Cells and viruses
The DEV strain CHv (GenBank No. JQ647509.1) was procured from Avian Disease Research Center of Sichuan Agricultural University. Duck embryo fibroblast (DEF) cells were maintained in modified Eagle’s Minimum Essential Medium (MEM) (Thermo Fisher, USA) supplemented with 10% bovine serum at 37°C in a 5% CO2 atmosphere. Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco’s Modified Eagle’s Minimum Essential Medium (Thermo Fisher, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, USA), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a 5% CO2 atmosphere.
Antibodies and vectors
Rabbit anti-UL21 polyclonal antibodies were generated in this study, and rat anti-UL16 polyclonal antibodies were provided by He Qin (31). The following monoclonal antibodies were used in this study: rabbit anti-GRP78 BiP (Abcam, UK), mouse anti-TGN46 (Abcam, UK), goat anti-rabbit IgG (Thermo Fisher Scientific, USA), rabbit anti-Myc tag (Beyotime, CHN), mouse anti-Flag tag (Transgen Biotech, CHN), Alexa Fluor 594 Goat anti-Rabbit IgG (Thermo Fisher Scientific, USA), Alexa Fluor 488 goat anti-mouse IgG (Thermo Fisher Scientific, USA), Alexa Fluor 488 goat anti-rat IgG (Abcam, UK), Alexa Fluor 594 goat anti-rabbit IgG (Life Technologies, USA) , and mouse anti-β-actin (Beyotime, CHN). Normal rabbit IgG was obtained from Beyotime, and normal rat IgG was obtained from Thermo. The pCAGGS (32) and pCMV-Myc (33) plasmids were provided by the Sichuan Agricultural University Avian Diseases Research Center.
Preparation and identification of polyclonal antibodies
pUL21 was expressed and purified via gel and electrioi elution. Approximately 1 mg of UL21 was emulsified in complete Freund’s adjuvant (Sigma, GER) and used to immunize rabbits through intradermal injections. Subsequent booster doses of 1 mg, 1.5 mg and 0.5 mg were prepared in incomplete Freund’s adjuvant, and the protein was administered after 2 and 3 weeks by subcutaneous injection. To collect the antibodies, the rabbits were bled through an ear vein 1 week after the last immunization. The antiserum was harvested, and preliminary purification was conducted using saturated ammonium sulfate. Antibody production followed the Sigma polyclonal antibody production method.
Western blotting
For western blotting (WB), lysates were separated by SDS-PAGE, and then the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA), which was subsequently blocked with blocking buffer (5% skim milk and 0.1% Tween 20 in PBS) for 1 h at room temperature. The membrane was incubated overnight at 4°C with rat anti-UL16 or rabbit anti-UL21 monoclonal antibodies at dilutions of 1:100 or with rabbit anti-Myc or mouse anti-Flag polyclonal antibodies at dilutions of 1:1000. The membrane was then washed three times with PBST and incubated with HRP-conjugated goat anti-rabbit IgG, goat anti-mouse IgG or goat anti-rat IgG (1:3000) secondary antibodies for 1 h at 37°C. The membrane was then washed three times with PBST, and the signals were developed using an enhanced chemiluminescence (ECL) kit (Takara, JPN).
Quantitative reverse transcription PCR
Total RNA was isolated from DEV-infected DEFs at different time points (3, 6, 13, 17, 24, 36, 48 and 60 hpi), and then, reverse transcription was performed; an uninfected control was included. The primers were designed with Oligo 7 (Table 1). Quantitative reverse transcription PCR was performed in a 20-µL reaction volume containing 10 µL of SYBR Green mix (Takara, JPN), 1 µL of each primer, 1 µL of cDNA, and 7 µL of RNase-free water. Triplicate experiments were performed to analyse UL21, UL54, UL13, US2 and β-actin gene expression, and the relative transcription levels were calculated using the 2-ΔCt method (27).
Table 1. Sequence and characteristics of RT-qPCR primers.
Primer
|
Primer sequence (5’-3’)
|
Gene
|
Product size (bp)
|
P1
|
TACGCCAACACGGTGCTG
|
β-actin
|
178
|
P2
|
GATTCATCATACTCCTGCTTGCT
|
|
|
P3
|
GCCCAGGAACACCAGTCT
|
DEV UL21
|
106
|
P4
|
CAGTGCGTATTGCCGTCT
|
|
|
P5
|
GCCACCAACCCTACCAAG
|
DEV UL13
|
131
|
P6
|
GTCGTCAGCCCATCACCA
|
|
|
P7
|
AGACGGTTCCGA-AAGTACAG
|
DEV US2
|
111
|
P8
|
TCGGCAGCACCAATAATCC
|
|
|
P9
|
GAACAACCGCCGAACAC
|
DEV UL54
|
127
|
P10
|
TCAAACATCCGCCTCAA
|
|
|
Pharmacological inhibition
Pharmacological inhibition was performed to confirm DEV UL21 gene expression patterns. Three flasks of DEFs were prepared and inoculated with DEV: one was prepared without any drug, and others contained either 300 μg/mL ganciclovir (GCV, a DNA polymerase synthesis inhibitor) or 100 μg/mL cycloheximide (CHX, a protein synthesis inhibitor). Total RNA was isolated from DEV-infected DEFs incubated with GCV or CHX (Meilunbio, CHN) at 24 hpi and subsequently reverse-transcribed into cDNA. The cDNA was then used for PCR analysis.
Immunofluorescence analysis
Cells grown on coverslips were washed three times with PBS and fixed overnight with 4% paraformaldehyde in PBS at 4°C. For indirect immunofluorescence analysis (IFA), the fixed cells were permeabilized with 1% Triton X-100 in PBS for 30 min at 4°C and incubated with 200 μL of blocking buffer (3% bovine serum albumin in PBS) in a humidified chamber for 1 h at 37°C. The cells were then incubated with primary antibodies (rabbit anti-UL21 and rat anti-UL16 at a dilution of 1:200) and Alexa Fluor-conjugated secondary antibodies (at a dilution of 1:1000) in blocking buffer were incubated for 60 min at 37°C. The samples were examined using a Nikon H550L fluorescence microscope.
Transfection
Cells were transfected at 90 to 95% confluence with 2.5 μg of plasmid DNA added to 125 μL of MEM and mixed well; then, 3.75 μL of Lipofectamine 3000 (Thermo Fisher Scientific, USA) in 125 μL of MEM was added,and the cells were gently mixed and incubation at room temperature for 5 min. The DNA suspension and 4 μL of p3000 were mixed together and incubated at room temperature for 15 min, after which the mixture was added to a 6-well plate. The plate was shaken gently and placed in a 37°C cell incubator.
Coimmunoprecipitation
DEFs were infected with DEV strain CHv at a multiplicity of infection (MOI) of 0.2. The infected DEFs were washed twice with cold PBST, and PMSF was added to the immunoprecipitation (IP) cell lysis buffer (Beyotime, CHN) at a final concentration of 1 mM. Precooled IP cell lysis buffer at 100 μL/mL was added to the cells, which were scraped from the plates, placed on ice, and shaken slowly on a horizontal shaker for 15 min until they were fully lysed. The cells were centrifuged at 14,000×g for 15 min at 4°C, and the supernatant was collected. Protein A + G agarose (Bio-Rad, USA) was washed three times with PBST. Rat anti-UL16 IgG and rabbit anti-UL21 IgG (rat anti-UL16 and rabbit anti-UL21 monoclonal antibodies at dilutions of 1:10) were added to the agarose beads. Rabbit anti-Myc or mouse anti-Flag polyclonal antibodies were also used at dilutions of 1:100. The samples were gently rotated at room temperature for
30 min. Afterwards, the complexes were washed three times with PBST. The lysates containing the target proteins were then added, and the mixture were incubated at 4°C overnight with gentle rotation. The samples were washed using PBST, the complexes were rapidly centrifuged for 30 s, and the supernatants were collected. Finally, 1×SDS loading buffer was added, and the samples were heated for 10 min at 70°C.
Mass spectrometry
SDS-PAGE was used to separate purified virion samples. The products were stained with Coomassie brilliant blue (Bio-Rad, USA) and then sent to Sangon Biotech Company (Sangon Biotech, CHN) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The methods used for in-gel trypsin digestion, LC-MS/MS, and database searches have been described in detail by Loret et al. (34).
Virion purification
DEFs were infected with DEV strain CHv at an MOI of 5. At 2 hpi, the cells were washed twice with PBS, and the medium was replaced with Opti-MEM. At 72 hpi, the medium was collected and clarified by centrifugation at 2000×g for 20 min at 4°C to remove the cell debris. The DEV virions were harvested by ultracentrifugation (40,000 × g, 2 h, 4°C) through a 30% (wt/vol) sucrose cushion and then banded by isopycnic gradient ultracentrifugation in a continuous 30 to 60% (wt/vol) potassium tartrate gradient in TBS (40,000 × g, 2 h, 4°C). The band containing the virions was collected, diluted tenfold in TBS, and pelleted by ultracentrifugation (20,000 × g, 30 min, 4°C). The pellets were resuspended in TBS and stored at -80°C (20).