Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication I.J. Fernández-Sainz a , E. Largo b , D.P. Gladue a , P. Fletcher a ,V.O’Donnell a,c , L.G. Holinka a , L.B. Carey d ,X.Lu c , J.L. Nieva b , M.V. Borca a, n a Plum Island Animal Disease Center, ARS, USDA, USA b Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao, Spain c Plum Island Animal Disease Center, DHS, Greenport, NY 11944, USA d Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain article info Article history: Received 6 February 2014 Returned to author for revisions 25 February 2014 Accepted 4 March 2014 Available online 4 April 2014 Keywords: Fusion peptide Classical Swine Fever Virus CSFV Classical swine fever Pestivirus abstract E2, along with E rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, 818 CPIGWTGVIEC 828 , containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β -type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP 818 CPIGWTGVIEC 828 indicates a membrane fusion activity and a critical role in virus replication. Published by Elsevier Inc. Introduction Classical 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. CSFV is classified as a member of the pestivirus genus within the Flaviviridae family along with other viruses of economic importance, bovine viral diarrhea virus (BVDV) and border disease virus (BDV) (Becher et al., 2003). The approximately 12.5-kb CSFV genome contains a single open reading frame that encodes a polyprotein composed of 3898 amino acids that ultimately yields up to 12 final cleavage products (NH2-Npro-C-Erns-E1-E2-p7NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) through coand posttranslational processing of the polyprotein by cellular and viral proteases (Rice, 1996). Structural components of the virion include the capsid (C) protein and glycoproteins: E rns ,E1andE2.E rns , a secreted protein that demonstrates RNAse activity and is loosely associated with the viral envelope (Thiel et al., 1991; Weiland et al., 1990; Weiland et al., 1999), does not have a hydrophobic transmembrane anchor domain. E rns does, however, possess a C-terminal charged amphipathic segment that can mediate translocation of E rns across bilayer membranes (Langedijk, 2002). E1 and E2 are transmembrane proteins with an Nterminal ectodomain and a C-terminal hydrophobic anchor (Thiel et al., 1991). E2 is considered essential for CSFV replication, as virus mutants containing partial or complete deletions of the E2 gene are nonviable (van Gennip et al., 2002). E2 has been implicated, along with E rns (Hulst and Moormann, 1997)andE1(Wang et al., 2004), in viral adsorption to host cells (Liangetal.,2003;vanGennipetal., 2000). Modifications introduced into this glycoprotein appear to have an important effect on CSFV virulence (Risatti et al., 2005, 2006, 2007a, 2007b; Van Gennip et al., 2004). Using proteomic computational analysis, E2 has been characterized as a truncated class II fusion protein (Garry and Dash, 2003). Although the overall structures of class I and II fusion proteins are distinct, they may share structural/functional characteristics in the parts of the molecules that interact with and disrupt bilayer membranes. It is well established that class I fusion proteins have a fusion peptide at the amino terminus of the Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/yviro Virology http://dx.doi.org/10.1016/j.virol.2014.03.005 0042-6822/Published by Elsevier Inc. n Correspondence to: Plum Island Animal Disease Center, USDA/ARS/NAA, P.O. Box 848, Greenport, NY 11944-0848, USA. Tel.: þ1 631 323 3019; fax: þ1 631 323 3006. E-mail address: [email protected] (M.V. Borca). Virology 456-457 (2014) 121–130 molecule, or close to it, that is critical for fusion (Gallaher, 1987, 1996; Gallaher et al., 1989, 2001). Class II fusion proteins have an internal FP that is located after secondary structural folding at distal locations from the transmembrane anchor (Kuhn et al., 2002; Lescar et al., 2001; Rey et al., 1995). Garry and Dash described a putative FP located between amino acid residues 818–828 of CSFV E2. This putative FP contains a consensus sequence with aromatic and hydrophobic residues located between two cysteine residues. In addition, the FP is flanked by β sheets in class II fusion proteins. The cysteine residues as well as the sequences in between are highly conserved among pestiviruses, as is true of class I and II FPs from other enveloped RNA viruses (Garry and Dash, 2003). Recently the E2 protein of BVDV has been crystallized, revealing a three β -domain structure (El Omari et al., 2013). The putative FP appears to be partially accessible on the surface of the protein. However, it does not form a canonical fusion loop as those found in Class II proteins, which suggests that other mechanisms-structures might operate in pestivirus fusion. Here we evaluate the role of a specific E2 region, 818 CPIGWTGVIEC 828 , containing a putative FP sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent strain Brescia (BICv) was used to evaluate the role of individual as well as combined amino acid substitutions in the replication of BICv. Only double C818S/C828S or triple P819S/I820S/W822S substitutions resulted in replication-deficient viruses. Furthermore, we sought to establish a correlation between the functional effects induced by the amino acid substitutions and the capacity of synthetic FPs for inserting into membranes and breach the permeability barrier. At the outset we confirmed the retention of β -type extended conformations in membrane mimics for the native sequence and its variants harboring the double C818S/C828S or triple P819S/ I820S/W822S substitutions. Our data indicate that amino acid substitutions primarily affect the degree of FP penetration into the lipid bilayer, the triple substitution resulting in shallower insertion and reduced membrane activity. All in all, our results suggest that the putative FP 818 CPIGWTGVIEC 828 is involved in membrane fusion activity and plays a critical role in virus replication. Results Location of the putative fusion peptide in the CSFV E2 protein The putative E2 FP is formed by eleven amino acids situated between positions 818 and 828 of the CSFV polypeptide. Comparison of its amino acid sequence among different CSFV isolates reveals 100% identity among isolates (Fig. 1). Compared to other pestiviruses the amino acid identity ranges between 45 and 72% similar to BVDV isolates with substitutions mostly occurring at positions 820 and 825–827, 63–72% similar to BDV isolates with amino acid substitutions at positions 820, 822, 824, 825, and 81% similar to pestivirus giraffe-1 with substitutions at 823–825. This high degree of conservation within each of the pestivirus subgroups, compared to the differences between the subgroups, suggests the putative FP sequence may be involved in an important viral function such as virus tropism. Development of CSFV infectious clones harboring amino acid substitutions in the FP sequence To evaluate the role of the putative FP in the in vitro and in vivo replication of CSFV as well as in the production of disease in swine, a series of recombinant CSF viruses containing amino acid substitutions in the FP area were designed using the cDNA infectious clone of the Brescia strain (BICv) as a template. A total of eight cDNA constructs containing the amino acid substitutions described in Table 1 were constructed. Infectious RNA was in vitro transcribed from each mutated fulllength cDNA and used to transfect SK6 cells. Infectious virus containing individual substitutions of C818S, C828S, P819S, I820T, W822S or double substitution V825T/I826T was rescued from transfected cells by day 4 post-transfection. Partial nucleotide sequence of the rescued mutant E2 viruses was performed to ensure the presence of the predicted mutations (data not shown). Conversely, in three independent transfection events, constructs harboring a double, C818S/C828S, or triple, P819S/I820S/ W822S, substitutions were unvaryingly negative in terms of recovering infectious particles (data not shown). Immunohistochemistry analysis of transfected cell monolayers showed that cells transfected with either construct C818S/C828S or P819S/ I820S/W822S expressed E2 (Fig. 2A) although at a decreased level when compared to those found in cells transfected with the constructs producing viable virus particles. Thus, to determine if a gross defect in glycosylation of truncated expression was at the origin of the lethal phenotype, the expression of E2 glycoprotein in cells transfected with either C818S/C828S or P819S/I820S/W822S was additionally analyzed by Western blot. Cell extracts transfected with either of these two constructs demonstrated significant levels of E2 expression with a product showing an electrophoretic mobility similar to that observed with cell extracts transfected with an IC encoding for the parental Brescia virus (Fig. 2B). These results suggest that the double and triple substitution constructs produced a lethal replication virus phenotype, even though there is efficient expression of an apparently intact E2 product. Replication of the CSFV FP mutants in vitro in vitro replication characteristics of the FP E2 mutant viruses containing individual (C818S, C828S, P819S, I820T, W822S) or double substitutions (V825T/I826T) relative to parental BICv were evaluated in a single-step growth curve. Primary swine macrophage cell cultures were infected at a MOI of 0.01 TCID 50 per cell. Viruses were adsorbed for 1 h (time zero), and samples were collected at 72 h post-infection and titrated in SK6 cell cultures. All mutant viruses exhibited growth kinetics almost undistinguishable from that of the parental BICv (Fig. 3A). Additionally, when the FP E2 mutant viruses' plaque sizes were compared using SK6 cells, all mutants exhibited a plaque size similar to that of the parental BICv (Fig. 3B). Therefore, individual substitutions of most of the residues forming the putative FP do not significantly affect the ability of the virus to replicate in cell cultures. Virulence of CSFV FP mutants in vivo To examine whether alterations of different residues included in the putative E2 FP affect virulence, different groups of pigs were intranasally inoculated with approximately 10 5 TCID 50 of each of the E2 FP mutant viruses (C818S, C828S, P819S, I820T, W822S, V825T/I826T) and monitored for clinical disease, evaluated relative to parental BICv. All animals infected with BICv presented clinical signs of CSF starting 3–4 days post-infection (DPI), developing classic symptoms of the disease and dying around 7–8 DPI (Table 2). Total white blood cells, lymphocytes and platelet counts dropped by 4–6 DPI in animals inoculated with BICv and continued declining until death (data not shown). All mutant viruses presented a virulence phenotype almost indistinguishable from that of the parental BICv (Table 2). All animals infected with these viruses presented clinical signs of CSF starting at 3–5 DPI, with clinical presentation and severity similar to those observed in I.J. Fernández-Sainz et al. / Virology 456-457 (2014) 121–130122 animals inoculated with BICv. White blood cells, lymphocytes and platelet counts dropped by 4 DPI and continued declining until death (data not shown) at 6–9 DPI. Viremia in animals inoculated with E2 FP mutants in general accompanied the evolution of the clinical disease (data not shown), exhibiting viremia kinetics almost undistinguishable from that induced by parental BIC virus, presenting high titers that remained until death of the animal. Therefore, with the exception of the lethal substitutions C818S/ C828S or P819S/I820S/W822S, substitutions of any of the other 10 20 30 40 50 ....|....|....|....|....|....|....|....|....|....|....|... Bressia YDTSPVVKG-KYNTTLLNGSAFYLVCPIGWTGVIEC-TAVSPTTLRTEVVKTFRREKP AEA40484.1 F........-..........................-..................... AEA40482.1 F........-..........................-..................... AEA40481.1 F........-..........................-..................... AEA40487.1 F........-..........................-..................... AEA40478.1 F........-..........................-..................... AEA40477.1 F........-..........................-..A.................. AEA40480.1 F........-..........................-I.................... AEA40475.1 F........-..........................-..................... AGI98198.1 F........-..........................-..................... AFC37768.1 F..T.....-..........................-................K.... AEA40479.1 F........-..........................-..A.................. AGT80624.1 FN.......-..........................-..................... AGI98192.1 F....I...-..........................-..................... AGG91155.1 F..T.....-..........................-................K.... AEA40486.1 F........-.F........................-..................D.. AEJ37847.1 F..I.....-..........................-................K.... AFC37766.1 F..T.....-..........................-..................... BVDV AAW88355.1 C.A..LIR.-.F.......P..QM.S....P.PVSLYHWANKD.SA.A.GR.YK.PR. AAW88353.1 CYAR.LLR.-SL...Y..WP..QM..L...P.TVSVYHWAIKV..A.A..R.YK.PR. CAB45185.1 C.AK.I.R.-.........P..QM........TVS.-MLANRD..D.A..R.Y..SR. CAB45183.1 C.AK.I.R.-............QM........TVS.-MLANRD..D.A..R.Y..SR. AAA98608.1 C.SK..I..-.F.AS....P..QM...Q....R...-.L.NQD..D.T..R.Y..TT. AGV40886.1 C.AK.L.R.-.F.......P..QM...M....TVN.-ALANKD..DITI.R.YM.LE. AFX69713.1 S.AK.LGR.-.FKS.....A.SKK.W.T.G..TVS.-ALANKD..DVT..R.NT.L.. AAC06278.2 C.AR...R.-.F.A.....P..QM...T....S.S.-.L.NGD..S.T..R.Y..P.. NP_776264.2 C.AK.I.R.-.F.......P..QM........TVS.-.SFNMD..A.T..R.Y..S.. ABW38057.1 C.AK.L.R.-.F.......P..QM..H.....TVS.-ALANKD..ALT..R.YT.H.. BDV NP_777538.1 C.SR.....-............Q.I..Y..V.RV..-.T..KS..A.....IYKKT.. NP_777537.1 C.SR.....-............Q.I..Y..V.RV..-.T..KS..A.....IYKKT.. AAO26467.1 C.SR.....-............Q....Y..V.RV..-.T..KD..V....RIYKKT.. AAO26466.1 C.SK.....-...S........Q....Y....QV..-....RS..A.....IY..K.. AAO26471.1 C.SK.....-.F.......N..Q....L....QV..-.T..TS..A......Y..SR. AAO26470.1 C.SK.....-...A........Q....F..V.QV..-.T..TS..A......YK.ST. AAO26469.1 C.SK.....-...A........Q....F..V.RV..-.T..TS..A......Y.GST. AAO26468.1 C.SK.....-............QM...F....RV..-.T..TS..A....R.Y..ST. Giraffe-1 NP_777525.1 C.SR.....-NF....I.H...Q.......V.T...-.L.NTD..A.T...RYT.TT. NP_620053.1 C.SR.....-NF....I.H...Q.......V.T...-.L.NTD..A.T...RYT.TT. Fig. 1. Sequence alignment to show putative FP sequences (highlighted) of E2 from different Pestiviruses (CSF, BVD, BD and Giraffe-1). Partial amino acid sequence surrounding polypeptide residues 818–828 are presented. Table 1 Nucleotide sequence of primers used for the production of E2FP recombinant viruses (only forward primers are presented). Mutant name Forward primer sequence C818S 5 0 GGTAGTGCATTCTACCTAGTTTGCCCAATAGGGTGGACGGGTGTT 3 0 C828S 5 0 GGGTGGACGGGTGTTATAGAGTGCACGGCAGTGAGCCCGACAACT 3 0 C818S/C828S a P819S 5 0 AGTGCATTCTACCTAGTTTGCTCAATAGGGTGGACGGGTGTTATA 3 0 I820T 5 0 GCATTCTACCTAGTTTGCCCAACAGGGTGGACGGGTGTTATAGAG 3 0 W822S 5 0 TACCTAGTTTGCCCAATAGGGTCGACGGGTGTTATAGAGTGCACG 3 0 P819S/I820S/W822S 5 0 GCATTCTACCTAGTTTGCTCAACAGGGTCGACGGGTGTTATAGAGTGC 3 0 V825T/I826T 5 0 TGCCCAATAGGGTGGACGGGTGATACAGAGTGCACGGCAGTGAGCCCG 3 0 a Primers C818S and C828S were consecutively used to create mutant C818S/C828S. I.J. Fernández-Sainz et al. / Virology 456-457 (2014) 121–130 123 residues under study within the FP area do not significantly affect virus growth in cell culture or pathogenesis in swine. Evaluation in vitro of the putative fusion peptide in terms of structure and membrane interaction To determine the effect of the amino acid substitutions on the adopted conformations and the interactions with membranes, three FP variant peptides were synthesized and comparatively analyzed. One of the synthetic peptides represents the native sequence of the E2 FP (designated as WT) while the other two harbor a double (C818S/C828S) or a triple substitution (P819S/ I820S/W822S) and were designated as mut-1 and mut-2, respectively (Fig. 4A). All three sequences were flanked by additional lysine residues to confer solubility, and tagged at the N-terminus with the NBD fluorescent probe for assessment of interactions with lipid bilayers. The secondary structures adopted by these synthetic peptides are displayed in Fig. 4B. Circular dichroism measurements disclosed comparable amounts of β -type extended structures that were predominant for the three variants, and overall retained in the membrane-mimicking environments provided by SDS and DPC micelles. Thus, the amino acid substitutions did not seem to affect the secondary structure of the E2 FP, which otherwise performed comparably in solution and the low-polarity membrane environment. Membrane interactions of WT, mut-1 and mut-2 variants were next assessed in lipid monolayer and unilamellar vesicle models (Fig. 5). The three sequences were first compared in the lipid monolayer system, which allows monitoring peptide penetration into membranes as an increase of the monolayer lateral pressure (see Largo et al. 2014 for a description of the system). The system also allows adjustment of the initial lateral pressure ( π 0 ). Thus, the FP capacity of raising the monolayer lateral pressure upon injection at high π 0 s(i.e., with tightly packed phospholipids) will correlate with its capacity for inserting into the viral target membrane (Nieva and Agirre, 2003; Rafalski et al., 1990). Penetration levels of the synthetic FPs depending on π 0 are shown in Fig. 5A. The monolayer exclusion pressures, π ex , or the maximum initial lateral pressures at which membrane-association was accompanied by peptide integration into the monolayer, were Fig. 2. Expression of E2 glycoprotein in SK6 cells transfected with the indicated constructs (detection was performed using anti-E2 mAb WH303) by (A) immunocytochemistry, and (B) Western blot. I.J. Fernández-Sainz et al. / Virology 456-457 (2014) 121–130124 comparable for WT and mut-1, and consistent with their penetration into membranes with the lipid packing density existing at the target cell membrane ( π c Z30 mN/m, (Marsh, 2007). In contrast, the mut-2 variant was unable to efficiently penetrate into monolayers compressed to those levels. To determine degrees of association with lipid bilayers, we next compared partitioning of the three sequences into vesicles by monitoring changes in the emitted NBD-fluorescence (Fig. 5B). The partitioning coefficients, estimated upon titrating the peptides with increasing quantities of vesicles, were all in the range of 10 5 , therefore denoting comparable degrees of association for the three variants. However, NBD intensity increased by a factor of 10 in the case of the mut-1 peptide, while this factor was reduced to 3 for the mut-2 peptide. The higher increase in emission intensity was consistent with deeper penetration of the fluorophore into the hydrocarbon core of the lipid bilayer in the former case. The WT NBD emission increased by a factor of 5, consistent with an intermediate level of insertion. In summary, lipid monolayer and NBDfluorescence results suggest that the triple substitution interferes with FP penetration into the target membrane, while replacing the CC residues results in deeper insertion into the low-polarity region. Finally, results displayed in Fig. 5C revealed that WT inserted into membranes was capable of perturbing the lipid bilayer C818S C828S I820T P819S V826S/I827T W822S BIC 0 1 2 3 4 5 6 7 8 2hr 24hr 48hr 72hr Log10 TCID 50 /ml Hours post-infection C818S C828S P818S I819S W822S V826T/I827T BIC Fig. 3. in vitro growth characteristics of FP mutants and parental BICv. (A) Primary swine macrophage cell cultures were infected (MOI¼0.01) with each of the FP virus mutants or BICv and virus yield titrated at times post-infection in SK6 cells. Data represent means and standard deviations from two independent experiments. Sensitivity of virus detection: Z1.8 TCID50/ml. (B) Plaque formation of the FP virus mutants and BICv. SK6 monolayers were infected, overlaid with 0.5% agarose and incubated at 37 1C for 3 days. Plates were fixed with 50% (vol/vol) ethanol-acetone and stained by immunohistochemistry with mAb WH303. Table 2 Swine survival and fever response following infection with E2FP mutants and parental BICv. Virus No. of survivors/ total no. Mean time to death (days7SD) Fever No. of days to onset (days7SD) Duration (days7SD) BIC 0/4 7.75 (1.5) 3.75 (0.9) 5 (1.4) C818S 0/4 7.25 (0.9) 4 (0.0) 4.25 (0.9) C828S 0/4 6.75 (2.1) 4 (0.0) 3.75 (0.9) P819S 0/4 7.75 (0.5) 5 (1.1) 4.75 (1.7) W822S 0/4 9.5 (3) 5.5 (2.3) 4.75 (1.7) V825T/ I826T 0/4 9.5 (2.3) 3.5 (1) 7 (2.1) I820T 0/4 8.5 (2.1) 3.5 (0.7) 6 (1.4) I.J. Fernández-Sainz et al. / Virology 456-457 (2014) 121–130 125 architecture more efficiently than the mutant-representing peptides. In these assays the integrity of the POPG membrane was monitored using the ANTS/DPX assay (Nieva et al., 1994; Rafalski et al., 1990). A peptide-to-lipid ratio of 1:700 was required to induce 50% of vesicle permeabilization by WT, whereas the ratio increased to 1:300 for mut-1 and this level of permeabilization was barely attained with the highest dose of mut-2 tested (1:100). Thus, for comparable amounts of peptide associated with the membrane, the WT sequence was more efficient than the mutants at destabilizing the integrity of the vesicles. Discussion This report attempted to characterize the role of the putative FP comprising the amino acid residues 818 CPIGWTGVIEC 828 of glycoprotein E2 in CSFV. Reverse genetics and an in vitro artificial membrane assays were used to assess the role of individual residues within the E2 FP in the process of virus replication and membrane insertion. Double (C818S/C828S) or triple (P819S/I820S/W822S) residue substitutions completely abrogate virus replication, additionally, the triple substitution led to a decreased capacity for membrane insertion. The characterization of the structure and membrane interactions of synthetic peptides representing the double and triple substitutions in the FP indicated that the conformations adopted by the WT and mutated peptides were compatible with the extended β -type structure of the FP. Such structures were not significantly altered in the presence of membrane mimetics, indicating the amino acid substitutions did not affect the overall conformation adopted by the FP in solution or the low-polarity membrane-like environment. In contrast, the three peptides displayed different patterns of interactions with membrane models. The ability of synthetic peptides and their mutant variants to penetrate into POPG monolayers was earlier described for the HIV-1 FP (Nieva et al., 1994; Rafalski et al., 1990). Those studies demonstrated that a polar substitution that inactivated the fusion glycoprotein also interfered with the penetration of the FP into the POPG monolayer. In our experiments, the triple substitution (P819S/ I820S/W822S) had a comparable blocking effect on the ability of the E2 FP. This mutant associated with POPG vesicles as efficiently as WT and double substitutions, but induced therein less permeabilization. Thus, we assume that the triple substitution causes a shallower association with the lipid bilayer, a possibility supported by the more polar environment sensed by the NBD fluorescent probe. In this regard, proline and tryptophan residues sustain insertion of the Ebola virus FP as an integral membrane hairpin, and are required for the fusogenic activity of the envelope glycoprotein. Structure and function of the complete internal fusion loop from Ebola virus glycoprotein 2 (Gregory et al., 2011). By analogy, the lack of FP insertion induced by the triple substitution might interfere with E2 fusogenic activity and prevent CSFV infection. By comparison the peptide harboring the double substitution inserted efficiently into monolayers and penetrated deeper into the lipid bilayer surrounding the vesicles (Nieva and Agirre, 2003). In this case it is possible that a di-Sulfide bridge stabilizing a functional integral haipin would be absent in the FP mutant, thereby allowing its deeper insertion but hampering its fusogenic activity. Nonetheless, this mutant was still less efficient than the WT at perturbing the membrane. It is postulated that the membrane restructuring effect will be more potent for peptides inserted at the level of the phospholipid glycerol backbone, than for peptides associated at the level of the headgroups of the phospholipids, or than for those inserted deeper into the acyl-chain region (Nieva and Agirre, 2003). We infer that, compared to the mutant derivatives, the WT peptide attains an intermediate depth of penetration to exert maximal lipid bilayer destabilization effects. This observation would support its role as a membrane-destabilizing anchor during fusion and explain its involvement in viral propagation. Recently the E2 protein of BVDV has been crystallized, revealing a three domain structure. Domains I and II are similar to Ig-like domains and domain III is a series of three small β -sheet modules; this structure is believed to be similar to CSFV E2 by prediction analysis (Iourin et al., 2013; Li et al., 2013). Using the crystal structure for BVDV-1 E2 at ph 8.0 (pdb:2yq2) (El Omari et al., 2013), the putative FP appears to be partially accessible, however the critical residues P819S/ I820S/W822S appear to be mostly internal situated (Fig. 6). In summary, this preliminary characterization of the functionality of the putative FP comprising the amino acid residues 818 CPIGWTGVIEC 828 of glycoprotein E2 in CSFV demonstrated that altering this area although do not significantly alter protein expression, results critical in the process of virus replication and the ability of the protein to be inserted in cell membranes. Materials and methods Viruses and cells Swine kidney cells (SK6) (Terpstra 1990), free of BVDV, were cultured in Dulbecco's minimal essential media (DMEM) (Gibco, Grand Island, NY) with 10% fetal calf serum (FCS) (Atlas Biologicals, Fort Collins, CO). CSFV Brescia strain was propagated in SK6 cells Fig. 4. Designation and structure of E2 FP-derived synthetic peptides. (A) Designation and sequences of the peptides used in this study. Substituted residues are indicated in bold. (B) Secondary structures. The structural components were calculated for the CD spectra obtained in buffer (black bars), or in the presence of 100 mM DPC (white bars) or 100 mM SDS (gray bars). Means7SD for the fraction values estimated with CONTIN-LL, CDSSTR and SELCON3 programs are plotted for the peptides as indicated in the panels. H, helix; S, strand; TþU, turnsþunordered. I.J. Fernández-Sainz et al. / Virology 456-457 (2014) 121–130126 and was used for the construction of an infectious cDNA clone (Risatti et al., 2005). Growth kinetics was assessed using primary swine macrophage cell cultures prepared as described by Zsak et al. (1996). 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 monoclonal antibody WH303 (mAb WH303) (Edwards et al., 1991) and the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Titers were calculated according to the method of Reed and Muench (1938) and expressed as TCID 50 /ml. As performed, test sensitivity was Zlog 10 1.8 TCID 50 /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 mutations in the FP area were obtained using the QuickChange XL Site-Directed Mutagenesis kit (Stratagene) performed per manufacturer's instructions using full-length pBIC as template and the primers described in Table 1. The product was then digested with Dpn1, leaving only the newly amplified plasmid, transformed into XL10Gold ultracompetent cells, and grown on Terrific Broth Agar Plates with ampicillin (Teknova). Positive colonies were selected for by sequence analysis of the E2 gene and grown for plasmid purification using a Maxiprep kit (Qiagen Sciences, MD). Each of the IC constructs were completely sequenced to verify that only sitedirected mutagenesis-induced changes were present. in vitro rescue of CSFV Brescia and FP mutants Full-length genomic clones were linearized with SrfI and in vitro transcribed using the T7 Megascript system (Ambion, Austin, TX) (Risatti et al., 2005). RNA was precipitated with LiCl and transfected into SK6 cells by electroporation at 500 V, 720 Ω , 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 1C Fig. 5. Interactions with model membranes of E2 FP-derived synthetic peptides. (A) Penetration into POPG monolayers. Maximum increase in surface pressure induced upon injection of 0.4 μM peptide into the subphase was measured as a function of the initial surface pressure of the phospholipid monolayers. Monolayer exclusion pressures are indicated in the panels. The dotted line begins at 30 mN/m. (B) Partitioning curves as estimated from the fractional change in NBD-fluorescence in the presence of increasing amounts of POPG LUV. The solid lines correspond to the best fittings of the experimental values to equation [1]. The estimated K x values are displayed in the panels. (C) Final extents of ANTS leakage (percentage after 30 min) from POPG LUV as a function of the lipid-to-peptide mole ratio. Lipid concentration (100 mM) was fixed. The amount of peptide bound to membrane was corrected according to the partition coefficients in panel B. EC 50 values are displayed in the panels. I.J. Fernández-Sainz et al. / Virology 456-457 (2014) 121–130 127 and 5% CO 2 . Virus was detected by immunoperoxidase staining as described above, and stocks of rescued viruses were stored at 70 1C. DNA sequencing and analysis Full-length clones and in vitro rescued viruses were completely sequenced with CSFV-specific primers by the dideoxynucleotide chain-termination method (Sanger et al., 1977). Viruses recovered from infected animals were sequenced in the region of the genome that contained the desired mutations. Sequencing reactions were prepared with the Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Reaction products were sequenced on a PRISM 3730xl automated DNA sequencer (Applied Biosystems). Sequence data was assembled using Sequencher 4.7 ™ software (Genes Codes Corporation, Ann Arbor, MI). The final DNA consensus sequence represented, on average, a threeor four-fold redundancy at each base position. Animal infections Virulence of FP mutant viruses relative to BICv was initially assessed in 10–12 weeks old, forty-pound commercial-breed pigs inoculated intranasally (IN) with 10 5 TCID 50 of each virus. Pigs were randomly allocated into 7 groups of 4 animals each and were inoculated with a 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 and scored as previously described (Mittelholzer et al., 2000). Blood was collected at times postinfection from the anterior vena cava into EDTA-containing tubes (Vacutainer) for total and differential white blood cell counts (performed using a Beckman Colter ACT, Beckman, Colter, CA) and quantification of viremia by virus titration as described above. Peptides Peptides representing the CSFV E2 FP (WT) and its derived mutants (mut-1 and mut-2, sequences displayed in Fig. 2A) were commercially synthesized (Thermo Scientific) and tagged with the 7-nitrobenz-2-oxa-l,3-diazole-4 (NBD) fluorophore at the N-terminus. The purified peptides were dissolved in dimethyl sulfoxide (DMSO, spectroscopy grade) and their concentrations determined by the bicinchoninic-acid microassay (Pierce, Rockford, IL, USA). Small, diluted aliquots (typically 20 μ L, 1 mg/mL) were stored frozen and were thawed only once, upon use. 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) was purchased from Avanti Polar Lipids (Birmingham, AL). The 8-aminonaphthalene-1,3,6-trisulfonic acid sodium salt (ANTS) and p-xylenebis (pyridinium)bromide (DPX) were obtained from Molecular Probes (Junction City, OR). Dodecylphosphocholine (DPC) was from Anatrace (Maumee, OH, USA). Circular dichroism srudies Circular dichroism (CD) measurements were obtained from a thermally-controlled Jasco J-810 circular dichroism spectropolarimeter calibrated routinely with (1S)-(þ)-10-camphorsulfonic acid, ammonium salt. Samples consisted of lyophilized peptides dissolved at concentrations of 0.03 mM in 2 mM Hepes (pH 7.4) buffer. Spectra were measured in a 1 mm pathlength quartz cell initially equilibrated at 25 1C. Data were taken with a 1 nm bandwidth at 100 nm/min speed, and the results of 20 scans were averaged. Lipid monolayer penetration Changes in surface pressure were monitored as a function of time in a fixed-area circular trough ( μ Trough S system, Kibron, Fig. 6. Location of putative FP into schematic representation of a tridimensional structure of BVDV E2 (El Omari et al., 2013). Critical cystines are presented in green, the PI.W residues in blue, and the remainder of the fusion peptide in yellow. The N-terminus is colored red for orientation. (A) A ribbon model is shown with the fusion peptide colored and the surrounding β-sheets are colored in orange (B) The location of the putative FP it is shown a space filling model. I.J. Fernández-Sainz et al. / Virology 456-457 (2014) 121–130128 Helsinki) measuring 2 cm in diameter and with an aqueous volume of 1 ml (5 mM Hepes, 100 mM NaCl (pH 7.4)). Lipids, dissolved in chloroform, were spread over the surface and the desired initial surface pressure ( π 0 ) was attained by changing the amount of lipid applied to the air-water interface. Peptides were injected into the subphase to a final concentration of 0.4 μ M with a Hamilton microsyringe. Lipid vesicle assays Large unilamellar vesicles (LUV) made of POPG were prepared according to the extrusion method in 5 mM Hepes, 100 mM NaCl (pH 7.4) using membranes with a nominal pore-size of 0.1 mm. Partitioning into membranes was evaluated by monitoring the change in the emitted NBD-fluorescence. Corrected spectra were recorded using a FluoroMax-3 (Jobin Yvon Horiba, Longiumeau, France) with excitation set at 460 nm and 2-nm slits. Partitioning curves were subsequently computed from the fractional changes in emitted NBD-fluorescence when titrated with increasing lipid concentrations. The apparent mole fraction partition coefficients, K x(app) , were determined by fitting the experimental values to a hyperbolic function: F=F 0 ¼1þ½ðF max =F 0 Þ1½L Kþ½Lð1Þ where [L] is the lipid concentration and Kis the lipid concentration at which the bound peptide fraction is 0.5. Therefore, K x(app) ¼[W]/ Kwhere [W] is the molar concentration of water. tlsb-0.009w?>Vesicle permeabilization was assayed by monitoring the release to the medium of encapsulated fluorescent ANTS as described (Nieva et al., 1994). In brief, LUV containing 12.5 mM ANTS, 45 mM DPX, 20 mM NaCl and 5 mM Hepes were obtained by separating the unencapsulated material by gelfiltration in a Sephadex G-75 column that was eluted with 5 mM Hepes and 100 mM NaCl (pH 7.4). Fluorescence measurements were performed by setting the ANTS emission at 520 nm and the excitation at 355 nm. A cutoff filter (470 nm) was placed between the sample and the emission monochromator. The baseline leakage (0%) corresponded to the fluorescence of the vesicles at time 0, while 100% leakage was the fluorescence value obtained after addition of Triton X-100 (0.5% v/v). Acknowledgments This study was in part supported by Spanish MINECO and Basque Government grants (BIO2011-29792 and IT838-13 to J.L.N.). 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