). For the membrane association assays, giant unilamellar vesicles (GUVs) were prepared according to the electrofor-mation method. In brief, 4␮l of the lipid mixture stock (1 mM) in chlo-roform was spread on the platinum wires of the electchlo-roformation cham-ber. After solvent evaporation, the wires were immersed in 200 mM sucrose buffer, and electric pulses of 10 Hz were provided for 2 h, followed by 2-Hz pulses for 30 min. Confocal fluorescence microscopy of GUVs was performed on a commercial Nikon Declipse C1 inverted fluorescence microscope (Nikon Inc., Melville, NY) with a total internal reflection fluorescence 60⫻oil immersion objective. To perform these experiments, synthetic peptides spFPII.wt, spFPII.2, and spFPII.5 were tagged with the fluorophore 7-nitrobenz-2-oxa-l,3-diazole-4 (NBD) at the N terminus and membranes were labeled by inclusion of PE-Rho in the lipid compo-sition.

RESULTS

Prediction of internal fusion peptide II of CSFV E2 and design of mutations.Viral IFPs of class II/III glycoproteins form connect-ing loops in ␤-domains (25). These loops are enriched in Gly residues and often stabilized through DiS bridges. A more defini-tive feature is the configuration of a solvent-exposed apex con-taining hydrophobic-at-interface aromatic residues (prominently Trp) that enable insertion of the viral glycoprotein into the target cell membrane. Calculation of mean interfacial hydrophobicity using Wimley-White (WW) free-energy scales (26–28) disclosed CSFV E2 regions with the potential for favorably associating with the membrane interface (blue and red plots inFig. 1). The most prominent WW peak was found within a region encompassing residues 864 to 881 (FPII inFig. 1). The range and intensity of the WW peak were augmented after protonation of Asp and Glu res-idues (red plot), in accordance with the activation of the glycopro-tein at acidic pH (26). Moreover, the corresponding Kyte-Doolittle (KD) hydropathy plot was almost flat within this region and set at negative values close to 0 (black plot inFig. 1). Thus, WW hydrophobicity, i.e., the tendency for partitioning into mem-brane interfaces, specifically gathered within the E2 sequence from residues 864 to 881, which was not overall hydrophobic according to the KD scale. Subsequent location in the closely related BVDV1 E2 crystal structure (29,30) confirmed that the region from resi-dues 864 to 881 embodies a turn stabilized through DiS bridge formation and partially exposed to solvent (Fig. 1, top). Together, these observations support an internal FP role for the CSFV E2 sequence from residues 864 to 871 (designated henceforth FPII). We note, however, that in contrast to other internal fusion pep-tides, FPII is not found at the predicted target membrane-proxi-mal tip of the structure.

To test the FPII implication in the biological function of E2, we devised a series of PFII mutations (Fig. 2). Nonconservative changes to render FPII.1 (W875D-V878T) and FPII.2 (W871T-W875D-V878T) variants were first selected to abate hydrophobic-ity (i.e., reducing interfacial hydropathy index) on solvent-ex-posed E2FPII positions. Further changes, FPII.3 (G873D), FPII.4 (N874D) and FPII.5 (N874D-W875D), were executed to increase polarity at the loop tip. Finally, implication of the DiS bridge in the IFP function was assessed by inducing the FPII.6 (C869S-C877S) double substitution. All replacement residues were selected to minimize the impact on global stability of the protein, based on

the BVDV E2 crystal structure and using the Prediction of Protein Mutant Stability Changes server (31). The resulting mutants are predicted to fold properly upon translation but are incapable of insertion into the target cell membrane.

Development of CSFV infectious clones harboring amino acid substitutions in the FPII sequence.To evaluate the role of the putative FPII in thein vitroandin vivoreplication of CSFV as well as in the production of disease in swine, a series of recombi-nant CSF viruses containing amino acid substitutions in the FPII area were designed using the cDNA infectious clone of the Brescia strain (BICv) as a template. Amino acid substitutions in FPII were selected based on the premises already discussed. Therefore, a to-tal of six cDNA constructs (FPII.1 to FPII.6) containing the de-sired amino acid substitutions were constructed (Fig. 2).

Infectious RNA wasin vitrotranscribed from each mutated full-length cDNA and used to transfect SK6 cells. Infectious vi-ruses were rescued from cells transfected with all the constructs by day 4 posttransfection, although with different efficiencies since constructed virus yields of FPII.2 and FPII.5 are 100 to 1,000 times lower than for any of the other FPII constructs or the parental pBIC (data not shown). Full-length nucleotide sequencing of the rescued mutant E2 viruses was performed to ensure the presence of the predicted mutations (data not shown).

Replication of the CSFV FP mutantsin vitro.In vitro replica-tion characteristics of the FPII mutant viruses relative to those of parental BICv were evaluated in a multistep growth curve using SK6 cells. SK6 cell cultures were infected at a multiplicity of infec-tion (MOI) of 0.01 TCID50per cell. Viruses were adsorbed for 1 h

FIG 1Hydropathy plots corresponding to the E2 protein sequence spanning residues 690 to 1063 of CSFV polypeptide (Brescia strain). The plots (mean values for a window of 11 amino acids) were produced using the WW interfa-cial hydrophobicity scale for individual residues (blue) and KD hydropathy index (black). The WW plot in red was rendered using interfacial hydropho-bicity values for protonated side chains of Asp and Glu residues. The desig-nated peaks correspond to the predicted IFPs, namely, FPI and FPII. The ectodomain structure of BVDV1 E2 (PDB accession code 2YQ2) is depicted in ribbon representation at the top. Backbone stretches spanning FPI and FPII sequences are colored red and green, respectively. Side chains of residues W871 and W875 are displayed in space-filling representation.

on November 7, 2019 by guest

http://jvi.asm.org/

(4)

(time zero), and samples were collected at 72 h postinfection and titrated in SK6 cell cultures. All mutant viruses, with the exception of FPII.2 and FPII.5, exhibited growth kinetics almost undistin-guishable from that of the parental BICv (Fig. 3A). In contrast, FPII.2 and FPII.5 produced approximately 100 and 1,000 times less virus, respectively, than did BICv. Therefore, combined sub-stitutions W871T-W875D-V878T, in mutant virus FPII.2, and N874D-W875D, in mutant virus FPII.5, inside the putative FPII significantly affect the ability of the virus to replicate in cell cul-tures.

Virulence of CSFV FPII mutantsin vivo.To examine whether alterations of different residues included in the putative FPII affect virulence, different groups of pigs were inoculated i.n. with ap-proximately 105 TCID50s of each of the FPII mutant viruses

(FPII.1 to FPII.6) and monitored for clinical disease, evaluated relative to that with parental BICv. All animals infected with BICv presented clinical signs of CSF starting 3 to 4 dpi, developing clas-sic symptoms of the disease and dying around 7 to 9 dpi (Table 2). All mutant viruses, with the exception of FPII.2, presented a vir-ulence phenotype almost indistinguishable from that of the pa-rental BICv (Table 2). All animals infected with these viruses pre-sented clinical signs of CSF starting at 3 to 5 dpi, with clinical presentation and severity similar to those observed in animals inoculated with BICv. Conversely, animals inoculated with FPII.2 virus did not present any clinical signs associated with CSF during the 21-day observation period.

Viremia in animals inoculated with mutant viruses FPII.1, FPII.3, FPII.4, FPII.5, and FPII.6 generally accompanied the evo-lution of the clinical disease, with viremia kinetics almost undis-tinguishable from that induced by parental BIC virus, presenting high titers that remained until death of the animal (Fig. 4A). In contrast, animals infected with FPII.2 virus present almost unde-tectable levels of viremia during the 21-day observation period. Therefore, combined substitutions W871T-W875D-V878T in mutant virus FPII.2 drastically affect the ability of the virus to produce disease in swine.

Comparative growth of mutant virus FTII.2 in swine macro-phage cell cultures.Cells derived from the macrophages are the main target for CSFV replication during the infection in swine. Since the FPII.2 mutant virus clearly replicates with much lower

efficiency than BICv during swine infection, it was important to assess the comparative ability of FPII.2 virus to replicate in swine macrophages.In vitrogrowth characteristics of the FPII.2 mutant virus relative to those of parental BICv were evaluated in a multi-step growth curve. Primary swine macrophage cultures were in-fected at an MOI of 0.01 TCID50per cell. Viruses were adsorbed

for 1 h (time zero), and samples were collected at 72 h postinfec-tion. Samples were titrated in SK6 cells and the presence of virus was detected by immunoperoxidase staining. The growth of FPII.2 virus was significantly less than that of BICv, showing titers approximately 10-fold lower than those of the parental virus, de-pending on the sampling time considered (Fig. 3B). Therefore, attenuated FPII.2 virus has an evident disadvantage replicating in swine macrophages compared with the parental virulent BICv.

Mutant virus FPII.2 protects pigs against lethal CSFV chal-lenge.The ability of attenuated FPII.2 virus to induce protection against virulent BICv was assessed in early- and late-vaccination-exposure experiments. Groups of pigs (n⫽5) were inoculated i.n. with 105TCID

50s of FPII.2 virus and challenged i.n. at 3 or 21 dpi

with 105TCID50s of virulent BICv. Mock-vaccinated control pigs

receiving BICv (n⫽5) developed anorexia, depression, and fever by 3 to 4 days postchallenge (dpc) (Table 3) and died or were euthanizedin extremisby 8 dpc. FPII.2 virus induced complete clinical protection in animals challenged at 21 days postimmuni-zation. All pigs survived infection with the virulent parental BICv and remained without fever or demonstrating any other CSF-associated clinical signs during the 21-day observation period (

Ta-ble 3). Only two out of five animals in the group challenged at 3

days after FPII.2 infection showed a transient and mild elevation of body temperature.

Viremia in vaccinated and challenged animals was examined at different times postchallenge. As expected, in mock-vaccinated control animals, viremia was observed within 4 dpc, with virus titers remaining typically high at the last time point tested before animals died or were euthanized (Fig. 4B). Conversely, animals inoculated with FPII.2 virus and challenged with BICv at either 3 or 21 dpi did not produce detectable BICv viremia, with the ex-ception of very low titers by 4 dpc in animals challenged at 3 dpi

(Fig. 4B). These results indicate that protection induced by FPII.2

virus was complete, preventing both the presentation of CSF-re-FIG 2Representation of amino acid substitutions in each of the FPII constructs. Nucleotide and amino acid residues changed are in bold italics and have a gray background. Amino acid positions in the CSFV polypeptide are indicated at the top.

on November 7, 2019 by guest

http://jvi.asm.org/

(5)

lated clinical signs and the replication of the challenge virus when challenge was conducted at 21 days after initial infection.

Structure and membrane interactions of FPII-based syn-thetic peptides.FPII-membrane interactions and effects of muta-tions in the process were assessed in peptide-based assays (Fig. 5

and6). To determine the structuring degree in membrane mimet-ics, secondary structures adopted by spFPII.wt, spFPII.2, and spFPII.5 were measured by CD (Fig. 5A). The spectroscopic mea-surements in solution disclosed comparable␤-type and disor-dered conformations for the three variants (left), which were over-all preserved in the membrane-mimicking environment provided

FIG 3In vitrogrowth characteristics of FPII mutants and parental BICv. (A) SK6 cell cultures were infected (MOI⫽0.01) with each of the FPII virus mutants or BICv, and virus yield was titrated at times postinfection in SK6 cells. (B) Primary swine macrophage cultures were infected (MOI⫽0.01) with either FPII.2 virus or BICv, and virus yield was titrated at times postinfection in SK6 cells. Data in both panels represent means and SDs from three independent experiments. Sensitivity of virus detection:ⱖ1.8 log10TCID50s/ml. Asterisks indicate statistical significance at aPvalue of 0.05 (ttest). In panel A, significant differences are as follows: *, BICv and FPII.1 are significantly different from FPII.3, FPII.4, and FPII.6; **, FPII.2 and FPII.5 are significantly different from the rest of the tested viruses; and ***, FPII.2 is significantly different from FPII.5. In panel B, a single asterisk indicates significant difference between the two viruses.

TABLE 2Swine survival and fever response following infection with FPII virus mutants and parental BICv

Virus No. of survivors/ total

Fever

Maximum avg temp, °F (SD) Mean time

to death, days (SD)

No. of days to onset (SD)

Duration, days (SD)

BICv 0/5 8.4 (1.17) 3.6 (0.57) 4.8 (1.1) 105.08 (0.54) FPII.1 0/5 9.4 (1.67) 4 (0.7) 5.4 (3.7) 105.08 (0.54) FPII.2 5/5 103.36 (0.57) FPII.3 0/5 9.4 (3.99) 3 (3.6) 5 (2.12) 106.25 (0.7) FPII.4 0/5 9.4 (3.05) 3.8 (0.45) 5.6 (2.97) 105.98 (0.18) FPII.5 0/5 9.2 (4.09) 3.8 (0.45) 5.66 (2.97) 106.14 (0.4) FPII.6 0/5 9 (2.22) 4 (3.6) 5.2 (2.18) 105.9 (0.38)

on November 7, 2019 by guest

http://jvi.asm.org/

(6)

by dodecylphosphocholine micelles (right). These structural fea-tures are further consistent with the retention of the IFP-like, short-loop conformation upon insertion into the low-polarity membrane environment.

Membrane interactions of E2 FPII peptides were next evalu-ated in lipid monolayer and vesicle membrane models. Peptide penetration into membranes was first inferred from the increase of the monolayer lateral pressure as a function of the initial com-pression (⌸0) (Fig. 5B). Enhancement of monolayer lateral

pres-sure upon injection at high⌸0-s positively correlates with FP

ca-FIG 4(A) Viremia detected in pigs inoculated with FPII virus mutants (FPII.1 to FPII.6) or parental BICv. Groups of animals (n⫽5) were i.n. inoculated with 105TCID

50s of the corresponding virus and blood samples were obtained at indicated times postinoculation. (B) Viremia detected in pigs inoculated with FPII.2 and challenged with BICv. Two groups of animals (n⫽5) were intramuscularly (i.m.) inoculated with 105TCID

50s of FPII.2 virus and i.n. challenged either 3 or 21 days later with 105TCID

50s of virulent BICv. Virus titrations were performed in SK6 cells. Each point represents the mean log10TCID50per milliliter and SD from five animals. Sensitivity of virus detection:ⱖ1.8 log10TCID50s/ml. Asterisks indicate statistical significance at aPvalue of 0.05 (ttest). **, FPII.2 and FPII.6 are significantly different from the rest of the tested viruses; *, FPII.2 is significantly different from the rest of the tested viruses.

TABLE 3Swine survival and fever response in FPII.2-infected animals after challenge with parental virulent BICv

Challenge group

No. of survivors/ total

Mean time to death, days (SD)

Fever No. of days to onset (SD)

Duration, days (SD)

Maximum daily temp, °F (SD) Mock 0/5 8 (0.0) 3.5 (0.58) 4.5 (0.58) 105.7 (0.57) FPII.2, 3 dpi 5/5 3 (1.41)a

4.5 (2.12)a

104.6 (0.85)a

FPII.2, 21 dpi 5/5 103.5 (1.62)

a

Only two out of the five inoculated animals presented an increase in body temperature to over 104°F.

on November 7, 2019 by guest

http://jvi.asm.org/

(7)

pacity for inserting into the viral target membrane (32,33). The exclusion pressures derived from the plots (⌸ex) or the maximum

initial lateral pressures at which membrane association was ac-companied by peptide integration into the monolayer (indicated by the arrows) suggested higher penetration for the wild type (WT) (33 mN/m) than for FPII.2 (19 mN/m) or FPII.5 (23 mN/m) mutants.

Emission fluorescence of NBD-labeled FPII peptides was next measured to infer their level of penetration into lipid bilayers

(Fig. 5C). The maximum emission of the NBD-spFPII.wt peptide

in the presence of lipid vesicles was shifted to lower wavelengths and the intensity was higher than for the NBD-spFPII.5 sequence (black and blue spectra, respectively). In the case of NBD-spFPII.2 peptide, neither the intensity nor the maximum emission under-went major changes in comparison with the peptide in solution (red and gray spectra, respectively). These data suggest that the NBD probe was immersed in a low-polarity environment in the case of the spFPII.wt peptide, indicative of deep penetration into the bilayer. spFPII.5 insertion was shallower, while spFPII.2 seemed to remain mostly in solution dissociated from vesicles.

To obtain more direct evidence of membrane association, the NBD-labeled FPII peptides were incubated with GUVs (POPG– POPC–PE-Rho with a 1:1:0.01 molr ratio) and imaged by confocal microscopy (Fig. 5D). The confocal micrographs correspond to equatorial sections of GUVs incubated with peptides (green color) and labeled with the lipophilic probe PE-Rho (depicted in red). spFPII.wt and spFPII.5 could be detected in association with membranes as revealed by the colocalization of peptide and lipid in the samples (top and bottom rows). In the spFPII.2-containing samples, NBD stain appeared as a uniform bright background, against which the vesicles were seen as dark objects (center row). This fluorescence pattern confirmed that the spFPII.2 did not as-sociate with GUVs under these conditions.

Measurements of vesicle permeability were next carried out to compare the capacities of perturbing the POPG lipid bilayer ar-chitecture (Fig. 6). In these assays, only the spFPII.wt sequence destabilized the integrity of the vesicles, while the spFPII.2 and spFPII.5 sequences had no effect (Fig. 6A). For comparison, the spFPI.wt sequence was also included in these experiments (Fig. 6A, right, green symbols). In general, peptides spFPI.wt and spFPII.wt adopt similar structures and insert into membranes ef-ficiently (18), but spFPI displays higher membrane activity. Fi-nally, further consistent with the biological relevance of the inter-action, the pH dependence supported activation of the spFPII.wt membrane activity at a pH ofⱕ6.0 (Fig. 6B).

In summary, the data displayed inFig. 5and6support the capacity of the E2 FPII sequence for inserting into the target mem-brane and destabilizing its architecture. In comparison, the spFPII.2 mutant retains the same secondary structure but loses the capacity for inserting efficiently into membranes. The spFPII.5 mutant appears to associate more peripherally with membranes, an interaction pattern that preserves the lipid bilayer organiza-tion.

DISCUSSION

This report describes and functionally characterizes as a potential FP (FPII) an amino acid stretch of the structural glycoprotein E2 in CSFV within residues869CKWGGNWTCV878. In addition, it

presents the effect of critical residue substitutions within FPII on virus replication in cell line cultures and swine and virus virulence in the natural host.

Calculation of mean interfacial hydrophobicity using WW al-gorithms (28) disclosed CSFV E2 regions with the potential for favorably associating with the membrane interface at pHs 7.4 and 5.0 (Fig. 1). The most hydrophobic-at-interface area, encompass-ing residues 864 to 881 (FPII inFig. 1), was found within a region where the corresponding Kyte-Doolittle hydropathy plot was flat. This trend is typically observed within class II IFPs, which insert into the external leaflet of the target membrane (25,32). The class FIG 5Structure and membrane interactions of E2 FPII-derived synthetic

peptides. (A) Secondary structures measured by CD. The structural compo-nents of spFPII.wt, spFPII.2, and spFPII.5 (black, red and blue bars, respec-tively) were calculated for the CD spectra obtained in buffer (left) or in the presence of 100 mM dodecylphosphocholine (right) with the SELCON3 pro-gram. (B) Penetration of spFPII.wt, spFPII.2, and spFPII.5 into lipid mono-layers made of POPG (black, red, and blue plots, respectively). Maximum increase in surface pressure induced upon injection of 0.4␮M peptide into the subphase was measured as a function of the initial surface pressure of the phospholipid monolayers. Monolayer exclusion pressures are indicated by the arrows. (C) NBD emission spectra of labeled spFPII.wt, spFPII.2, and spFPII.5 peptides in the presence of POPG vesicles (black, red, and blue spec-tra, respectively). Peptides (0.5␮M) were incubated for 30 min with saturating concentrations of lipid (1 mM) before collection of the spectra. Maximum NBD emission in solution was normalized to 1 for the 3 peptides (gray spec-tra). (D) Imaging of peptide binding to GUVs. Confocal microscopy of GUVs (equatorial sections) electroformed from a mixture of POPG–POPC–PE-Rho (1:1:0.01, molar ratio), incubated with NBD-labeled FPII peptides (0.1␮M) for 1 h. Peptide (green) staining and lipid (red) staining are shown in the left and center columns, respectively. The right column displays merging of both detection channels.

on November 7, 2019 by guest

http://jvi.asm.org/

(8)

II fusion glycoproteins share a fold composed of␤-domains I, II, and III (34). The class II IFPs, also known as “fusion loops,” em-body two␤-strands connecting loops at the tip of domain II, distal to the transmembrane region.

In comparison with most class I FPs, class II IFPs are shorter, less hydrophobic sequences (usually lacking aliphatic residues) and are comparatively enriched in aromatics (25). Besides these features, class II IFPs follow common patterns of interaction with membranes. These interactions are defined by the stabilization of the short loop conformation upon membrane association, shal-low insertion, and a high dose requirement for generating lipid bilayer perturbations (25). In this regard, the conformations ad-opted by representative synthetic peptides (compatible with

␤-type structures), their capacities for inserting into lipid mono-layers and for partitioning from water solution into lipid bimono-layers, their membrane penetration depth, and their scored membrane-perturbing activity levels (Fig. 5and6) all support an IFP role for the CSFV E2 FPII sequence. The effects on these processes of the sequence alterations that interfere with virus production and in-fectivity add biological relevance to that notion.

However, CSFV E2 glycoprotein does not adopt a canonical class II fold, and the FPII loop, although exposed, is not located at the tip of the molecule (29,30). Thus, in a more general sense, our findings here and in our previous work (18) support cumulative evidence indicating that the members of the different genera of the Flaviviridaefamily do not share a fusion mechanism (35, 36). Differences in the crystal structures of the BVDV1 and HCV E2 glycoproteins illustrate that common genome arrangement among members of theFlaviviridaefamily does not translate to structurally similar fusion glycoproteins (29, 30). Conversely, structural comparisons indicate that sequences showing marginal

homology can display a common class II fold (e.g., dengue virus E and Semliki Forest virus E1 proteins) (37,38). It has been sug-gested that the use of arthropod vectors may require conservation of the class II fold by distant members of theTogaviridaeand Flaviviridae families, while phylogenetically closer viruses that strictly infect mammals may have evolved to develop specialized entry machineries (39).

A common theme in those machineries, however, could be the generation, upon fusion activation, of extensive fusion surfaces, involving several loops for association with the target membrane, as has been described for the rubella virus (39). In the particular case of the HCV fusion glycoproteins, experimental evidence sup-ports the idea that regions in both the E1 and E2 subunits might contribute to such a fusion surface (40,41). The CSFV FPII was located consecutive to the previously described E2 FP (FPI inFig. 1), both in the sequence and in comparison with the tridimen-sional structure of BVDV E2 glycoprotein (29,30). We surmise that these regions, alone or in combination with other sequences of the E1 subunit, might, upon fusion activation, assemble a mem-brane-interactive surface (42).

The biological relevance of FPII in CSFV functioning was evi-denced by the decreased replication of FPII.2 virusin vitroandin vivo. In particular, amino acid substitutions W871T, W875D, and V878T in FPII.2 virus significantly reduced virus yields compared with those of the parental virus both in SK6 cell cultures and in primary swine macrophage cultures. More drastic is the reduction in FPII.2 virus replication during infection in swine, with almost undetectable levels of viremia. The effect caused by the mutations is still more dramatic when measured in terms of virulence: FPII.2 virus is completely attenuated in swine. Therefore, amino acid substitutions that specifically alter the interaction of spFPII.2 with FIG 6Membrane destabilization induced by E2 FPII-derived synthetic peptides. (A) Membrane permeabilization induced by spFPII.wt, spFPII.2, and spFPII.5 peptides (black, red, and blue plots, respectively). (Left) ANTS leakage from POPG LUVs as a function of time. Peptides were added at the time indicated by the arrow to a 1:10 peptide-to-lipid molar ratio. (Right) Extents after 5 min of incubation with peptide have been plotted as a function of the lipid-to-peptide mole ratio. The green plot corresponds to samples containing the spFPI.wt sequence. Lipid concentration (100␮M) was fixed. (B) pH dependence of spFPII.wt-induced membrane destabilization. In both panels, plotted leakage extents are means⫾SDs of three independent measurements.

on November 7, 2019 by guest

http://jvi.asm.org/

(9)

artificial membranesin vitrohave a profound effect on virus phe-notype both in cell cultures and in infection in the natural host. Interestingly,in vivoattenuation of FPII.2 virus was accompanied by the ability to induce protection against challenge with the pa-rental virulent BICv. Therefore, our results provide the proof of principle that supports discovery and manipulation of these po-tential fusion surfaces as a promising strategy for vaccine develop-ment.

In this report, we present novel data regarding the discovery and the functional characterization of a potential FP located within E2 that, along with a previously published report from our group (18), constitute the only data available so far describing the presence of functional FP structures in pestiviruses (BVDV, bor-der disease virus, and CSFV). Here are described thein silico ana-lytical discovery of a putative FP sequence, the functionality of FPIIin vitroas determined using different independent artificial membrane models, the identification of specific amino acid resi-dues within FPII mediating the interaction with membranesin vitro, the critical effect of replacing the same amino acid residues within FPII on virus replication in primary and cell line cultures and in the natural host and in virus pathogenesis in swine, and the development of experimental CSFV vaccine virus based on the genetic manipulation of the FPII sequence in E2 glycoprotein.

ACKNOWLEDGMENTS

This study was in part supported by the Agricultural Research Service of the United States (ARS-USDA project 8064-32000-056-18S to E.L. and J.L.N.) and the Basque Government (project IT838-13 to J.L.N.).

We thank the Plum Island Animal Disease Center animal care unit staff for excellent technical assistance. We especially thank Melanie Prarat for editing the manuscript.

FUNDING INFORMATION

This work, including the efforts of Eneko Largo and Jose L. Nieva, was funded by USDA | Agricultural Research Service (ARS) (8064-32000-056-18S).

REFERENCES

1.Becher P, Avalos Ramirez R, Orlich M, Cedillo Rosales S, Konig M, Schweizer M, Stalder H, Schirrmeier H, Thiel HJ.2003. Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification. Virology311:96 –104.http://dx.doi.org/10.1016/S0042 -6822(03)00192-2.

2.Rice CM.1996. Flaviviridae: the viruses and their replication, p 931–959. InKnipe DM, Fields BN, Howley PM, Chanock RM, Melnick JL, Monath TP, Roizman B, Straus SE (ed), Fields virology, 3rd ed. Lippincott Wil-liams & Wilkins, Philadelphia, PA.

3.Weiland E, Stark R, Haas B, Rumenapf T, Meyers G, Thiel HJ.1990. Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer. J Virol64:3563–3569.

4.Weiland F, Weiland E, Unger G, Saalmuller A, Thiel HJ.1999. Local-ization of pestiviral envelope proteins E(rns) and E2 at the cell surface and on isolated particles. J Gen Virol80(Part 5):1157–1165.

5.Thiel HJ, Stark R, Weiland E, Rumenapf T, Meyers G. 1991. Hog cholera virus: molecular composition of virions from a pestivirus. J Virol

65:4705– 4712.

6.Langedijk JP.2002. Translocation activity of C-terminal domain of pes-tivirus Erns and ribotoxin L3 loop. J Biol Chem277:5308 –5314.http://dx .doi.org/10.1074/jbc.M104147200.

7.van Gennip HG, Bouma A, van Rijn PA, Widjojoatmodjo MN, Moor-mann RJ.2002. Experimental non-transmissible marker vaccines for classical swine fever (CSF) by trans-complementation of E(rns) or E2 of CSFV. Vac-cine20:1544 –1556.http://dx.doi.org/10.1016/S0264-410X(01)00497-2. 8.Hulst MM, Moormann RJ.1997. Inhibition of pestivirus infection in cell

culture by envelope proteins E(rns) and E2 of classical swine fever virus:

E(rns) and E2 interact with different receptors. J Gen Virol78(Part 11):

2779 –2787.

9.Wang Z, Nie Y, Wang P, Ding M, Deng H.2004. Characterization of classical swine fever virus entry by using pseudotyped viruses: E1 and E2 are sufficient to mediate viral entry. Virology330:332–341.http://dx.doi .org/10.1016/j.virol.2004.09.023.

10. van Gennip HG, van Rijn PA, Widjojoatmodjo MN, de Smit AJ, Moormann RJ.2000. Chimeric classical swine fever viruses containing envelope protein E(RNS) or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable anti-body response. Vaccine 19:447– 459. http://dx.doi.org/10.1016/S0264 -410X(00)00198-5.

11. Liang D, Sainz IF, Ansari IH, Gil LH, Vassilev V, Donis RO.2003. The envelope glycoprotein E2 is a determinant of cell culture tropism in rumi-nant pestiviruses. J Gen Virol84:1269 –1274.http://dx.doi.org/10.1099 /vir.0.18557-0.

12. Risatti GR, Borca MV, Kutish GF, Lu Z, Holinka LG, French RA, Tulman ER, Rock DL.2005. The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. J Virol79:3787–3796.http://dx .doi.org/10.1128/JVI.79.6.3787-3796.2005.

13. Risatti GR, Holinka LG, Carrillo C, Kutish GF, Lu Z, Tulman ER, Sainz IF, Borca MV. 2006. Identification of a novel virulence determinant within the E2 structural glycoprotein of classical swine fever virus. Virol-ogy355:94 –101.http://dx.doi.org/10.1016/j.virol.2006.07.005. 14. Risatti GR, Holinka LG, Fernandez Sainz I, Carrillo C, Kutish GF, Lu

Z, Zhu J, Rock DL, Borca MV.2007. Mutations in the carboxyl terminal region of E2 glycoprotein of classical swine fever virus are responsible for viral attenuation in swine. Virology364:371–382.http://dx.doi.org/10 .1016/j.virol.2007.02.025.

15. Risatti GR, Holinka LG, Fernandez Sainz I, Carrillo C, Lu Z, Borca MV.

2007. N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine. J Virol81:924 –933.

http://dx.doi.org/10.1128/JVI.01824-06.

16. Van Gennip HG, Vlot AC, Hulst MM, De Smit AJ, Moormann RJ.2004. Determinants of virulence of classical swine fever virus strain Brescia. J Virol

78:8812– 8823.http://dx.doi.org/10.1128/JVI.78.16.8812-8823.2004. 17. Garry RF, Dash S.2003. Proteomics computational analyses suggest that

hepatitis C virus E1 and pestivirus E2 envelope glycoproteins are trun-cated class II fusion proteins. Virology307:255–265.http://dx.doi.org/10 .1016/S0042-6822(02)00065-X.

18. Fernández-Sainz IJ, Largo E, Gladue DP, Fletcher P, O’Donnell V, Holinka LG, Carey LB, Lu X, Nieva JL, Borca MV. 2014. Effect of specific amino acid substitutions in the putative fusion peptide of struc-tural glycoprotein E2 on classical swine fever virus replication. Virology

456-457:121–130.

19. Terpstra C, Woortmeyer R, Barteling SJ.1990. Development and prop-erties of a cell culture produced vaccine for hog cholera based on the Chinese strain. DTW. Dtsch Tierarztl Wochenschr97:77–79.

20. Zsak L, Lu Z, Kutish GF, Neilan JG, Rock DL.1996. An African swine fever virus virulence-associated gene NL-S with similarity to the herpes simplex virus ICP34.5 gene. J Virol70:8865– 8871.

21. Edwards S, Moennig V, Wensvoort G.1991. The development of an international reference panel of monoclonal antibodies for the differenti-ation of hog cholera virus from other pestiviruses. Vet Microbiol29:101– 108.http://dx.doi.org/10.1016/0378-1135(91)90118-Y.

22. Reed LJ, Muench H.1938. A simple method of estimating fifty percent endpoints. Am J Hyg27:493– 497.

23. Sanger F, Nicklen S, Coulson AR.1977. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A74:5463–5467.http://dx .doi.org/10.1073/pnas.74.12.5463.

24. Mittelholzer C, Moser C, Tratschin JD, Hofmann MA.2000. Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains. Vet Microbiol74:293–308.http: //dx.doi.org/10.1016/S0378-1135(00)00195-4.

25. Apellániz B, Huarte N, Largo E, Nieva JL.2014. The three lives of viral fusion peptides. Chem Phys Lipids181:40 –55.http://dx.doi.org/10.1016 /j.chemphyslip.2014.03.003.

26. Nieva JL, Suarez T.2000. Hydrophobic-at-interface regions in viral fu-sion protein ectodomains. Biosci Rep20:519 –533.http://dx.doi.org/10 .1023/A:1010458904487.

27. Suárez T, Gallaher WR, Agirre A, Goni FM, Nieva JL.2000. Membrane interface-interacting sequences within the ectodomain of the human im-munodeficiency virus type 1 envelope glycoprotein: putative role during

on November 7, 2019 by guest

http://jvi.asm.org/

(10)

viral fusion. J Virol74:8038 – 8047.http://dx.doi.org/10.1128/JVI.74.17 .8038-8047.2000.

28. Wimley WC, White SH.1996. Experimentally determined hydrophobic-ity scale for proteins at membrane interfaces. Nat Struct Biol3:842– 848.

http://dx.doi.org/10.1038/nsb1096-842.

29. Iourin O, Harlos K, El Omari K, Lu W, Kadlec J, Iqbal M, Meier C, Palmer A, Jones I, Thomas C, Brownlie J, Grimes JM, Stuart DI.2013. Expression, purification and crystallization of the ectodomain of the en-velope glycoprotein E2 from bovine viral diarrhoea virus. Acta Crystallogr Sect F Struct Biol Cryst Commun69:35–38.http://dx.doi.org/10.1107 /S1744309112049184.

30. Li Y, Wang J, Kanai R, Modis Y.2013. Crystal structure of glycoprotein E2 from bovine viral diarrhea virus. Proc Natl Acad Sci U S A110:6805– 6810.http://dx.doi.org/10.1073/pnas.1300524110.

31. Dehouck Y, Kwasigroch JM, Gilis D, Rooman M.2011. PoPMuSiC 2.1: a web server for the estimation of protein stability changes upon mutation and sequence optimality. BMC Bioinformatics12:151.http://dx.doi.org /10.1186/1471-2105-12-151.

32. Nieva JL, Agirre A.2003. Are fusion peptides a good model to study viral cell fusion? Biochim Biophys Acta1614:104 –115.http://dx.doi.org/10 .1016/S0005-2736(03)00168-8.

33. Rafalski M, Lear JD, DeGrado WF.1990. Phospholipid interactions of synthetic peptides representing the N-terminus of HIV gp41. Biochemis-try29:7917–7922.http://dx.doi.org/10.1021/bi00486a020.

34. White JM, Delos SE, Brecher M, Schornberg K.2008. Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme. Crit Rev Biochem Mol Biol43:189 –219.http://dx.doi .org/10.1080/10409230802058320.

35. Khan AG, Miller MT, Marcotrigiano J.2015. HCV glycoprotein struc-tures: what to expect from the unexpected. Curr Opin Virol12:53–58.

http://dx.doi.org/10.1016/j.coviro.2015.02.004.

36. Sabahi A, Uprichard SL, Wimley WC, Dash S, Garry RF. 2014. Unexpected structural features of the hepatitis C virus envelope

pro-tein 2 ectodomain. J Virol88:10280 –10288.http://dx.doi.org/10.1128 /JVI.00874-14.

37. Kuhn RJ, Zhang W, Rossmann MG, Pletnev SV, Corver J, Lenches E, Jones CT, Mukhopadhyay S, Chipman PR, Strauss EG, Baker TS, Strauss JH.2002. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell108:717–725.http://dx.doi.org /10.1016/S0092-8674(02)00660-8.

38. Lescar J, Roussel A, Wien MW, Navaza J, Fuller SD, Wengler G, Wengler G, Rey FA.2001. The Fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH. Cell105:137–148.http://dx.doi.org/10.1016/S0092 -8674(01)00303-8.

39. DuBois RM, Vaney MC, Tortorici MA, Kurdi RA, Barba-Spaeth G, Krey T, Rey FA. 2013. Functional and evolutionary insight from the crystal structure of rubella virus protein E1. Nature493:552–556.http: //dx.doi.org/10.1038/nature11741.

40. Lavillette D, Pecheur EI, Donot P, Fresquet J, Molle J, Corbau R, Dreux M, Penin F, Cosset FL.2007. Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus. J Virol

81:8752– 8765.http://dx.doi.org/10.1128/JVI.02642-06.

41. Perin PM, Haid S, Brown RJ, Doerrbecker J, Schulze K, Zeilinger C, von Schaewen M, Heller B, Vercauteren K, Luxenburger E, Baktash YM, Vondran FW, Speerstra S, Awadh A, Mukhtarov F, Schang LM, Kirschning A, Muller R, Guzman CA, Kaderali L, Randall G, Meuleman P, Ploss A, Pietschmann T.2016. Flunarizine prevents hepatitis C virus membrane fusion in a genotype-dependent manner by targeting the po-tential fusion peptide within E1. Hepatology63:49 – 62.http://dx.doi.org /10.1002/hep.28111.

42. El Omari K, Iourin O, Harlos K, Grimes JM, Stuart DI.2013. Structure of a pestivirus envelope glycoprotein E2 clarifies its role in cell entry. Cell Rep3:30 –35.http://dx.doi.org/10.1016/j.celrep.2012.12.001.

on November 7, 2019 by guest

http://jvi.asm.org/

Figure

TABLE 1 Nucleotide sequences of primers used for the production of FPII recombinant virusesa

TABLE 1

Nucleotide sequences of primers used for the production of FPII recombinant virusesa p.2
FIG 1 Hydropathy plots corresponding to the E2 protein sequence spanningresidues 690 to 1063 of CSFV polypeptide (Brescia strain)

FIG 1

Hydropathy plots corresponding to the E2 protein sequence spanningresidues 690 to 1063 of CSFV polypeptide (Brescia strain) p.3
FIG 2 Representation of amino acid substitutions in each of the FPII constructs. Nucleotide and amino acid residues changed are in bold italics and have a graybackground

FIG 2

Representation of amino acid substitutions in each of the FPII constructs. Nucleotide and amino acid residues changed are in bold italics and have a graybackground p.4
TABLE 2 Swine survival and fever response following infection withFPII virus mutants and parental BICv

TABLE 2

Swine survival and fever response following infection withFPII virus mutants and parental BICv p.5
FIG 3 In vitroas follows: *, BICv and FPII.1 are significantly different from FPII.3, FPII.4, and FPII.6; **, FPII.2 and FPII.5 are significantly different from the rest of the tested growth characteristics of FPII mutants and parental BICv

FIG 3

In vitroas follows: *, BICv and FPII.1 are significantly different from FPII.3, FPII.4, and FPII.6; **, FPII.2 and FPII.5 are significantly different from the rest of the tested growth characteristics of FPII mutants and parental BICv p.5
TABLE 3 Swine survival and fever response in FPII.2-infected animalsafter challenge with parental virulent BICv

TABLE 3

Swine survival and fever response in FPII.2-infected animalsafter challenge with parental virulent BICv p.6
FIG 4 (A) Viremia detected in pigs inoculated with FPII virus mutants (FPII.1 to FPII.6) or parental BICv

FIG 4

(A) Viremia detected in pigs inoculated with FPII virus mutants (FPII.1 to FPII.6) or parental BICv p.6
FIG 5 Structure and membrane interactions of E2 FPII-derived syntheticpeptides. (A) Secondary structures measured by CD

FIG 5

Structure and membrane interactions of E2 FPII-derived syntheticpeptides. (A) Secondary structures measured by CD p.7
FIG 6 Membrane destabilization induced by E2 FPII-derived synthetic peptides. (A) Membrane permeabilization induced by spFPII.wt, spFPII.2, and spFPII.5induced membrane destabilization

FIG 6

Membrane destabilization induced by E2 FPII-derived synthetic peptides. (A) Membrane permeabilization induced by spFPII.wt, spFPII.2, and spFPII.5induced membrane destabilization p.8

References