Construction of plasmids and BACs
Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA) was used for PCR reactions to amplify DNA sequences for subsequent cloning. Takara ExTaq polymerase (Takara, New York, NY) was used for all analytical PCR. The authenticity of all constructs throughout this study was confirmed by sequencing using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Plasmid DNA was isolated using Qiawell or Qiaprep Plasmid prep (Qiagen Inc., Valencia, CA). Electrocompetent RR1 cells were prepared using ice-cold water and 10% glycerol essentially as described in [29
]. BAC DNA was isolated from a large culture (1.5 liters) using Qiagen's Large-Construct kit. Routine molecular biology procedures used in the course of plasmid constructions and characterization were based on those described in [29
]. Restriction enzymes, calf intestinal phosphatase (CIP), and Klenow fragment used to blunt DNA fragments were obtained from NEB (Beverly, MA).
Antibiotics and supplements in solid agar plates and liquid media were used at the following concentrations: zeocin (Zeo), 25 μg/ml; kanamycin (Kan), 50 μg/ml; chloramphenicol (Cam), 20 μg/ml; Ampicillin (Amp), 100 μg/ml; Sucrose (Suc) 5% (w/v).
Vector pKO5.2-C.1 is a derivative of pKO5, modified for use as a destination vector by the insertion of a gene cassette containing attR recombination sites into the multiple cloning region. To achieve that, pKO5 was digested with SalI and NotI and treated with calf intestinal phosphatase (CIP). Reading frame cassette C.1 (Gateway vector conversion system, Invitrogen) was ligated with the linearized pKO5 and transformed into E. coli DB3.1 cells (Invitrogen). DB3.1 cells are resistant to the effects of the product of ccdB gene located between the attR flanks of C.1 reading frame in pKO5.2-C.1.
This vector is based on a previously described plasmid pENTR-1A-gD5678 [17
]. To construct pENTR-gD-EGFP, pEGFP (Clontech, Mountain View, CA) was digested with Pvu
II and Stu
I to obtain an ~1 kb fragment harboring the gene coding for the Enhanced Green Fluorescent Protein (EGFP
) downstream of a prokaryotic (Plac
) promoter. The fragment was cloned into a Kpn
I-digested, Klenow-blunted pENTR-1A-gD5678, to obtain pENTR-gD-EGFP. The gD
flanks with the inserted EGFP
were transferred by LR recombination to the BAC insertion shuttle vector pKO5.2-C.1, to generate pKO5.2-C.1-gD-EGFP.
To construct the UL41-insertion vector, two regions of the HSV-2 (Genbank accession number NC_001798) UL40-UL42 region were amplified by PCR using the following primers:
The resulting BglII-EcoRI and HindIII-XhoI fragments containing approximately 2 kb downstream and upstream flanking sequences of the UL41 gene, respectively, were cloned into pSP72 (Promega, Madison, WI) to generate pUL41. pUL41 was digested with enzymes HindIII and EcoRI and treated with Klenow. Reading frame cassette A (Gateway vector conversion system, Invitrogen) was ligated into the linearized vector, giving rise to plasmid vector pUL41-attR-ccdB-cat. The plasmid was maintained in E. coli DB3.1 cells.
Plasmid pUL41-attR-ccdB-cat was digested with SalI, treated with Klenow, and was partially digested with NotI. The product was ligated with a PvuII/NotI fragment from plasmid pEGFP, containing the EGFP gene with a prokaryotic promoter (Plac). The resulting construct, pUL41-attR-EGFP, was digested with BglII/XhoI. A fragment containing the UL41 flanks with attR sequences and EGFP was transferred to BglII/XhoI digested pKO5.2, to obtain pKO5.2-UL41-attR-EGFP, the shuttle vector for allele replacement into UL41 locus of HSV2-BAC. DEST-BAC was generated from HSV2-BAC by allele replacement of the HSV2-BAC UL41 gene with the disrupted UL41 gene containing the attR flanks and EGFP. Successful allele replacement was detected visually by expression of EGFP, and confirmed by PCR analysis, using three sets of HSV2-specific primer pairs, UL41 primers flanking the insertion site, and primers amplifying a product within EGFP gene. The resulting recombinant BAC DNA was transfected into Vero cells. Presence of plaques on a Vero cell monolayer confirmed that the recombinant BAC is infectious.
DsRed2, A33R, and B5R allele replacement vectors
A set of allele replacement vectors were designed to study aspects of the LR recombination reaction and to investigate transfer of a suitable transgene to a viral BAC. These vectors are based on pENTR-1A (Invitrogen) and contained either DsRed2 or the vaccinia virus A33R or B5R genes. To insert the DsRed2 gene, pENTR-1A was digested with SalI/NotI, followed by treatment with Klenow to fill in overhangs, and ligation of the PvuII/StuI fragment containing DsRed2 and the lac promoter (pDsRed2, Clontech). To insert the B5R gene, plasmid pDNA3-B5R-HA (Clement Meseda, manuscript in preparation) was digested with restriction enzymes BglII (blunted with Klenow) and PvuII to obtain an expression cassette of vaccinia virus B5R gene fused to an HA-tag under the control of the CMV promoter. The fragment was than cloned into the blunted, SalI/NotI digested pENTR-1A vector. Similarly, the plasmid pcDNA3-A33R-HA (Clement Meseda, manuscript in preparation) was digested with restriction enzymes NruI and PvuII, to obtain an expression cassette of the A33R gene with an HA-tag under the control of the CMV promoter, and ligated with SalI/NotI digested, Klenow-treated pENTR-1A vector. Transformed E. coli harboring entry vectors were grown on LB Kan medium.
In vitro LR recombination
LR recombination reactions were performed using the Gateway LR Clonase enzyme mix (Invitrogen) as described in the manufacturer's protocol. Modifications are described. Briefly, the enzyme mix contains the bacteriophage lambda recombination proteins Integrase (Int) and Excisionase (Xis), and the E. coli-encoded protein Integration Host Factor (IHF). Each reaction was performed in a 20 μl volume, using 150 ng of DEST-BAC DNA and 100 ng of entry vector DNA. Reactions were incubated overnight at room temperature. After Proteinase K treatment, ten-fold and 100-fold dilutions were transformed into electrocompetent DH10B cells (Invitrogen). The electroporation conditions employed 0.1 cm cuvets and 1.7 kV/25 μF/200 Ω settings on a BioRad GenePulser II. Clones were confirmed by PCR with primer pairs targeting sequences in the inserted genes (DsRed2, A33R, B5R), and with HSV-2 UL41-specific primers (UL_41_F; 5'GGGGGTCTTCTTCGTAGTCG and UL_41_R; 5'ACATCAGCACCGGCTACATT) hybridizing close to the site of insertion whereby generating fragments of different size according to the inserted gene. A primer pair that amplifies a fragment in the EGFP gene was used to show loss of EGFP in recombinant clones.
Mutagenesis of HSV2-BAC was performed as described previously, employing a BAC replacement vector containing a temperature-sensitive origin of replication and marker genes for positive and negative selection during the BAC mutagenesis procedure [4
]. Briefly, electrocompetent RR1 E. coli
cells were transformed with HSV2-BAC DNA and selected for chloramphenicol resistance. The electroporation conditions employed 0.1 cm cuvets and 1.7 kV/25 μF/200 Ω settings on a BioRad GenePulser II. Clones were confirmed by PCR with 3 independent HSV-2 specific primer pairs and a primer pair that amplifies a fragment in the chloramphenicol acetyltransferase (cat
) gene. A 620 bp fragment in the cat
gene was amplified with the following primers: MM507 (5'GCCCATGGTGAAAACGGGGGC) and MM508 (5'GATCGGCACGTAAGAGGTTCC).
Electrocompetent RR1 cells harboring the HSV2-BAC were prepared as previously described [29
], and were electroporated with 10 ng of the respective pKO5.2-derived shuttle vector (pKO5.2-C.1-gD-EGFP or pKO5.2-UL41 att
R-EGFP), and plated onto chloramphenicol/zeocin (Cam/Zeo) plates in serial dilutions at 30°C. The following day, multiple colonies were picked into 1 ml of LB broth and plated in serial dilutions onto Cam/Zeo plates and incubated at 43°C. Colonies at 43°C were analyzed by picking several into 1 ml LB each and plating out 20 μl onto sucrose/chloramphenicol (Suc/Cam) plates at 30°C. Approximately 10 colonies from each plate, representing an original 43°C colony, were transferred to grids on Suc/Zeo and Cam LB plates at 30°C. Colonies that were Cam+, Zeo/Suc- were picked into 100 μl of 10 mM Tris pH 8.0 for colony PCR using HSV-2 specific primers. along with either a primer pair flanking the UL41
coding region (for insertion of the att
cassette) or a primer pair flanking the gD
coding region (for insertion of EGFP
to delete US6
). Positive colonies were streaked on Cam plates and grown at 37°C. Large scale BAC vector DNA isolation was performed using the Large Construct kit (Qiagen Inc., Valencia, CA).
Cells and viruses
Vero cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco's Minimal Essential Medium (DMEM) supplemented with 10% fetal bovine serum, FBS (HyClone, Logan, UT); 2 mM Glutamax-1; and 0.05 mg/ml Gentamicin (Gibco/Invitrogen, Grand Island, NY). VD60 cells, a Vero cell line that expresses the HSV-1 gD [30
], were a gift from Dr. David C. Johnson, Department of Molecular Microbiology & Immunology, Oregon Health Sciences University. VD60 cells were maintained as suggested in Eagle MEM lacking histidine (MEM-his) supplemented with 1 mM histidinol (Sigma Chemical Co., St. Louis, MO) and 5% FBS. Prior to infection, VD60 cells were passaged two times in DMEM containing 10% FBS. Cells were seeded in 6-well plates at 8 × 105
/well one day before infection or transfection. The HSV-2 (MS) BAC used as the parental clone for subsequent recombinant BACs described in this study has been described previously [17
Transfection of BAC DNA
Vero cells were transfected using Lipofectamine (Invitrogen, Carlsbad, CA), at a ratio of five μl per μg of plasmid or BAC DNA. At 5 h after transfection, an equal volume of complete media was added; media was changed again the next day. Infected cell lysates were harvested and used to infect Vero cells seeded at 2 × 106/flask in 25 cm2 tissue culture vessels.
Infection of cells
For Western blot, cells were infected at a multiplicity of infection (moi) of 5; for immunostaining, virus stocks were titered to get a sufficient number of separated plaques per well. Virus was added to the cell sheet in about 1 ml of complete media per well. Plates were incubated at 37°C for 2 hours, with gentle mixing of the media by shaking the plate every 15 minutes. After that, cells were overlaid with complete media (Western blot) or methylcellulose (immunostaining).
Immunostaining of plaques
Infected cell sheets were fixed using a 1:1 mixture of methanol/acetone. All wash steps were performed using Tris buffered saline (TBS) with 0.1% Triton-X. Fetal bovine serum at a concentration of 20% in TBS was used for blocking. Mouse anti-HA monoclonal antibody was used as primary antibody (diluted 1:1000). Detection was performed with goat anti-mouse polyclonal antibodies conjugated to alkaline phosphatase (Promega, Madison, WI) in combination with Western Blue stabilized substrate for alkaline phosphatase (Promega). Pictures were taken with a Canon G3 digital camera equipped with a HOYA +4 macro lens.
Two days after infection, infected cells were harvested and resuspended in lysis Buffer (50 mM Tris Cl pH 7.5; 150 mM NaCl; 1% Triton X-100) with EDTA-free Protease Inhibitor Cocktail Tablets (Roche, Mannheim, Germany). Proteins were fractionated on precast NuPAGE 12% Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred electrophoretically onto PVDF membrane (Bio-Rad Laboratories, Hercules, CA) for subsequent Western blot. Mouse anti-HA monoclonal antibody (Covance, Richmond, CA) at a dilution of 1:1000 was used to detect HA-tagged A33R and B5R. Protein bands were detected by chemiluminescence, using horseradish peroxidase conjugated sheep anti-mouse antibody (Amersham Biosciences, Piscataway, NJ) at a dilution of 1:30000, and SuperSignal West Dura detection reagents (Pierce, Rockford, IL). Digital pictures were captured with a Fuji LAS-3000 CCD camera and software supplied by the manufacturer.