Materials, cells and viruses
African green monkey kidney BSC40 cells and human TK- 143B cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 5% fetal calf serum (FCS, Invitrogen, Carlsbad, CA) at 37°C in the presence of 5% CO2; human diploid fibroblasts (kindly supplied by S. Terhune, Medical College of Wisconsin, Milwaukee, WI) were cultured similarly except that the medium contained 10% FCS. Viral stocks (WR strain of vaccinia virus) were prepared by ultracentrifugation of cytoplasmic lysates through 36% sucrose; titration was performed on confluent monolayers of BSC40 cells, which were fixed and stained with 0.1% crystal violet in 3.7% formaldehyde at 48 hpi. Restriction endonucleases, T4 DNA ligase, calf intestinal alkaline phosphatase (CIP) and Taq polymerase were purchased from Roche Applied Sciences (Indianapolis, IN). Geneticin (G418 sulfate), Lipofectamine 2000, monoclonal V5 antibody, protein molecular weight markers and DNA molecular weight standards were purchased from Invitrogen (Carlsbad, CA). 32P-orthophosphate and 35S-methionine were purchased from Perkin-Elmer Life and Analytical Sciences, Inc. (Boston, Mass.). Ultra pure chemicals, Protein A sepharose and Protein G agarose were from Sigma Aldrich (St. Louis, MO). DNA oligonucleotides were synthesized by IDT (Coralville, Iowa).
Construction of recombinant viruses
Recombinant viruses were prepared as described below, using the primers described in Table .
Generation of the vI5V5 virus
The overlapping products of two initial PCR reactions (1: primers PN 5'+ I5V5 3'; 2: primers I5V5 5'+ PN3') were used together as the template for a second round of PCR performed with primers PN 5' and PN 3'. The final product was digested with Bam HI and cloned into pUCNEO [26
], forming pUCneo:I4-I5V5-I6. B) Isolation of the virus by transient dominant selection with G418
. Cells were infected with wt virus and transfected with pUCneo: I4-I5V5-I6; at 15 hpi G418 was added to select for viruses in which the plasmid had been inserted into the viral genome. Cells were harvested at 48 hpi and two rounds of plaque purification were performed to purify G418R
viruses; insertion of the plasmid was confirmed by PCR with primers specific for NEO. Two sequential rounds of plaque purification in the absence of G418 were then performed to allow recombinational resolution of the tandem repeats present in this virus. To distinguish viruses containing only the wild-type allele from those containing the V5-tagged locus, plaque isolates were screened by PCR using primers that flank the I4 3' and I5 5' junction (I5 SCRN 5' or 3').
Generation of the vΔindI5V5 virus
The overlapping products of two initial PCR reactions (1: primers 1 +B; 2: primers C +3') were used together as the template for a second round of PCR performed with primers 1+3'; this product was digested with Hind
III and Cla I
and cloned into pJS4-tetR (final product pJS4:tetR ↔ opI5) [27
]. The final plasmid contains two transcriptional cassettes. One drives constitutive expression of the TET repressor (tetR), and the other contains the V5I5 ORF under the regulation of the TET operator and the I5 promoter. These two cassettes are flanked by the left and right halves of the TK gene, which enables insertion into the genome by homologous recombination. B) Isolation of the virus by BrdU and G418 selection
. Cells were infected with wt virus and transfected with linearized pJS4:tetR ↔ opI5 DNA. TK-
virus was isolated by two rounds of plaque purification on human TK-
cells in the presence of BrdU (25 μg/ml). Plaques of the correct genotype were expanded. To generate the final inducible virus, the endogenous I5 allele was then replaced with a NEO cassette using the I5 KO plasmid as described below.
Generation of the vΔI5 virus
A) Cloning: generation of the I5KO plasmid
Two PCR reactions were performed, one with primers I4 pUCneo Asp + I4 pUCneo Bam, and one with primers I6pUCneo Bam/Hind + I6 pUCneo Xba. The products were digested with Asp718 + BamHI and BamHI + XbaI, respectively, and then ligated simultaneously into pBSIIKS plasmid DNA that had been digested with Asp718 and XbaI. The resultant plasmid, pBSIIKS:I4-I6, was used as the recipient in the next round of cloning. A 1.3 kb fragment containing the NEO gene under the regulation of a constitutive viral promoter was released from pUCneo by digestion with BamHI and HindIII, and the 5' overhangs were filled in with the Klenow fragment of E. coli DNA polymerase. The plasmid pBSIIKS:I4-I6 was linearized at the internal BamHI site and treated with calf intestinal alkaline phosphatase; the 5' overhangs were filled in as described above. This linearized plasmid was then ligated with the NEO insert; the resulting plasmid, in which portions of the I4 and I6 gene flank NEO, was designated pI5KO. B) Isolation of the virus by G418 selection. Cells were infected with vI5V5 and transfected with pI5KO; G418R virus was obtained by two rounds of plaque purification in the presence of G418; correct insertion of the NEO cassette and deletion of the I5 locus were confirmed by PCR.
Metabolic labeling and immunoprecipitation
Confluent monolayers of BSC40 cells were infected with vI5V5 or wt virus (MOI 5) and metabolically labeled with 35S-methionine (100 μCi/ml) from 3–9 hpi or with 32PPi (100 μCi/ml) from 4–8 hpi in DMEM lacking methionine or phosphate, respectively, along with 5% FCS that had been dialyzed against TBS. Cells were lysed in phospholysis buffer (10 mM NaPO4 [pH 7.4], 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate), and clarified lysates were incubated with either monoclonal V5 or polyclonal F18 antisera for 4 hr on ice followed by the addition of Protein A for 1.5 hours. Immune complexes were washed, resolved electrophoretically, and visualized by autoradiography using a Kodak low emission screen.
BSC40 cells were infected with vI5V5 (MOI 2) for 17 hr and then fixed in situ with 4% paraformaldahyde/0.1% glutareldahyde in PBS for 60 min at room temp. Cells were then processed for immunoelectron microscopy and embedded in Lowicryl K4M; grids were stained using the V5 antibody followed by incubation with a secondary antibody conjugated to 5 or 10 nm gold.Intact virions: Purified vI5V5 virions (or control virions encoding a wt I5 protein lacking the V5 epitope) were applied to grids and probed with either a control antibody (anti-A17) or the anti-V5 antibody and a secondary antibody conjugated to 10 nm gold particles. Grids were post-stained with uranyl acetate. All images were obtained on a Hitachi H-600 microscope.
BSC40 cells were infected with wt virus or vI5V5 for 8 h prior to being fixed with 4% paraformaldehyde; cells were then incubated with the monoclonal anti-V5 antibody and a secondary antibody conjugated to Alexafluor 594. DAPI was included to visualize the nucleus and viral replication factories.
NP40 and DTT fractionation
wt and I5V5 virions purified by sedimentation on 25–40% sucrose gradients were treated with NP40 (1%) or NP40 and DTT (1% and 50 mM, respectively) [5
]. The soluble (S) and particulate (P) fractions, representing the membrane and core components, respectively, were resolved by sedimentation (16,000 × g, 30 min, room temperature) and analyzed by immunoblot analysis with anti-V5 and antibodies against known membrane (A17) and core (F18) proteins.
I5V5 virions (6 μg) were subjected to treatment with chymotrypsin (Chymo) or trypsin (Tryp) (10 μg/ml, 30 min at 37°C) or with proteinase K (ProtK; 50 μg/ml) for 10 or 30 min at 4°C. After sedimentation at 14,000 × g, 5 min, the soluble (S) and pellet (P) fractions were resolved and analyzed by immunoblot analysis using anti-D8 or anti-V5 antibodies.