Cells and viruses
CaSki cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% non-essential amino acids, 50 μg/ml streptomycin, 100 U/ml penicillin, and 10% fetal bovine serum (FBS).
HSV-1 (F) is the prototype HSV-1 strain used in this study. The recombinant virus R3659, described previously , lacks the BglII -SacI subfragment of the BamHI Q fragment, encoding the tk and UL24 genes. In R3659 the BstEII-StuI subfragment of the BamHI S fragment, encoding the γ134.5 and ORF P genes, is replaced by a chimeric gene consisting of the coding domain of the tk gene fused to the α 27 promoter (see Fig. 1).
The virus R3616 has a 1 kb deletion in both copies of the γ134.5 gene [43, 44], and the rest of the genome is intact. R3617 is similar to R3616 but is thymidine kinase negative, having a 501 bp deletion in its tk gene similarly as in Fig. 1, line 1.
The plasmid pRB4878 has been described elsewhere . Briefly, it contains the egr-1 promoter, polylinker site and the hepatitis B virus polyadenylation signal cloned within the BspEI-BstEII deletion of the γ134.5 gene (Fig. 1).
Plasmid pRB5225, containing the first 100 bases of the HPV-16 E7 gene in antisense orientation under the egr-1 promoter, was made by insertion of a 100-bp PCR product, digested with KpnI, into the KpnI site in the polylinker sequence of the pRB4878 plasmid (Fig. 1). The PCR reaction was performed using HPV-16 DNA-containing CaSki cellular DNA as template and PfuI DNA polymerase (New England Biolabs, Beverly, MA, USA). The cloning PCR primers E7ASp-FP and E7ASp-RP are shown in Table 1. An additional deoxythymidine nucleotide was inserted in the E7ASp-FP primer in order to introduce a frameshift at the nucleotide position 571 of the HPV-16 E7 ORF, introducing two stop codons in the sense orientation of the HPV-16 E7 ORF. The correct sequence of the insertion in the pRB5225 plasmid and in the viruses was verified by sequencing (Table 1).
Construction of recombinant viruses
The antisense E7-RNA-expressing virus R5225 was constructed by cotransfection of rabbit skin cells with the pRB5225 plasmid and R3659 viral DNA, using protocols described previously . The selection for the resulting recombinant tk- viruses was performed on 143TK- cells overlaid with DMEM containing 5% fetal bovine serum and 100 μg/ml of bromodeoxyuridine (BUdR). The selection was done by three successive rounds of plaque purification under BUdR, followed by preparation of viral DNA and stocks from Vero cell cultures infected with selected viral plaques, as described elsewhere . The corresponding tk+ virus R5226 was constructed according to methods described previously . The purified DNA of the virus R5225 was cotransfected into rabbit skin cells with the plasmid pRB165 containing the entire BamHI-Q fragment. The selection for tk+ viruses was done by successive rounds of HAT selection in 143TK- cells overlaid with HAT medium (DMEM supplemented with 5% FBS, hypoxanthine, aminopterin, and thymidine), followed by plaque purifications. The viral stocks and DNA were prepared from Vero cell cultures infected with plaque purified viruses.
Analysis of viral DNA
The large scale preparation of viral DNA was done from roller cultures of Vero cells, infected with selected plaque stocks of the recombinant viruses. The DNA was purified using NaI gradients . The correct structure of the insert was verified by hybridization of the electrophoresed, Southern blotted NcoI-digested viral DNA fragments with digoxigenin-labeled 1.8 kb NcoI fragment of the BamHI S fragment (derived from the plasmid pRB4794; see ) (Fig. 2) or with digoxigenin-labeled oligonucleotide E7ASp-FP used for cloning the HPV-16 E7 insert (data not shown). The presence of the DNA insert in the recombinant genome was also verified by PCR of the isolated viral DNA preparates using the insert-specific primers E7ASp-FP and E7ASp-RP. The DNA preparates of R5226, digested with BamHI, were analyzed for the presence of the intact BamHI Q fragment by Southern hybridization with the digoxigenin-labeled pRB165 plasmid, containing the BamHI Q fragment of HSV-1 (F) DNA.
Infection of CaSki cells and production of cDNA
Trypsinized CaSki cells were plated on 6-well tissue culture plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA) and let reach 80% confluency. The cells were infected with either R3617, R3659, R5225, R3616, or R5226 using either 0.1, 1.0, or 3.2 plaque forming units (p.f.u.) per cell. After adsorption (1 h, 37°C), monolayers were washed with PBS and overlaid with DMEM containing 10% FBS. CaSki cells were harvested at 16 h post infection and total RNA was extracted using the TRIZOL Reagent and treated with DNase I for 15 minutes in room temperature as described by the manufacturer (Life Technologies, Paisley, UK). Reverse transcription was performed with 1 μg of total RNA using the "1st Strand cDNA Synthesis Kit" (Amersham Pharmacia Biotech Inc., Uppsala, Sweden) and random hexamer primers for 1 h at 37°C. In time series experiments the level of transgene expression was found to be highest at 16–20 h.p.i., and the effects on the E7 mRNA in CaSki cells were most evident at 16 h.p.i. Therefore the 16 h.p.i was selected as a timepoint for further experiments. The HSV vectors caused cytopathogenicity in the cell monolayer cultures at late timepoints, beyond 24 hpi.
Real-time PCR using the cDNA samples was performed with the "ABI Prism 7700 Sequence Detection System" and the "TaqMan Universal PCR Master Mix" (Applied Biosystems, Foster City, CA, USA). The amplification conditions were: initial incubations for 2 min at 50°C and for 10 min at 95°C, followed by PCR cycling using a two step cycle at 95°C for 15 sec and 60°C for 60 sec for a total of 40 cycles. The primers and probes are shown in Table 1. The primers and probes for this experiment except for HBVAS-FP were designed using the "Primer Express" software (Applied Biosystems). Specific detection of anti-E7 cDNA was achieved with an upstream primer (HBVAS-FP) specific for the hepatitis B virus polyadenylation sequence present only in the antisense RNA. A standard curve for anti-E7 cDNA was obtained by amplification of a dilution series of cDNA produced from RNA transcribed in vitro from a pGEM-T plasmid containing the insert of pRB5225. A standard curve for E7 cDNA was obtained by amplification of a dilution series of cDNA from CaSki cell total RNA. All samples were also analyzed with the 18 S rRNA Kit (PE Biosystems, Warrington, UK), and the anti-E7 and E7 values were corrected using the values from these amplifications. At least three "no template control" reactions were included in each run. As a control for cellular mRNA changes during the HSV vector infections we studied the cellular beta-actin mRNA by quantitative RT-PCR as described previously . The statistical analyses were performed with SAS software using the Dunnett generalized linear model procedure.
Protein electrophoresis and immunoblotting
CaSki cells were grown as described above in 20 cm2 cell culture dishes (Nalge Nunc International, Roskilde, Denmark) to 80% confluency. A similar culture of HPV negative HaCaT cells (immortalized human keratinocytes) was grown to yield a control sample not containing E7 protein. CaSki cells were infected with R3659, R5225 and R5226 at a multiplicity of infection (m.o.i.) of 1.0 and 3.2 and harvested at 16 hours post infection. The cells were lysed by incubation in 50 μl of buffer containing 150 mM NaCl, 50 mM Tris (pH 8.0), 5 mM EDTA, 1% Nonidet-P 40, 2 mM dithiothreitol, and 2 mM PMSF for 30 minutes on ice. The lysate was centrifuged at 12000 × g for 30 minutes at +4°C. The supernatant with proteins was removed and stored at -70°C.
The samples, containing 30 μg of protein, were suspended in a 2 × sample buffer (100 mM Tris-HCl (pH 6.8), 200 mM dithiothreitol, 4% SDS, 0,2% bromophenol blue, 20% glycerol), boiled for 4 minutes and run on a 15% SDS-PAGE. The proteins were transferred electrophoretically to "Hybond-P" PVDF membrane (Amersham Life Science, Buckinghamshire, UK) in a running buffer containing 25 mM Tris, 192 mM glycine, and 20% v/v methanol. Equal loading was verified with Ponceau S staining. The membranes were blocked with a buffer containing 1 × PBS, 0,1% Tween-20, and 5% non-fat dried milk for 1 h and then probed with a rabbit polyclonal anti-HPV-16 E7 antibody (a gift from Dr. Massimo Tommasino). The membranes were washed with PBS containing 0.1% Tween-20 before and after incubation with the primary antibody, 2 times for 10 min and 5 times for 5 min, respectively. The membranes were then incubated with an HRP-conjugated anti-rabbit-Ig secondary antibody (Dako, Glostrup, Denmark) for 1 h and detected using ECL+Plus Western blotting detection reagents (Amersham Pharmacia Biotech, Uppsala, Sweden). Computerized image analysis was performed to quantitate the intensities of the signals with "Microcomputer Imaging Device version M4" (Imaging Research, St. Catharines, Ontario, Canada).