Monolayer cultures of BSR-T7 cells (2
) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 10% tryptose phosphate broth (TPB), and 400 μg/ml G418. Monolayer cultures of Vero cells and L929 cells were maintained in DMEM containing 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin. All cells were incubated at 37°C, 5% CO2
. Virus-infected cells were grown in DMEM containing 2% FBS. Plaque assays were performed on Vero cells.
Construction of recombinant viruses.
A complete cDNA of the 18,954-nucleotide JPV genome was constructed from plasmids carrying the genes for N, P, M, F, SH, TM, G, and L. PCRs were applied to provide adaptor DNAs over some of the intercistronic junction, leader, and trailer sequences using a backbone plasmid from a parainfluenza virus 5 infectious cDNA clone (13
). Plasmids were constructed using standard molecular biology techniques. A NotI sequence tag was introduced in the 3′ noncoding region of ORF-X. The construct containing the complete JPV genome was designated pJPV. An enhanced green fluorescent protein (EGFP) gene was inserted between the F and the SH genes. To transcribe the extra gene, gene end (GE) and gene start (GS) sequences were inserted into the 5′ noncoding region of the SH gene after the ORF of the EGFP gene. The proposed F-SH end/start sequences [TAAATAAAAA (intercistronic 3 nucleotides CTT) AGGACAAAAG] were used. The construct containing EGFP was designated pJPV-EGFP. The ORF of the SH gene was replaced with the Renilla
luciferase (Rluc) gene. The construct lacking the SH gene and containing the Rluc gene was designated pJPVΔSH.
The plasmids, pJPV carrying the full-length genome of JPV, pJPV-EGFP carrying the full-length genome of JPV with the EGFP gene insertion, or pJPVΔSH carrying the full-length genome of JPV but with the SH gene replaced with the extra Rluc gene, and three helper plasmids pJPV-N, pJPV-P, and pJPV-L carrying genes for the N, P, and L proteins, were cotransfected into BSR-T7 cells at 95% confluence in 6-cm plates with Plus and Lipofectamine (Invitrogen). The amounts of plasmids used were as follows: 5 μg pJPV/pJPV-EGFP/pJPVΔSH, 1 μg pJPV-N, 0.3 μg pJPV-P, and 1.5 μg pJPV-L. After 3 h of incubation, the transfection medium was replaced with DMEM containing 10% FBS and 10% TPB. After 72 h of incubation at 37°C, 1/10 of the BSR-T7 cells were passed into a T-75 (75 cm2) flask containing 1 × 106 Vero cells. The mixed cells were cocultured for 2 weeks with passaging at 3- or 4-day intervals. The medium was harvested, and cell debris was pelleted by low-speed centrifugation (3,000 rpm for 10 min). Plaque assays were used to purify single clones of the recombinant viruses. Recombinant viruses recovered from cDNA were designated rJPV, rJPV-EGFP, or rJPVΔSH.
RT-PCR and nucleotide sequencing.
Total RNAs from rJPV-, rJPV-EGFP-, or rJPVΔSH-infected Vero cells were purified using an RNeasy kit (Qiagen, Inc., Valencia, CA). cDNAs were prepared using random hexamers, and aliquots of the cDNA were then amplified in reverse transcription (RT)-PCRs using appropriate oligonucleotide primer pairs. Primers p70 (GCCAATTAGTCCCTGCGATT) and p71 (ACACGGGTTCTTGCACAACT) were used to identify rJPV. Primers p80 (CTGGGACGAGAACGGTCTTA) and p146 (CAGCTTGCCTGTGACTATGG) were used to identify the NotI sequence tag of rJPV. Primers p61 (CAACGAGTCGATCAACAAGTCTCATG) and p94 (CATCTTCTAGGTAATGCTGGTAACCC) were used to identify rJPVΔSH. The improved rapid amplification of cDNA ends (RACE) PCR was used to amplify the leader and trailer sequences. The sequences of all primers for sequencing of the complete genomes of rJPV, rJPV-EGFP, and rJPVΔSH are available on request. DNA sequences were determined using an Applied Biosystems sequencer (ABI, Foster City, CA).
To confirm the rescued rJPV, Vero cells were mock infected with or infected with rJPV. At 2 days postinfection (d.p.i.), the cells were washed with phosphate-buffered saline (PBS) and then were fixed in 0.5% formaldehyde. The cells were permeabilized in 0.1% PBS-saponin solution and incubated for 30 min with polyclonal anti-TM rabbit serum at a 1:100 dilution (Genscript USA, Inc., Piscataway, NJ), and then fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit antibody was added to the cells. The cells were incubated for 30 min and were examined and photographed using a Nikon FXA fluorescence microscope.
To confirm the rescue of rJPV-EGFP, Vero cells were infected with rJPV or rJPV-EGFP. At 2 d.p.i., the cells were photographed using a Nikon FXA fluorescence microscope.
To confirm the rescue of rJPVΔSH, Vero cells were mock infected or infected with rJPV or rJPVΔSH. At 2 d.p.i., the cells were treated as described above. The permeabilized cells were incubated with polyclonal anti-TM or SH rabbit serum and then examined and photographed using a Nikon FXA fluorescence microscope.
The p65 subunit of NF-κB was examined as described previously (23
). Briefly, L929 cells were mock infected or infected with rJPV or rJPVΔSH. At 1 d.p.i., cells were processed as described above. The cells were incubated with rabbit monoclonal antibody specific for the p65 subunit of the NF-κB transcription factor (Santa Cruz Biotechnology, Santa Cruz, CA). The cells were examined and photographed using a Nikon FXA fluorescence microscope.
Vero cells in 12-well plates were infected with rJPV or rJPVΔSH at an MOI of 5 or 0.1. The cells were then washed with PBS and maintained in DMEM-2% FBS. The medium was collected at 0, 24, 48, 72, and 96 hours postinfection (h p.i.). The titers of viruses were determined by plaque assay on Vero cells.
Immunoprecipitation of polypeptides.
Vero cells were mock infected or infected with rJPV or rJPVΔSH. At 22 h p.i., the cells were labeled for 2 h with 35S-Met/Cys Promix (100 μCi/ml). The cells were lysed in radioimmunoprecipitation buffer, and aliquots immunoprecipitated using polyclonal anti-P C-terminal or anti-V C-terminal rabbit serum (Genscript USA, Inc., Piscataway, NJ). The precipitated proteins were resolved by 15% SDS-PAGE, and then the proteins were examined by autoradiography using a Storm phosphorimager (Molecular Dynamics, Inc., Sunnyvale, CA).
The rJPVΔSH genome contains the Renilla luciferase gene in the place of the SH gene. To examine Rluc expression in virus-infected cells, 24 wells of Vero cells were mock infected or infected with rJPV or rJPVΔSH. At 1 d.p.i., the cells were washed and lysed with 100 μl of 1× passive lysis buffer. Ten microliters of lysate from each well were used to examine the Renilla luciferase activity with a luciferase assay system (Promega Corporation, Madison WI).
To examine whether JPV SH protein can inhibit TNF-α-induced NF-κB activation, 24 wells of L929 cells were transfected with an empty pCAGGS vector, pCAGGS-PIV5 SH, or pCAGGS-JPV SH plus pκB-TATA-Luc and pRL-TK. The cells were incubated at 37°C with 5% CO2 for 18 to 24 h, and then the medium was replaced with either 250 μl of Opti-MEM alone or 250 μl of Opti-MEM containing 10 ng/ml TNF-α (catalog no. 522-009; Alexis, San Diego, CA) or 250 μl of Opti-MEM containing 50 ng/ml of the phorbol ester phorbol 12-myristate 13-acetate (PMA) (catalog no. p1585; Sigma, St. Louis, MO), and cells were incubated for 4 h at 37°C with 5% CO2. The luciferase activity, expressed as the ratio of firefly luciferase activity to Renilla luciferase activity, was measured using a Veritas microplate luminometer (Turner Biosystems) to indicate the expression levels of the reporter gene under the control of the NF-κB element. The fold increase and the ratio of the amount of luciferase activity of TNF-α-treated cells to that of untreated cells were used to indicate the effect of SH on TNF-α signaling.
UV inactivation of viruses.
L929 cells were mock infected or infected with rJPV or rJPVΔSH at an MOI of 5. At 2 d.p.i., the plate was uncovered, placed inside a Fisher Hamilton biological safety cabinet class II, and UV treated for 30 min. The medium was then filtered through a 0.22-μm filter to remove cell debris. The effectiveness of the UV treatment in inactivating JPV was confirmed by plaque assay.
Enzyme-linked immunosorbent assay (ELISA) of TNF-α.
L929 cells were mock infected or infected with rJPV or rJPVΔSH at an MOI of 5. The medium was collected at different time points postinfection. The amounts of TNF-α were measured by using a murine TNF-α detection kit purchased from Amersham Pharmacia (Piscataway, NJ) according to the manufacturer's instructions. Amounts of 50 μl of medium from infected cells or standards in duplicate and 50 μl of biotinylated antibody against TNF-α were added to strips prelabeled with antibody against TNF-α. The strips were incubated at room temperature for 2 h. After the strips were washed three times with wash buffer provided by the manufacturer, 100 μl of streptavidin-horseradish peroxidase conjugate was added, and they were incubated at room temperature for 30 min. The strips were then washed three times, and 100 μl of 3,3′,5,5′-tetramethylbenzidine substrate solution was added to each well. The strips were incubated in the dark at room temperature for 30 min, and 100 μl of stop solution was added to each well. The optical density at 450 nm was measured within 30 min. The amounts of TNF-α were calculated by using standard curves generated from known concentrations of TNF-α provided by the manufacturer.
Fragmented DNAs were purified as described previously. Briefly, confluent L929 cells were mock infected or infected with rJPV or rJPVΔSH at an MOI of 5. At 2 d.p.i., L929 cells were washed twice with PBS without Mg2+ or Ca2+ and incubated in 0.5 ml of TTE buffer (0.2% Triton X-100, 10 mM Tris, 15 mM EDTA, pH 8.0) at room temperature for 15 min. Cell lysates were harvested and centrifuged at 14,000 rpm for 20 min. Supernatants were digested with 100 μg of RNase A/ml at 37°C for 1 h. Samples were purified by phenol-chloroform extraction, precipitated, and washed with 70% ethanol. Pellets were air dried and resuspended in 10 μl of Tris-EDTA. Electrophoresis was performed on 2% agarose gels with size markers.
For terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, L929 cells were trypsinized and combined with floating cells in the medium at different time points. The harvested cells were centrifuged and washed with PBS. The cells were fixed and permeabilized. The cells were then incubated with 25 μl of TUNEL reaction mixture (cell death detection kit; Roche Diagnostics Corp., Mannheim, Germany) for 2 to 3 h in the dark at 37°C. The cells were analyzed by flow cytometry (Invitrogen Corporation, Carlsbad, CA).
Antibody treatment of infected cells.
Confluent L929 cells were mock infected or infected with rJPV or rJPVΔSH at an MOI of 5 and were incubated in 0.5 ml of DMEM-2% FBS with neutralizing antibody against TNF-α (BD Pharmingen, San Jose, CA) or isotype control at 50 μg/ml. At 2 d.p.i., the cells were photographed using a light microscope. The cells were collected, and TUNEL assays were carried out as described above.
Infection of mice with JPV.
All animal experiments were carried out strictly following the protocol approved by the IACUC. To study the pathogenesis of JPV in mice, 6-week-old wild-type BALB/cJ mice (Jackson Laboratories) were infected with 50 μl of PBS or 105 PFU of rJPV or rJPVΔSH intranasally. The weight of the mice was monitored daily up to 7 days postinfection. Mice were euthanized at 1, 3, and 7 days postinfection to collect sera and tissues, including lungs. To preserve the morphology for histology studies, lungs were inflated with 4% paraformaldehyde. Tissues were fixed in 4% paraformaldehyde at 4°C.
BALB/cJ mice from the infection study were euthanized by asphyxiation. The lungs were inflated with 4% paraformaldehyde and collected. Samples were routinely processed, embedded, and sectioned for hematoxylin-and-eosin (H&E) staining. Alveolar infiltrates and perivascular cuffing were scored from 1 (minimal) to 4 (severe) in a blinded fashion by a board-certified veterinary pathologist. Photomicrographs were taken using an Olympus BX41 microscope with an Olympus DP70 microscope digital camera and DP Controller imaging software.