Sequence analysis
The complete DNA sequence coding for gG was determined by the dideoxy chain-termination method. Raw sequence data were assembled using the ContigExpress function of Vector NTI suite 9.1 software (Invitrogen, Carlsbad, CA, USA). The DNA sequence coding for gG was translated using the same software and nucleotide and amino acid sequences were compared to EHV-3 sequence databases using Basic Local Alignment Search Tool (BLAST). The SignalP-6.0, TMHMM-2.0 and ProtPi online tools were used for signal peptide, protein topology and molecular weight predictions, respectively. N-and O-linked sites for glycosylation were predicted from its deduced amino acid sequence using NetOglyc 4.0 and NetNglyc 1.0, online tools respectively.
Viral and BAC DNA extraction, PCR and sequencing
Viral DNA for PCR was extracted using EasyPure Viral DNA/RNA Kit (TransGen Biotech) from 200 µl of cell culture supernatants of infected cells according to the manufacturer's instructions. Primers were designed based on the published data for EHV-3 strain AR/2007/C3A (GenBank Acc. Nº KM051845). PCRs reported in the study were carried out in a 50 μl reaction mixtures containing 5 µl 10x HiFi buffer, 0.5 μl of TransTaq®HiFi DNA Polymerase (Transgen Biotech), 2 µl of 2.5 mM each dNTPs, 1 µl each of 10 μM forward and reverse primers, 38.5 μl of H2O and 2 μl of purified DNA. The temperature profile, unless otherwise stated, consisted of an initial denaturation at 95°C for 2 min, followed by 30 cycles of DNA denaturation at 94°C for 15 s, primer annealing for 30 s at 53°C and an elongation step at 72°C for 1 min. The final extension step was carried out at 72°C for 5 min. For sequencing of PCR products, amplified DNA was purified with Wizard® SV Gel and PCR Clean-Up System (Promega). Sanger sequencing was performed in both directions and carried out by Cerela – CONICET, Argentina. BAC DNA was extracted by a column-based NucleobondBAC 100 kit(Clontech Laboratories) following the manufacturer's instructions. Viral DNA extraction for restriction fragment length polymorphism (RFLP) analysis was done as previously described [9]. Two micrograms of purified DNA was digested with a restriction enzyme for 3 h, separated by 0.8% agarose gel electrophoresis at 45 V for 18 h.
BAC mutagenesis and virus reconstitution
The recombinants were generated from an EHV-3 BAC (referred to as parental in this work) derived from EHV-3 strain C175 containing a restored gE coding region, as the latter strain codes for a truncated version of the gE protein that impact negatively in viral cell-to-cell spread in vitro [9]. For all genetic manipulations, E. coli strain GS1783 harboring a temperature-sensitive red recombination system and a gene encoding the endonuclease I-SceI in its genome was used (a kind gift from Dr. Greg Smith, Northwestern University, Chicago, IL, USA). For the manipulation of the EHV-3 BAC, the two-step red recombination protocol was followed as previously described [9, 10]. gG-minus and -tagged versions of EHV-3 were constructed respectively by deleting completely ORF70 using primers P1/P2 or adding an HA tag either after the predicted cleavage of the gG signal sequence or before the stop codon using primers P3/P4 and P5/P6, respectively (Table 1). Generated BACs pEHV-3_gGtagC, pEHV-3_gGtagN and pEHV-3ΔgG were characterized by RFLP and PCR (primers P11-P12) followed by sequence of the amplified product using primers P11 to P16 (Table 1).
For the construction of the revertant v_ΔgGr virus, sequence coding for gG and flanking nucleotide sequences were PCR amplified from parental EHV-3 BAC using primers P7/P8 which contained appropriate restriction sites to allow cloning into pcDNA3, resulting in the plasmid pcDNA_gG. Then a PCR product containing a 40-bp homologous sequence (nucleotides 191 to 294 of ORF 70), a Kr/I-SceI cassette and NotI sites at both ends was obtained using primers P9/P10 and using a Kr cassette containing plasmid [10] as a template. The 1038-bp amplified fragment was cloned into pUC_gG by using a unique NotI restriction site present in the gG ORF region (nucleotides 225 to 232 of ORF 70). Digestion of the generated plasmid with EcoRI and BamHI released a 2577-bp fragment containing flanking sequences, the gG sequence, the 40-bp replicated sequences and the Kr/I-SceI cassette. The reaction was treated with DpnI, purified by column gel purification and used for electroporation into GS1783 harboring pEHV-3ΔgG. The two-step mediated recombination protocol was then followed [9] to obtain a pEHV-3ΔgGr BAC.
Cells, viruses and transfection protocol
Equine dermis (EDerm) cells (NBL-6, ATCC; CCL-57) were used throughout the study. Cells were propagated in Dulbecco’s modified essential medium (DMEM) (ThermoFisher Scientific) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin.
Parental EHV-3 virus reconstitution from parental BAC was described previously [9]. Here, to produce BAC-derived viruses v_tagC, v_tagN, v_ΔgG and v_ΔgGr, EDerm cells were transfected with lipofectamine 2000 (ThermoFisher Scientific) as follow. Ten microliters of lipofectamine 2000 and 2 μg of column purified BAC DNA were each diluted in 50 μl of Opti-MEM I Reduced Serum Media (ThermoFisher Scientific). The DNA solution was then added to the lipofectamine solution and incubated for 15 min at room temperature. In the meantime, EDerm cells were trypsinized, washed once with Opti-MEM I and resuspended in the same medium at a concentration of 3x105 cells/100 μl. DNA- lipofectamine complexes were then added to the cells and further incubated at room temperature for 20 min. At the end of the incubation period, cells were plated in 1 well of a 12-well plate containing 500 μl of DMEM containing FBS and antibiotics. Transfected cells were incubated for 5-7 days until characteristic cytopathic effect (CPE) reached 80%. Virus stocks, produced after 2 more passages, were titrated by standard plaque assay using an overlaid medium consisting in complete MEM supplemented with 1.5% of carboxymethyl cellulose (SIGMA, medium viscocity). Generated viruses were characterized by RFLP and PCR (primers P11-P12) followed by sequence of the amplified product using primers P11 to P16 (Table 1).
Immunofluorescence
EDerm cells, grown on six-well plates, were infected with v_parental, v_tagC or v_tagN viruses at a multiplicity of infection (moi) of 0.1. At 8 h p.i., cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Samples were washed 3 times for 10 min with PBS, incubated in 50 mM NH4Cl in PBS for 30 min, rinsed 3 times for 10 min each in a washing solution (0.05 % saponin, 0.2 % BSA in PBS), and incubated overnight at 4°C with mouse anti-HA tag 6E2 monoclonal antibodies (Cell Signaling Technology) at 1/100 dilution. Finally, the samples were rinsed with wash solution (3 times for 10 min) and incubated with Cy3-conjugated anti-mouse IgG Ab (1/500 dilution, Jackson) for 1 h at 37°C. Fluorescent images were viewed and recorded with a Leica DMi8 imaging system.
Preparation of cell lysates
EDerm cells were seeded in 6-well plates that were either mock-infected or infected with appropriate viral stock (v_parental, v_tagC and v_tagN). When CPE reached 80%, cells and supernatants were harvested with the help of a scraper and centrifuged at 13,000 x g for 15 min. Pellets were resuspended in PBS, further centrifugated at the same speed and supernatants discarded.
Virus purification and protein precipitation from supernatans of infected cells
EDerm cells, grown on 10-cm cell culture dishes, were infected with v_ΔgG, v_tagC or v_tagN viruses at a moi of 3 in a serum-free medium. When CPE reached 80%, supernatants were harvested and subjected to two subsequent centrifugation steps using 13,000 x g for 15 min and 80,000 × g for 90 min spins. At the end of the second centrifugation, supernatant was carefully taken and the viral pellet washed with PBS. For protein purification from supernatants, a trichloroacetic acid (TCA) method was used. Briefly, the organic solvents used here, including acetone and 50% TCA/acetone, were precooled at -20˚C. TCA solution was added to the protein extract (1:1, v/v, with a final 25% TCA/50% acetone), the mixture was placed on ice for 10 min, centrifuged at 13,000 x g for 5 min and the supernatant was discarded. The protein precipitate was washed with 200 µl cold acetone and centrifuged as above. The washing step was repeated once. The protein precipitate was dried by placing tubes in a 95°C heat block for 5 min to drive off acetone and finally treated for immunoblotting as described below.
Immunoblotting
Precipitated supernatant’s proteins, viral pellets and cell lysates were treated as described [9], subjected to a 12% SDS-PAGE and transferred onto a nitrocellulose membrane (GE Healthcare) by a wet transfer at 30 V overnight. Membranes were blocked using 5% nonfat milk and then probed with antibodies diluted in PBS-0.1% Tween-20. Expression of EHV-3 gG was detected with an anti-HA tag monoclonal antibody (1/400 dilution, Cell Signaling Technology) followed by an anti-mouse IgG-peroxidase conjugate (1/2500 dilution, Sigma). Beta actin (β-actin) was detected using β-Actin (8H10D10) Mouse mAb (1/1000 dilution, Cell Signaling Technology). Reactive bands were visualized by enhanced chemiluminescence (ECL plus - GE Healthcare) in an ImageQuant LAS 4000 (GE Healthcare).
In vitro characterization of BAC-derived EHV-3
To assess virus replication in vitro of v_parental, v_ΔgG and v_ΔgGr generated viruses, plaque size measurements and one-step growth kinetics were determine as described [9].For plaque size measurements, briefly, confluent monolayers of EDerm cells in 6-well plates were infected with 20 PFU/well of the respective virus, overlaid with a semi-solid medium and after 2 days post-infection (p.i.) plaques were fixed and stained with a solution containing 2 mg/ml of crystal violet, 40% methanol and 10% formaldehyde for 2 h. Ten plaques for each independent experiment (30 in total) were photographed and the plaque area was determined using the ImageJ software (http://rsb.info.nih.gov/ij/). For one-step growth kinetics, EDerm cells grown in 24-well plates were infected at moi of 3 and treated at 0, 4, 8, 12, 24 and 36 h p.i. exactly as detailled [9].
Statistical Analysis
All data was analyzed with GraphPad Prism 5 software. The Shapiro–Wilks test was used to assess for normality. An unpaired Student’s t-test (two-tailed) was used to determine significant differences in virus titers in growth kinetics and plaque size. The data represent a mean ± standard error of the mean (SEM) of three independent experiments. The differences were only considered significant when p < 0.05.