Viruses and Cells
Feline calicivirus strain vR6, derived from the infectious cDNA clone of the Urbana strain designated pR6, was described previously (Sosnovtsev et al., 2005
), and will be referred to as wild-type (wt). Crandell-Rees feline kidney (CRFK) cells were grown in Dulbecco's modified Eagle's medium (designated as maintenance medium, Lonza Inc., Allendale, NJ) containing amphotericin B (0.25 μg/ml, Mediatec, Inc, Manassas, VA), penicillin (250 U/ml, Mediatec), streptomycin (250 μg/ml, Mediatec) and L-Glutamine (2mM, Mediatec) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen Corporation, Carlsbad, CA)
Construction of Intermediate Plasmid
A plasmid designated Topo-VP1/VP2 was generated by cloning DNA fragments derived by PCR from pR6 as template into Invitrogen's Topo TA vector. The primer pair: sense primer, 5′-GGCATGACCGCCCTACACTGT (designated URB5273F) and antisense primer, 5′- GCGTTGTTGTCCAAGCGCAGCC (designated URB7391R) amplified the entire ORF2 and the first 96 nt of ORF3. The amplified and cloned FCV sequences included the unique restriction sites BstBI and AvrII, which were used for directional cloning back into the pR6 backbone following transposon mutagenesis.
Construction of cDNA Library of FCV Clones Containing a Randomly Inserted 15-bp Sequence
A cDNA library of Topo subclones with random transprimer insertions was created by using the GPS-LS linker scanning system according to the manufacturer's instructions (New England Biolabs, Beverly, MA). The GPS-LS insertion mutagenesis is performed in three major steps: first, a 1383 bp transprimer sequence, that is flanked by PmeI restriction sites and encodes the chloramphenicol resistance gene, is randomly inserted into a DNA target; second, all but 10 bp of the transprimer is removed by PmeI digestion; and finally, the ligation of the mutagenized DNA target results in a 15 bp insertion (10 bp from the transprimer which includes the PmeI site, and a 5 bp duplication of target DNA created by the transposition reaction).
Briefly, upon producing the GPS reaction mixture of an unamplified library of transprimer insertions, 5 μl of the reaction were transformed into competent cells. Competent cells were grown for 1 hour at 37°C with 600 μl of S.O.C. media (Invitrogen). Two hundred μl of the cell mix were added to 3 plates containing both chloramphenicol and kanamycin to select for transformants, and incubated overnight at 37°C. The first library of random mutations consisted of approximately 2,763 colonies. Ten ml of LB broth were added to the plates and the colonies were scraped and collected. Plasmid DNA was purified from the resuspended colonies using a DNA Mini-Prep kit (Qiagen, Valencia, CA). The three samples of DNA plasmid were then proportionally combined into a pool that contained approximately 3 μg of DNA. One μg of pooled DNA was digested using the restriction enzyme that flanked the region of interest (BstBI and AvrII), and separated on a 1% agarose gel. The scanned region of interest (the 2027 bp sequence of FCV bordered by the restriction sites BstBI and AvrII) plus the transprimer insertion was identified based on its size, and gel purified. The region of interest plus the transprimer insertion was ligated back into the parental full-length cDNA backbone of the pR6 plasmid, and transformed into competent cells. Identical to the previous pooling step, competent cells were grown for 1 hour at 37°C with 600 μl of S.O.C. media (Invitrogen). Two hundred μl of the cell mix were added to 3 plates containing both chloramphenicol and carbenicillin to select for transformants, and incubated overnight at 37°C. The second library of random insertion consisted of approximately 1,093 colonies. Ten ml of LB broth were added to the plates and the colonies were scraped and collected. Plasmid DNA was purified from the resuspended colonies as described above and three samples of DNA plasmid were then proportionally combined into a pool that contained approximately 3 μg of DNA. One μg of the pool was digested using the restriction enzyme PmeI. The digestion reaction was separated on a 1% agarose gel, and the parental backbone, minus the transprimer, was excised and gel purified. The pool of mutagenized parental backbone was religated, and transformed into competent cells. Identical to the previous pooling steps, competent cells were grown for 1 hour at 37°C with 600 μl of S.O.C. media (Invitrogen). The cell mix (200 μl) was added to 3 plates containing carbenicillin to select for transformants, and incubated overnight at 37°C. The final library of random mutations consisted of approximately 2,348 colonies. Ten ml of LB broth were added to the plates and the colonies were scraped and collected. Plasmid DNA was purified from the resuspended colonies as above, and the three samples of DNA plasmid were then proportionally combined into a pool that contained approximately 5 μg of DNA. Two μg of the final pool of mutagenized full-length FCV, containing 15-nt insertions randomly distributed in ORF2 and the first 12 nucleotides of ORF3 (the region of FCV bordered by the restriction sites BstBI and AvrII) were used to test for recovery of virus.
Recovery of Virus
Virus was recovered from plasmid DNA by using the MVA/T7 expression system, described previously (Sosnovtsev, Garfield, and Green, 2002
). Briefly, confluent CRFK cell monolayers in 6-well plates (approximately 2 × 106
cells) were infected with MVA/T7 (a gift of Dr. Bernard Moss) at an MOI of 10 and incubated for 1 h at 37°C. The supernatant was removed, and 2 ml of maintenance media were added to the cells. Transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Following incubation for 24-48 hours at 37°C, medium from the transfected cell monolayer was transferred to a fresh cell monolayer, which was monitored for the development of viral CPE. If CPE was observed, the supernatant was used to perform a plaque assay, and individual plaques were analyzed.
To confirm that recovered virus originated from the engineered constructs, reverse-transcriptase (RT)-PCR products derived from viral RNA were analyzed by direct sequencing. RNA was purified using the RNeasy mini kit (Qiagen). As a control for the presence of DNA from the original plasmid used to synthesize the RNA for transfection, PCR was performed on the isolated viral RNA in the absence of RT.
Construction of Recombinant FCV Infectious Clones
To reconstruct the recovered transposon-modified virus, the RNA extracted from the plaque of a mutagenized virus that contained a 15-nt insertion in the LC (encoding a PmeI restriction site; ) was used for RT-PCR amplification using the following primer pair that is complementary to the FCV genome: sense primer 5′-GAGTTGCATCTTGAGCCGCC (UrbanaFL5174F) and antisense primer 5′-GCGTTGTTGTCCAAGCGCAGCC (UrbanaFL7391R). This DNA fragment was treated with BstBI and AvrII and subsequently cloned back into the infectious cDNA clone pR6 using the same restriction sites to reconstruct the mutagenized LC viral sequence. The reconstructed plasmid containing the 15-nt insertion in the LC protein was designated pR6-LC-15.
Two sites within the ORF2 and 3 region of FCV can tolerate a 15-nt insertion
The multiple cloning site was then inserted into pR6 clone using a QuikChange XL Site-Directed Mutagenesis kit (Stratagene's, La Jolla, CA) and PCR mutagenesis. The sequences of oligonucleotides used to mutagenize the cDNA clone are available upon request. The infectious clone with the multiple cloning site was designated pR6-LC ().
The DsRed and GFP monomeric proteins were PCR-amplified using the pDsRed-Monomer-N1 and pAcGFP1-Monomer-N1 vectors as templates (Clontech), respectively, to include bordering KpnI and AflII sites that flanked a ‘linker’ amino acid (GGS) sequence. The PCR amplified DsRed or GFP fragments were digested, purified, and ligated into the KpnI and AflII sites of the pR6-LC clone a Rapid Ligation kit (Roche). The resulting plasmids were designated pR6-LC-GFP and pR6-LC-DsRed ().
Viruses were titered on CRFK cells. CRFK cells were seeded onto 6-well plates, and upon confluency, the monolayers were infected with serial dilutions of virus prepared in Dulbecco's modified Eagle's medium. Plates were incubated for 60 min (37°C, 5% CO2), with gentle agitation every 15 min. The inocula were removed, and monolayers were overlaid with 2 ml maintenance media containing 1% agarose gel (Invitrogen). Plates were then incubated for approximately 48 hr at 37°C, in humidified 5% CO2. Cells were fixed with 10% formaldehyde and the agarose overlay was removed. The fixed cells were stained with 1% (w/v) crystal violet solution, washed, and viral plaques were counted.
Western Blot Analysis
Confluent monolayers of CRFK cells in 150 cm2 flasks were infected with vR6, vR6-LC-GFP, and vR6-LC-DsRed respectively, and following complete lysis (approximately 48 hours), the flasks were submitted to three freeze-thaw cycles. The insoluble material was pelleted by centrifugation at 2,655 × g for 10 min and the pellet was resuspended in 500 μl PBS. A 10 μl aliquot was mixed with an equal volume of 2× Tris-glycine SDS sample buffer (Invitrogen) containing 5% β-mercaptoethanol. The sample was subjected to SDS-PAGE and Western blot analysis as described below.
For VP1 detection over time, CRFK cells were infected at an MOI of 1 with vR6, vR6-LC-GFP, and vR6-LC-DsRed. At the desired time point, approximately 300 μl of Tris-glycine SDS sample buffer was added directly to the monolayer to disrupt the cells, and the lysate was stored at -70°C until further use.
Samples were heated at 95°C for 5 min in 1× Tris-glycine SDS sample buffer (Invitrogen) containing 5% β-mercaptoethanol. Western blot analysis was performed using 4-20% Tris-glycine SDS-PAGE gels (Lonza). Proteins were transferred by dry blotting to nitrocellulose membranes using Invitrogen's iBlot apparatus, and membranes were incubated with guinea pig anti-FCV virion hyperimmune serum, rabbit anti-LC hyperimmune serum, rabbit polyclonal anti-DsRed (Clontech), or mouse monoclonal anti-GFP (Clontech). The binding of the primary antibodies was detected with goat anti-guinea pig (Kirkegaard & Perry Laboratories, Gaithersburg, MD), anti-rabbit or anti-mouse secondary antibodies (Thermo Scientific, Waltham, MA) conjugated with horseradish-peroxidase, followed by development with the SuperSignal Chemiluminescent Substrate (Thermo Scientific).
Western blots were stripped using the Restore Plus Western Blot Stripping Buffer (Thermo Scientific), and incubated with monoclonal HRP-conjugated anti-β-actin antibody. SeeBlue Plus2 Pre-Stained Standard (Invitrogen) was used to estimate molecular weights of the proteins detected by Western blot.
Subgenomic Expression Plasmids
To construct vectors expressing the FCV capsid precursor protein for trans-cleavage analysis, the entire FCV subgenomic RNA region present in pR6, pR6-LC-GFP, or pR6-LC-DsRed was cloned into the pCI expression vector (Promega, Madison, WI). The following primer pair was used to amplify the subgenomic region of each construct (including ORF2, ORF3, the 3′-NTR, poly-A tail, and a unique NotI restriction site downstream of the poly-A tail), and to introduce a SalI restriction site (boldface) upstream of the subgenomic transcription initiation site: sense primer 5′-CGCCCTACACTGTGAGTCGACTGTGTTCGAAGTTTG (pR6-SubGen-SalI F) and antisense primer that is downstream of the NotI restriction site 5′-CCCAGTCACGACGTTGTAAAAC (pR6-Dnstream PolyA R). The DNA fragments were treated with SalI and NotI, and cloned into pCI vectors (Promega) using the same restriction sites. The plasmids were designated pCI-vR6, pCI-vR6-LC-GFP, and pCI-vR6-DsRed (). All plasmids were confirmed by sequence analysis.
Coupled In Vitro Transcription and Translation Reaction and Immunoprecipitation Assay
One to 5 μg of plasmid DNA were used as template in a coupled transcription and translation reaction (TNT T7 Coupled Reticulocyte Lysate System; Promega). For radiolabeling of synthesized protein, [35S] methionine (>1,000 Ci/mmol) from Amersham/GE Healthcare (Waukesha, WI) was added at a concentration of 1.5 mCi/ml.
Immunoprecipitation of the LC protein from the TNT reaction was performed as described previously (Sosnovtsev, Sosnovtseva, and Green, 1998
). Briefly, 20 μl of the TNT reaction were incubated with 40 μl of either mock-infected lysate or vR6 infected lysate at 37°C for 3 hr. The resulting mixture was diluted with 60 μl of radioimmunoprecipitation assay (RIPA) buffer. The mixtures were incubated with rabbit post-immunization serum (5 μl) raised against the FCV LC protein expressed in E. coli
, and the immune complexes were precipitated with protein A beads (Sigma Chemical Co., Saint Louis, MO). The binding and washing conditions were performed as described previously (Sosnovtsev, Sosnovtseva, and Green, 1998
). Following separation of the proteins in a 4-12% Tris-glycine SDS-PAGE gel, the gel was dried and exposed to Biomax MR film (Kodak, Rochester, NY) for autoradiography.
Multiple-cycle Growth Kinetics
CRFK monolayers were infected with vR6, vR6-LC-GFP, or vR6-LC-DsRed at a multiplicity of infection (MOI) of 0.01 and incubated at 37°C in 5% CO2. Cell lysates were collected at various times post-infection (p.i.) and frozen at -70°C. Following three freeze-thaw cycles, the titer at each time-point was determined by plaque assay in CRFK cells as described above. Results depicted in represent the mean value of each time point duplicate determined in two independent plaque titration assays, and the error bars represent one standard deviation. The graph was prepared using the software Prism from GraphPad (La Jolla, CA).
Recombinant FCV expressing GFP and DsRed fused to the LC are viable and show growth kinetics similar to wt FCV
Extraction of Viral RNA
CRFK monolayers (2 × 106 cells) were mock infected or infected with vR6, vR6-LC-DsRed or vR6-LC-GFP at a multiplicity of infection of 1 and incubated at 37°C in 5% CO2. At 8 h p.i. 300 μl of Trizol (Invitrogen) reagent was added directly to the monolayer, and RNA was extracted following the instructions of the manufacturer. The viral RNA of individual plaques was purified using Qiagen's RNeasy mini kit.
Preparation of RNA Transcripts and Biotinylated RNA Probes
An RNA probe was designed as described in Green et al. (Green et al., 2002b
). Briefly, using pR6 as a template, a forward primer that contained a T7 RNA polymerase promoter sequence and a reverse primer were used to generate a DNA fragment that was complementary to nt 5686 to 5987 of the ORF2. DNA fragments were agarose gel purified and extracted from the agarose (Qiagen). Approximately 1 μg of the DNA fragment was used as the template for in vitro transcription with the Ribomax Express T7 transcription kit (Promega). Transcribed RNA was purified with the RNeasy mini kit (Qiagen).
Purified RNA fragments were subsequently biotinylated with the reagents in the BrightStar psoralen-biotin nonisotopic labeling kit (Ambion, Carlsbad, CA). Briefly, 0.5 μg of RNA was cross-linked to the psoralen-biotin reagent on ice with a 365-nm UV light for 45 min. Unincorporated label was removed by 1-butanol extraction. The biotinylated RNA probes were stored at -70°C.
Northern blot analysis for the detection of FCV RNA was performed with the NorthernMax-Gly system (Ambion) as described by Green et al. (Green et al., 2002b
), with slight modifications. Briefly, the viral RNA was denatured with glyoxal loading dye for 30 min at 50°C, and separated in a 1% LE-agarose gel. The RNA was transferred to a Bright-Star-Plus membrane by capillary blotting. The membrane was treated with UV light to cross-link the RNA to the membrane, and was then incubated with Ultrahyb buffer at 68°C for 30 min. The membrane was then incubated overnight at 68°C with the sense biotinylated RNA transcript probe. Detection of bound biotinylated RNA probe was conducted following the instructions of the Bright Star BioDetect kit (Ambion).
Nucleotide Sequence Analysis
All plasmids were sequenced to confirm the presence of the desired engineered insertions. Nucleotide sequencing analysis was performed on an ABI 3730 automated sequencer (Applied Biosystems, Carlsbad, CA) with the Big Dye Terminator reagents, version 3.1.
Recovery of transposon mutagenized virus was confirmed by direct sequence analysis of RT-PCR products derived from viral RNA as described previously (Sosnovtsev, Sosnovtseva, and Green, 1998
). The sequences of oligonucleotides used to amplify and sequence virus-specific cDNA fragments are available upon request.
The following primer pair, that flanks the site of insertion into the LC coding sequence, was used in a “diagnostic” PCR to examine the stability of the insert: sense primer 5′- CTTGAGTCTATCCTGGGCGATG (UrbFL5500F), and the antisense primer 5′- ATCAGCAGCGGTTGACATTTG (UrbFL5758R).
Plaques were visualized using a Leica DMI4000 B microscope, and images were captured using a QImaging Retiga-2000R camera (Surrey, BC Canada). Images were processed using iVision 4.0.14 software (BioVision. Exton, PA).
For live-cell imaging of single infection, cells were infected with vR6-LC-PmeIDsRed at an MOI of 10. For the co-infection experiment, cells were infected with both vR6-LC-GFP and vR6-LC-DsRed at a multiplicity of infection (MOI) of 2 and 1, respectively. The infected cells were then transferred to a temperature-controlled chamber (37°C/5% CO2). Time-lapse confocal imaging was obtained using a Leica SP2-AOBS Confocal Microscope. Time-course images were processed using Imaris software (Bitplane, Zurich, Switzerland).