Reverse-phase high-performance liquid chromatography (rp-HPLC)
Separation of proteins was accomplished on a 2 × 50 mm Jupiter 300Å C4 column (Phenomenex, Torrance, California, USA) at 0.2 ml per minute, elution by a gradient from 20 to 90% of acetonitrile in water, and trifluoroacetic acid at 0.1% was included throughout. Analytical and semi-preparative scale rp-HPLC was performed with an 1100 series Agilent LC system. Injection volumes varied between 5 μL with a maximum of 40 μL depending on viral particle concentrations. Ultraviolet absorbance at 210 nm was collected continuously throughout the HPLC analysis. The absorbance at 210 nm was normalized to absorbance at 360 nm to eliminate anomalous absorbance due to scatter. The fractions corresponding to peaks in the UV absorbance traces were frozen on dry ice for a minimum of a half-hour. The fractions were then lyophilized in a rotary lyophilizer. Using the procedures applied here minor structural proteins such as IVa2 or protease were either not observed or identifiable.
Whole protein mass spectroscopy by MALDI-TOF
The residue in the rp-HPLC fraction tubes that remained after lyophilization was re-suspended in 50% acetonitrile and 0.1% TFA typically in a volume of less than 10% of the original collected volume. A small volume of the sample was mixed with 20 mg/ml sinnapinic acid at a 1-to-1 ratio and 2 μL of this mixture was spotted and dried on a MALDI target plate along with protein molecular weight standards such as equine myoglobin and enolase. A dedicated target plate was used that had been optimized in the mass spectrometer to correct for slight deviations in the flatness of the plate. The machine was run in linear mode and delayed extraction of ca. 50-600 ns and acceleration voltages as required for maximum signal-to-noise and resolution was utilized. The fractioned proteins were also analyzed by SDS-PAGE allowing for co-identification of the individual proteins on both SDS-PAGE and HPLC.
Tryptic peptide mass spectroscopy fingerprints by MALDI-TOF
The dried rp-HPLC fractions were re-suspended in 50 μL of 25 mM ammonium carbonate and incubated with 0.1 μg of TPCK-treated trypsin at 37°C for 2 hours. The peptide mixture was mixed 1-to-1 with 10 mg/ml of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% TFA in water, spotted onto a MALDI target plate with peptide standards (Bradykinin and Angiotensin) and analyzed for a peptide fingerprint following calibration. Typically, the machine was run in reflector mode and utilized a delayed extraction of ca. 50-200 ns and acceleration voltages as required for maximum signal-to-noise and resolution. Monoisotopic masses from the MALDI-TOF machine were matched to proteins by Aldente (version 18/10/2004.) Searches were generally limited to include only those proteins isolated from viral sources but not to any specific virus family. The SDS-PAGE, rp-HPLC, and mass spectrophotometry analyses determined that purified stocks of wild type Ad35 contained intact and fragmented protein II (hexon).
Ad35 virus description
A lysate of Ad35 was obtained from the American Type Culture Collection (ATCC, catalog # VR-718D) and a viral genome cloned into a plasmid by homologous recombination [25
]. The wild type virus and recombinant derivatives were generated from the cloned genome on 293-ORF6 cells when liberated from the plasmid backbone. Sequencing of the entire viral genome revealed it to be nearly identical with the published Holden sequence found at GenBank accession number AY128640
. All genome coordinates referenced in this manuscript are therefore based on the Holden sequence. Viral genomes were modified by homologous recombination in bacteria essentially as previously described [25
]. The CMV immediate early promoter and SV40 early polyadenylation signal expression cassette in the E1 region [27
] is oriented to direct transcription toward the left ITR of the virus except for those viruses with the d2 deletion in which case it is in the opposite direction. The junctions of the Ad35 deletions are indicated in the text. The P5 promoter which directs pIX transcription in the Ad35E1(d5) configuration correspond to AAV bp 143-309 (ATCC accession number AF043303). The fiber shaft modified virus (Figure ) is an E1(d2) configuration with a bovine growth hormone polyadenylation signal and expressing HIV glycoprotein 140B in which Ad35 sequences 31,222-31,797 are replaced with Ad5 sequences 32,239-32,787 (GenBank accession number M73260.1
) was compared to wild type virus.
Viral vector growth competitions
Virus with either E3 wild type (wt) or E3 deletions (X, HE) were competed pair wise against each other. All of the viruses contained a GFP expression cassette in the E1 (d8) deletion and are therefore E1-deleted or E1-, E3-deleted. Equal number of virus particles (pu) were mixed and 293-ORF6 cells were infected at 200 pu/cell total. 72 hours post infection cells were harvested and freeze thawed 3 times. Subsequently three sequential infections were carried out using 50 ul of crude lysate to infect a 60 cm plate of 293-ORF6 cells in a total volume of 500 ul before an additional 3.5 ml of DMEM with 10% FBS and 10 mM Zn Cl2 was added. The infections lasted 72 hrs. Relative levels of viral genomes were determined following the isolation of total DNA from the mixed input viral particles of a 200 μl aliquot of lysate using the High Pure Nucleic Acid Kit from Roche Applied Science (catalog # 11858874001). To distinguish the E3 wild type and E3 deletion X and wild type from deletion HE, the viral genomes were restricted with EcoRV and BlpI endonucleases (New England BioLabs), respectively, before being resolved on a 0.8% agarose gel using 0.5% TBE buffer.
Virion temperature lability
Wild type Ad35, an rAd35 with the E1 deletion d2 and GFP expression cassette, or an rAd35 with the E1 deletion d5 and HIV gp140BΔCFIΔV1V2 expression cassette were diluted in triplicate to 8 × 1010 particles/ml in 10 mM Tris pH7.5, 150 mM NaCl to a final volume of 800 ul in 1.5 ml microcentrifuge tubes on ice, then shifted to 48°C in a water bath. A 150 ul aliquot was removed at each time point, mixed with ice-cold 150 ul of DMEM + 5% FBS and kept on ice until tested for activity in the focus forming unit (FFU) assay. The data were presented as percentage of maximum activity because the input concentrations of the viruses were standardized to particle units and the three stocks of viruses had a 50-fold range in pu:FFU ratios.
Lambda cDNA library construction and cDNA sequencing
To construct the cDNA library total RNA was isolated using the PerfectPure RNA Cultured Cell Kit (catalog # 230240) from 5Prime using the human lung carcinoma cell line A549 24 hours post infection with Ad35 wild type at a moi of 5 FFU/cell. A lambda cDNA library was constructed by Lofstrand Labs Limited (USA) with a ZAP Express cDNA Synthesis Kit from Stratagene (catalog # 200403). Phage manipulation and screening were done essentially according to Current Protocols in Molecular Biology.
The sequence of the pIX and fiber cDNAs were determined. Phage containing pIX and fiber cDNA sequences were identified by probing plaque lifts using DNA labeled with the North2South Biotin Random Prime Labeling kit (catalog # 17075) from Thermo Scientific. The pIX and fiber DNA probes were generated by polymerase chain reaction (PCR) with primer pairs A35s3511 (GGAGTCTTCAGCCCTTATCTGACA) + A35a3853 (CGGCCACCTGCTGAGAAA) and A35s30987 (AACAACCACAGGCGGATCTCT) + A35a31619 (CTATCATAACTAGTCATGTAGTAACATATTCCATGAATGTAGTTTTC) respectively. The identified plaques were picked and phage expanded in culture. The phage lysates were used to prime PCRs whose products were gel purified and sequenced on an ABI 3100. The entire cloned cDNA was amplified using T3 (ATTAACCCTCACTAAAGGGA) and T7 (TAATACGACTCACTATAGG) flanking primers. In addition PCR products for the 5' and 3' portions of the cDNAs were also made and sequenced. The 5' specific reactions for pIX and fiber used A35a3853 and A35a31619 respectively along with the flanking primer CCGCGGATCCGCCCAGTACATGACCTTA. Similarly the cDNA 3' specific reactions used A35s3511 and A35s30987 in conjunction with the T7 primer for pIX and fiber, respectively.
Northern blot analysis
To generate viral RNA 293-ORF6 cells were infected at a moi of 5 FFU/cell when analyzing E1, pIX and fiber transcripts, and at a moi of 1 FFU/cell when analyzing E4 transcripts. Total RNA (5 ug) from viral and mock infected cells was harvested at 24 hours post infection except where noted in the manuscript, purified with the Versagene RNA cell Kit (Gentra Systems, catalog # VGR-0050CD), resolved on a 1 or 1.3% agarose gel containing 5% formaldehyde in 1 × MOPS buffer. The RNA was transferred to Immobilon-Ny+ (Millipore, catalog # INYC13750) rinsed sequentially in 6× SSC and water, air dried before being UV cross linked (UV Stratalinker 2400). The membrane was wetted with 5× SSPE and then prehybridized (50% Formamide, 2× SSC, 1% SDS, 5× Denhardt's and 0.5 mg/ml denatured salmon sperm DNA) at 42°C for 2-3 hours. Hybridization used the same buffer with 10% dextran sulphate overnight at 42°C along with the desired probe (see below) except for the strand specific probes which used the ULTRAhyb buffer (Ambion, catalog # AM8670). The membrane was washed sequentially twice briefly with 2× SSC, twice with 2× SSC, 0.1% SDS for 30 minutes at room temperature and three times with a solution of 0.5% SSC, 1% SDS at 56°C for 30 minutes each. The blot was air dried before being visualized using a Typhoon 9400 (Amersham Biosiences).
All probes were labeled with Redivue α-32P dCTP (Amersham, catalog # AA0075) using the Rediprime II Random Primer kit (Amersham, catalog # RPN1633) except for the strand specific probes which were DNA oligos labeled with γ-ATP using T4 kinase (New England BioLabs, catalog # M0201). The probes were: The description of the probes is as follows. The E1b and pIX probes 1-4 (Figure ) were generated by PCR with primer pairs AGGAAGACTAGGCAACTGT + AAAGTAAGAAAAGCCACAG (probe 1), GGTGGTAATAGATACTCA + GCGTTGATGGGAAACAATATGCACAGTAGC (probe 2), CAAGCATGCCAGGTTCC + TGCGGGCAATAACCAAATGAT (probe 3), A35s3511 + A35a3853 (probe 4). The probe for the 2nd intron was generated from two oligos AGTATTGGGAAAACTTTGGGGTGGGATTTTCAGATGGACAGATTGAGTAAAAATTTGTTTTTTCTGTCTTG + CAAGACAGAAAAAACAAATTTTTACTCAATCTGTCCATCTGAAAATCCCACCCCAAAGTTTTCCCAATAC. The fiber probe was generated by PCR with primer pair A35s30987 + A35a31619. The E4 probes generated by PCR used primer pairs ATAGTAGCCTGACGAACAGGT + GCTGATGAGGCTTTGTATGTG (E4 Orf1), GACAAAATATCTTGCTCCTGT + GCAGACTAATTCAAAACTAAC (E4 Orf2), ACGTGGTAACTGGGCTCTGGT + TGCTTGAAACGCAATCCAGTT (E4 Orf4).
CACCTTCCCATTTGACAGAATA + TTGGGTTTGGCGGGATT (E4 Orf6). The E4 Orf3 probe is a subcloned DNA fragment comprising Ad35 base pairs 33258 through 33611. The E4Orf6 probes were an NruI to DraIII DNA restriction fragment corresponding to Ad35 coordinates 32,008 through 32,883. The strand specific probes are A35a3853, A35a31619 and ACAACGTGGTAACTGGGCTCTGGTGTAAGGGTGATGTCTGGCGCATGATG for pIX, fiber and E4, respectively.
Virus growth analysis
To determine the onset of viral replication, A549 cells were infected with wild type virus with 5 FFU/cell in triplicate using 60 cm plates. At the times indicated cells were washed 3× in PBS and total DNA isolated using the DNeasy Blood and Tissue kit from Qiagen (catalog # 69506). Viral genomes were quantified in a qPCR on an ABI 7700 using manufacture recommended conditions with primers CCCCGAATGGAAGTGCC and CGCTCTTTGGCGGCG along with probe 6FAMTGTTGAGTTGGACATACTCGCGTGCCMGBNFQ purchased from Applied Biosystems. The level of viral genome was normalized to ng of total DNA used in the reaction. The standard curve was generated with a plasmid of the complete E1-deleted Ad35 viral genome.
E4 complementation assays were carried out on PC-3 cells (Human prostate adenocarcinoma) infected with E1-wild type, E4-deleted viruses with 3 FFU/cell and harvested 72 hours post infection. Cells were freeze thawed 3 times and levels of active viral progeny were quantified by an FFU assay [15
Generation of viral progeny was determined on 293-ORF6 cells which were infected with 1 FFU/cell. Cells were cultured in DMEM + 5%FBS + 100 um ZnCl2
and harvested 72 hours post infection. To quantify the number of particles cells were subjected to three freeze-thaw cycles, clarified through a 0.2 μm filter, and 100 ul volume was loaded onto a Poros 50D column (2 mm diameter, 10 cm length) equilibrated in 25 mM Tris pH 7.5. The column was washed with 25 mM Tris pH 7.5, vector eluted with 250 mM NaCl, 25 mM Tris pH 7.5, and particles detected by 214 nm absorbance. The number of viral particles was quantified by generating a standard curve using purified Ad35 particles standardized to A260
particle units [15
]. All of the viruses tested had either the GFP or enhanced GFP (eGFP) gene in the E1 expression cassette.
Vector packaging capacity
Limits on packaging capacity were assessed by testing for genetic rearrangements using a PCR. Viral genomes were liberated from its plasmid backbone by restriction digest, treated with a mixture of phenol:chlorophorm:isoamyl alcohol (24:24;1), ethanol precipitated and dissolved in water. A 60 mM plate of 293-ORF6 cells were transfected with 4 ug of viral genome using Poylyfect (Qiagen catalog # 301105). Five days post transfection cells were harvested, freeze thawed three times with 50 ul of lysate diluted to 500 ul with DMEM used to infect 60 cm plate of 293-ORF6 cells, rocked every 15 minutes for an hour before being fed with 4.5 ml DMEM + 5%FBS + 100 um ZnCl2. The virus was continued to be expanded using the same passaging regimen except cells were harvested at 72 hour post infection. To detect viral re-arrangement genomes were purified from 200 ul of lysates using the High Pure Viral Nucleic Acid kit (Roche, catalog # 11 858 874 001) in 20 ul of which 1 ul was used in a 25 ul PCR (94C 1 min, 30 cycles 94C 30 sec. × 55C 30 sec × 72C 3.5 min, with a final 72°C 10 min extension) with primers CGTACCGTGTCAAAGTCTTC and CGAGTTCTCAATGATCGAGA using Taq polymerase and Expand High Fidelity PCR buffer (Roch Diagnostics, catalog # 1435094 and 11732641001) and 1.7 ul resolved on an agarose gel.
Genetic stability analysis
293-ORF6 cells were transfected with the rAd35 genomic plasmids, cell - virus lysates were serial passaged 3 times (cytopathic effect appeared in the first passage), and viral DNA was extracted for a polymerase chain reaction (PCR) assay [27
] using primers CGTACCGTGTCAAAGTCTTC and CGAGTTCTCAATGATCGAGA.