3.5.2. Other viruses
Sendai virus (SeV), Cantell strain, was purchased from Charles River Laboratories and stored at -80oC. The rVSV-GFP virus stock used in the bioassay assay was generated by
transiently transfecting HEK293T cells in Opti-MEM with an expression plasmid encoding the Vesicular Stomatitis Virus (VSV) glycoprotein of surface (pVSV-G, kindly provided by Brian Willett) (Martinez-Sobrido et al., 2006) using TransIT-LT1 transfection reagent (Cambridge Biosciences) at 37oC, 5% CO
2 for 36h (100mm sterile dish format, 5x106 cells). At the end of
the transfection period, the cells were infected with a previous stock of rVSV-GFP virus (2.5x102 TCID50) for 4h at 37oC, 5% CO
2 humidified atmosphere in growth medium, then
washed with PBS and maintained at 37oC, 5% CO
2 humidified atmosphere in growth medium
for a further 16h. The supernatant was then collected and filtered through a 0.45μm filter (Fisher Scientific), before being aliquoted and stored at -80oC.
3.5.3. Virus rescue
Viruses were rescued using the eight-plasmid system of A/equine/Uruguay/1/1963 and A/equine/Ohio/1/2003 (Hoffman 2001). In this system, the cDNA of each eight influenza virus segments is inserted between the Polymerase I promoter (PolI h) and the Polymerase I
terminator (ti). This PolI transcription unit is flanked by the Polymerase II promoter of the
human cytomegalovirus (PolII CMV) and the polyadenylation signal of the gene encoding
bovine growth hormone (aII BGH). After transfection of the eight plasmids in a co-culture
(2:3) of HEK293T/MDCK cells (6-well plate format, 2x106 cells/well), two types of molecules
are synthesized: negative-sense vRNA by the cellular PolI, and transcription of mRNAs with
5’-cap structures and 3’-poly(A) tails by PolII. These mRNAs are translated into viral proteins.
(Figure 3-1). Co-cultures were seeded 24h prior to viral rescue, and reverse genetic viruses are generated within 2–3 days following transfection. For transfection, cells were incubated in Opti-MEM (Opti-Minimum Essential Medium, Life Technologies) and transiently co- transfected using TransIT-LT1 (Mirus, Cambridge Bioscience), with 2.5 g of seven-ambisense O/03 or U/63 plasmids (PB2-, PB1-, PA-, HA-, NP-, NA-, M-pDP2002 plasmids) plus ambisense O/03 NS- or U/63 NS-pDP2002 plasmids or the NS mutant-pDP2002 constructs (O/03-K186E, O/03-230 and O/03-K186E-230; U/63-E186K, U/63-219, U/63-E186K-219, respectively). After 24h, the medium was replaced by infection medium. Virus-containing tissue culture supernatants (passage 0, P0) were collected 3 days post-transfection, clarified, and stored at -80oC.
Figure 3-1: Eight-plasmid system for generation of Influenza A virus
Schematic representation of the IAV eight-plasmid rescue system. The left-hand side illustrates the co- culture system of HEK293T and MDCK cells. Cells are co-transfected with eight plasmids encoding for the eight segments of the IAV of interest. Reverse genetic viruses are generated within 2–3 days post transfection. On the right-hand side, a schematic representation of the polymerase (Pol) I–polymerase (Pol) II transcription system for synthesis of vRNA and mRNA is shown. The cDNA of each of the eight influenza virus segments is inserted between the Polymerase I promoter (PolI h) and the PolI terminator
(ti). This PolI transcription unit is flanked by the Polymerase II promoter of the human cytomegalovirus
(PolII CMV) and the polyadenylation signal of the gene encoding bovine growth hormone (aII BGH).
After transfection of the eight expression plasmids, two types of molecules are synthesized: negative- sense vRNA by the cellular PolI, and transcription of mRNAs with 5’ cap structures and 3’ poly(A) tails
by PolII. These mRNAs are translated into viral proteins. Of note, the start codon (ATG) of each viral
cDNA is directly following the PolII transcription start site.
3.5.4. Creating virus stocks
For experimental infection, a minimum of two viral stocks of each virus was rescued and grown independently. P0 stocks were used to infect fresh MDCK cells to generate a passage 1 (P1) stock. After 2 to 3 days of infection (when 80% of the monolayer was destroyed), supernatant was collected, cleared and stored at -80oC. Viral titres of P1 stocks
were determined as described below. The P1 stocks were then used to generate passage 2 (P2) viral stocks. To this end, MDCK cells were infected with P1 stocks at MOI 0.01 and after 2 to 3 days of infection (when 80% of the monolayer was destroyed), supernatants were
collected, cleared and stored at -80oC. The sequence of each genomic segment was checked
by Sanger sequencing and the size of each segment was checked by PCR and 1% agarose gel electrophoresis (Appendices 20 to 31). Viral titres of P2 stocks were determined as described
MDCK 293T pDP2002- PA pDP2002-HA pDP2002- PB2 pDP2002- PB1 pDP2002- M pDP2002- NS pDP2002- NP pDP2002-NA
ATG…...Viral cDNA …...TGA
PolIICMV aIIBGH PolIh tI Translation Viral proteins Cap A poly(A) (+) mRNAs
RNA polII(splicing)
RNA polI
ppp
below. For experimental infections, a minimum of two viral stocks for each virus were rescued and grown independently.
3.5.5. Experimental viral infections
Confluent monolayers of MDCK cells (12-well plate format, triplicates, 5x105
cells/well) or E.Derm cells (12-well plate format, triplicates, 2.5x105 cells/wells) were
infected (MOI 0.01 and 0.1, respectively) with the indicated viruses and placed at 37oC, 5%
CO2. After 1h incubation, cells were washed with PBS and infection medium was replaced
with 500 l of fresh growth medium. Tissue culture supernatants were collected at various times pi and stored at -80oC, and cells were fixed in 0.1% buffered formalin at 4oC for 16h
and kept for flow cytometry analysis. Each experiment was repeated three times independently. Viral titres were determined by immunofocus assay in MDCK cells. Titrations were repeated three times independently, and the mean value and standard error mean were calculated using GraphPad Prism7 (GraphPad Software Inc. San Diego, CA, USA). Of note, the presence of deficient interfering particles in viral stocks was not assessed. In addition, the level infectious particles versus genomic information was not compared between viruses. Thus, the differences observed throughout this work could either reflect differences due to the mutations introduced in NS1 of /03 and U/63 or NS segment swap between O/03 and U/63, or be due to differences in infectivity levels and ability to induce an IFN response between WT and mutant viruses.
3.5.6. Determination of viral titre and plaque phenotype by immunofocus
assay
Viral titres were determined by immunofocus assay (focus forming units, FFU/ml) in MDCK cells. Confluent monolayers of MDCK cells (48-well plate format, triplicates, 1.25x103
cells/well) were infected with serial dilution (1:10) of the viral stock of interest and placed at 37oC, 5% CO
2. Plates were gently rocked every 10 minutes. After 1h incubation, cells were
washed with PBS and infection medium was replaced with a 50:50 2.4% Avicell:2X MEM overlay for 48h. 2.4% Avicell solution was prepared as follow: 2.4% (w/v) of Avicell (Avicell RC/CL, Microcrystalline cellulose & Sodium carboxymethylcellulose1, Sigma-Aldrich) in
100ml of H2O. Autoclaved and stored at room temperature until use. At 48hpi, the overlay
was discarded, cells were washed 3 times with PBS and fixed with 80% ice-cold acetone solution (Acetone AR, >99.5%, Sigma-Aldrich) for 10 minutes at room temperature. The
plates were let to dry overnight at room temperature. then treated with 1% Triton X-100
PBS solution (TritonTM X-100, Sigma-Aldrich-Aldrich) for 10 minutes at room temperature,
followed by 1h of incubation with 10% NGS PBS solution at room temperature. This was followed by 3h of immunoblotting at room temperature in 10% Normal Goat Serum plus PBS with a monoclonal anti-influenza A virus nucleoprotein (NP) antibody (clone HB65, European Veterinary Laboratory) (Table 3-8). After a 3-step washing with PBS, a horseradish peroxidase-conjugated rabbit anti-mouse IgG antibody (AbD Serotec, UK) (Table 3-8) was used in PBS solution for a further 1h at room temperature. A color development method was used to reveal the immunofocus using the TrueBlue peroxidase substrate (Insight Biotechnology). 50 l of substrate per well was used under agitation for 10 minutes of incubation, and stop with water. Viral titres were calculated by counting the number of blue plaques and were expressed as log10 PFU per millilitre.
For plaque phenotype, a confluent monolayer of MDCK cells (6-well plate format, triplicates, 6.4x104 cells/well) were infected with serial dilution (1:2) of the viral stock of
interest and placed at incubation at 37oC, 5% CO
2, humidified atmosphere. The same
protocol as described above was then followed. For colour development, 2ml of TrueBlue peroxidase substrate was used per well.
3.5.7. Viral protein staining for flow cytometry and confocal microscopy
Cells were permeabilized with 1% Triton X-100 for 10 minutes, and blocked in PBS 10% Normal Goat Serum (Gibco, Life Technologies) for 1h. Cells were then incubated with anti-NP antibody, rabbit polyclonal anti-NS1 protein antibody (Genscript) or rabbit STAT1polyclonal antibody (Santa Cruz) overnight at 4oC. Cells were then washed twice with PBS
and incubated for 4h with a rabbit anti-mouse IgG Alexa fluor 488 (Cell signalling) or donkey anti-rabbit IgG Alexa fluor 555 (Cell signalling), before analysis by flow cytometry (Guava Flow Cytometer, Merck) or fixed in VECTASHIELD Mounting Medium with DAPI
(VECTASHIELD Hard+SetTM Mounting Medium with 1.5 g/ml DAPI, Vector laboratories) and
analysed by confocal microscopy.
3.5.8. Viral growth kinetics with Ruxolitinib or universal IFN
Ruxolitinib (Selleck Chemicals), a JAK1/2 inhibitor (Stewart et al., 2014), was prepared as 10mM stocks in dimethyl sulfoxide (DMSO). Several doses of Ruxolitinib were tested in the presence of 500 Units (U) of universal type I IFN alpha (uIFN, pbl Assay Science)
(Figure 3-2). To measure virus growth kinetics in cells rendered un-responsive to type I IFN or in the presence of exogenous type I IFN, E.Derm cells were treated with Ruxolitinib at a concentration of 4 M as determined in or with 500U of uIFN. Treatments were started 24h prior to infection and maintained at the same concentration for the whole experiment.
Figure 3-2: Test of Ruxolitinib concentrations
E.Derm cells were treated with different amounts of Ruxolitinib (0.5 to 40 M) or mock treated with 0.4% of DMSO for 24 h prior to treatment with universal IFN (uIFN). At the indicated time post infection (hpi) (A) Mx proteins expression was assessed by Western blot (78 and 76 kDa), and (B) cytopathic effect was evaluated using a confocal microscope (transmitted light mode).
3.6. Antiviral cytokine production and general protein shutdown.
E.Derm cells (12-well plate format, triplicates, 2.5x105 cells/well) were infected
(MOI 0.1) with the indicated viruses for a total of 72h. At the indicated times post-infection, supernatants were collected and stored at -80oC for further analysis by bioassay, while cells
were treated for 1h at 37oC, 5% CO
2 with Puromycin (20µg/ml in DMEM, 15% FBS) prior to
lysis in protein disruption buffer + -mercaptoethanol and stored at -80oC for further analysis
by western blot. Puromycin is a well-known antibiotic that competes against aminoacyl tRNA on the ribosome A site (Monro et al., 1968). As such, puromycin enables examination of total protein production without requiring transfection, radio-labeling, or the prior choice of a candidate gene (Starck et al., 2004). If cells are incubated with puromycin, lysed and immunoblotted using an anti-puromycin antibody, all the proteins being produced will be immunostained as puromycin will be incorporated at the C-terminus of all nascent proteins. For the IFN bioassay, supernatants were UV-inactivated for 5 minutes at room temperature and used to treat fresh E.Derm cells (48-well plate format, 6x104 cells/well, triplicates) for
24h. The cells were then infected with rVSV-GFP virus (2.5x102 TCID50) for 8h, then
trypsinized and fixed in 0.1% buffered formalin for 16h at 4oC. The percentage of GFP-
expressing cells were then analysed by flow cytometry. For controls, E.Derm cells were mock treated or treated with 500U of uIFN. GFP expression of mock-treated cells infected with rVSV-GFP was considered as 100% and GFP expression of uIFN-treated cells infected with rVSV-GFP was considered as 0%. Mean values and standard error means were calculated with GraphPad Prism7.
3.7. SDS-PAGE & Western blot analysis
Cells were lysed in protein disruption buffer (0.125 M Tris-HCl (pH 6.8), 4% (w/v) SDS, 25% (v/v) glycerol, 0.02% (w/v) bromophenol blue) + -mercaptoethanol and stored immediately at -80oC. Samples were boiled for 15 minutes at 95oC prior to polypeptide
separation by SDS-PAGE on NuPAGE Novex 4-12% Bis-Tris protein gels (ThermoFisher Scientific) using 20X NuPAGE MES SDS Running Buffer (Invitrogen), diluted in H2O. Proteins were detected by Western blotting following to transfer to nitrocellulose membranes using
20X NuPAGE Transfer Buffer (Novex, Life technologies) diluted in H2O. The membranes were
blocked for 1h at room temperature in 5% blotting-grade blocker (nonfat dry milk, Bio-Rad)
TBS 0.1% Tween-20 (TWEEN 20, Sigma-Aldrich) and immunoblotted overnight at 4oC in 5%
blotting-grade blocker TBS-0.1% Tween-20 with the antibody of interest. 10X TBS buffer (Tris base 48.4g, NaCl 160g, H2O up to 2L, pH adjusted at 7.6, Sigma-Aldrich) was diluted to
1X in H2O for use. Antibodies used in this study are summarized in table 3-8. The
chemiluminescent signal was detected using Amersham ECL Prime Western Blotting Detection Reagent (GE-Healthcare) and captured with ChemiDoc XRS+ System (Biorad).
3.8. RNA sequencing
Confluent monolayers of E.Derm cells (12-well plates, triplicates, 2.5x105
cells/wells) were infected with O/03 and revertant viruses (MOI 0.1 to 1) or mock infected at least three times independently. At 8 hpi cells were washed with PBS, immunostained with the anti-NP antibody and the proportion of infected cells was determined by flow cytometry. The samples containing a similar proportion of infected cells were selected for transcriptomic analysis, and cells were lysed with 500 l of TRIzol (ThermoFisher Scientific)
for further RNA extraction. Total RNA was extracted using the TRIzol method and further
purified using the RNeasy mini spin columns (Qiagen), including an on-column DNase I digestion step (Qiagen) according to the manufacturer’s protocol. RNA concentration was measure with Qubit and the Qubit RNA HS Assay Kit (ThermoFisher Scientific) following the manufacturer's protocol. The ribosomal (r)RNA integrity number was measured using an
Agilent 2100 BioAnalyzer (Agilent Technologies). Library preparation and sequencing were
carried out by the Viral Genomics and Bioinformatic team of the MRC University of Glasgow Centre for Virus Research. 4.5ug of total RNA was enriched by selectively depleting rRNA
using the RiboMinusTM Eukaryote Kit v2 (Ambion, Life Technologies). The sequence reads
were processed according to the Tuxedo pipeline (Trapnell et al., 2013). Read quality was assessed using FastQC, and TopHat2 and Bowtie2 were used to map short reads against the Equus caballus 2 genome (GCA_000002305.1). A list of differentially expressed genes (DEGs) to mock-infected samples was generated using CuffDiff2 (genes with Benjamini Hochberg-p value <0.05 were considered significant) (Ratinier et al., 2016). A complete list of DEGs in all samples can be found in Appendices 35 to 67.
3.9. Analysis of EIV NS1 amino acid sequences
For the phylogenetic analysis of EIV NS1, a total of 170 NS sequences were collected from the NCBI Influenza Virus Resource database. Four NS segments sequenced for this study were also added (42). SeaView (Version 4.6.1) (43) was used to align the NS1 coding regions and the final alignment (http://datadryad.org/review?doi=doi:10.5061/dryad.673bj) was edited manually to remove NEP coding regions. BEAST (Version 1.8.4) (44) was used to infer maximum clade credibility trees, as well as a strict molecular clock and HKY85+G model of nucleotide substitution. Each codon position was estimated with separate substitution rates and nucleotide frequencies. Two individual chains were run until convergence was achieved. To analyse NS1 single nucleotide polymorphisms (SNP), the 175 NS1 amino acid sequences were aligned with CLC genomic workbench 8 (QIAGEN Bioinformatics) (Appendices 1 and 2)
and the frequency of each amino acid found at each of the 230 composing the NS1 protein was recorded. A threshold of 7% was used in accordance with the “best practices for evaluating single nucleotide variant calling” (Olson Frontiers in Genetics 2015).
3.10. Graphing and statistical analysis
All statistical analyses were conducted using GaphPad Prism software. Unless stated otherwise, significance was calculated by Two-way ANOVA and a Bonferroni's multiple comparisons post hoc test.
3.11. Antibodies
All primary and secondary antibodies used in this work can be found in Table 3.8. Table 3-8. Antibodies
Target Antibody Dilution
NS1 Polyclonal rabbit, IgG, (GenScript)
1:1000 WB 1:500 IF NP Monoclonal mouse, IgG2 , clone HB65 (European Veterinary
Laboratory)
1:1000 WB 1:1000 IF MX1 Monoclonal mouse, IgG2 , clone M143
(kindly provided by Georg Kochs)
1:500
ISG15 Polyclonal rabbit, IgG, fusion protein ag8782 (Proteintech) 1:500
STAT1 (p84/p91)
Polyclonal rabbit, IgG, clone M-22 (Santa Cruz)
1:1000
Caspase 3 Polyclonal rabbit, IgG (Cell signaling)
1:1000
Cleaved caspase 3
Polyclonal rabbit, IgG, clone Asp175 (Cell signaling)
1:500
HA-tag Rabbit polyclonal, IgG (Sigma-Aldrich)
1:1000
Puromycin Monoclonal mouse, IgG2 , clone 12D10 (Millipore) 1:1000 -Tubulin Polyclonal rabbit, IgG, clone AK-15
(Sigma-Aldrich)
1:2000
Rabbit Polyclonal donkey, IgG, HRP-conjugate (GE-healthcare)
1:2000
Mouse Polyclonal rabbit, IgG, HRP-conjugated (Starlab, Biorad)
1:2000
Mouse Polyclonal donkey, IgG, Alexa Fluor 488-linked (ThermoFisher Scientific)
1:2000
Rabbit Polyclonal mouse, IgG, Alexa Fluor 647-linked (ThermoFisher Scientific)