Construction of hybrid GP64 envelope proteins
The Sendai Virus Fusion cDNA was kindly provided by Dr. Allen Portner (St. Jude Children's Research Hospital) in a mammalian expression vector. The GP64 cDNA was kindly provided by Marcel Westenberg, Wageningen University, and subsequently subcloned into the pCDNA 3.1p mammalian expression vector. An amino terminal truncation of GP64 was made by PCR to remove the native signal peptide (amino acids 1-21) retaining the native GP64 sequence starting at amino acid 25 using the following primers: ClaI GP64F 5'-GATCATCGATGAACGCGCAAATGAAGACGGGT-3' and GP64R 5'-TGCTGGATATCTGCAGAATT-3'. The resulting PCR product was cloned into the pCR2.1 TOPO TA vector (Invitrogen), pCR2.1 AA25GP64 and verified by sequencing. The Sendai-GP64 fusion construct was created by digestion of pCR2.1 AA25PG64 with ClaI and EcoRV to release a 1485 fragment containing the GP64 coding sequence. This fragment was cloned into a ClaI SmaI digest of the pCAG SV-F vector to create an in-frame F2-GP64 fusion cDNA. The resulting Sendai-GP64 fusion plasmid was verified by restriction digest and sequencing. A multiple cloning site containing a ClaI and AgeI restriction site was introduced immediately after the native GP64 signal peptide with the following primers as previously described .
The resulting PCR product was ligated into the pCR2.1 TOPO vector and sequenced. The GP64 coding sequence containing the MCS was released by a KpnI and SacII digest and subcloned into a KpnI and SacII digest of the pCDNA 3.1 GP64 to create pCDNA3.1 MCSGP64. The Sendai Virus F2 coding sequence was amplified with the following primers ClaISVF2f 5'-ATCGATATGACAGCATATATCCAGAGATC-3'
The resulting product was verified by sequencing and cloned into the ClaI site of MCSGP64 to create pCDNA 3.1 Sendai spGP64.
SDS-PAGE and western blotting
Concentrated viral supernatants of GP64, Sendai-GP64/wtGP64, Sendai spGP64, and Sendai spGP64/wtGP64 pseudotyped lentiviral vectors were run on 10% SDS-PAGE and blotted onto nitrocellulose using the Bio-Rad Miniprotean III system. An antibody directed against a C terminal epitope in the GP64 protein, AcV5 (eBioscience 14-6995), was used at a 1:1000 dilution. A 1:1000 dilution of Goat anti Mouse HRP (Bio-Rad 170-6516) was used as a secondary antibody on all blots. Detection of reactive bands on western blots was performed using the Lumi-Light western Blot Substrate (Roche 12 015 200 001) and blots were analyzed using a LumiImager F1 and LumiAnalyst 3.1 software (Roche).
Cell lines and culturing
HEK293T, HeLa, and HepG2 cells were grown in standard DMEM media supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 10% CO2. RAW mouse macrophages were grown in standard RPMI media supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 10% CO2
Lentiviral vector production
Lentiviral vectors with the phosphoglycerate kinase promoter driving eGFP expression were produced as described earlier . Briefly, lentiviral vectors were produced by transient transfection of 293T HEK cells using calcium phosphate precipitation. Sendai-GP64/wtGP64 pseudotyped lentiviral vectors were produced using 6.8 and 0.2 μg of plasmid per plate respectively. Sendai-spGP64 pseudotyped lentiviral vectors used 50 μg of envelope plasmid per plate and Sendai-spGP64/wtGP64 used 50 and 0.5 μg of envelope plasmid respectively. Viral supernatants were concentrated for animal experiments and western blotting by overnight centrifugation using a Hettich centrifuge (2230 g). Viral titers were determined by titration on both HeLa and HepG2 cells. Briefly transductions were performed for four hours (HeLa and HepG2) and overnight (RAW) in the presence of DEAE Dextran (10 μg/ml) and 72 hours later cells were analyzed by flow cytometry for GFP expression. GFP positive cells were not observed when viral transductions of HepG2 cells were performed in the presence of the HIV reverse transcriptase inhibitor AZT (Retrovir) indicating the absence of pseudotransduction.
Viral particles were measured using a commercial ELISA kit for the Gag p24 protein (Perkin Elmer NEK050). HepG2 titers for the concentrated lentiviral vectors ranged from 1.5 - 3.2 × 108 transducing units/ml and p24 levels ranged from 4-30 μg/ml.
Animals, viral injections, and tissue processing
Wild-type FVB male mice ages 6-10 weeks were used in all studies and were fed ad libitum on standard laboratory chow. All animal experiments were performed in accordance with the Animal Ethical Committee guidelines at the Academic Medical Center of Amsterdam.
Mice were anesthetized with an intraperitoneal injection of FFM mix (2.5 mg/ml Fluanisone/0.105 mg Fentanyl citrate/1.25 mg Midozalam HCl/kg in H2O, 7 ml/kg). Under deep anesthesia, the peritoneal cavity was opened and the mice were injected intraportally with identical amounts of infectious virions, the equivalent of 0.5 × 108 HepG2 transducing units, on day 0. The amount of HIV p24 injected was 6.3 μg for GP64, (0.25 ml of 2.0*108 TU/ml n = 5), 21.3 μg for Sendai-GP64/wtGP64, (0.35 ml of 1.5*108TU/ml) n = 7), and 4.6 μg for Sendai-spGP64/wtGP64, (0.16 ml, of 3.1*108TU/ml) n = 4. The peritoneal cavity was sutured and the animals received the analgesic Temgesic (20-30 μl, 0.03 m mg/ml) subcutaneously following recovering from FFM.
On day 7, the mice were killed by in vivo fixation. Under deep anaesthesia, the peritoneal cavity was opened and a ligature was place around the anterior right lobe of the liver, tightened, and the lobe was excised and snap frozen in liquid nitrogen for genomic DNA analysis. Subsequently, the animals were perfused intracardially with 20 ml of phosphate buffered saline (PBS), followed by 20 ml of 2% formaldehyde in PBS. Following perfusion, the liver and spleen were removed and further fixed for 4 hours in 4% formaldehyde in PBS at room temperature. The fixed tissues were then transferred to 30% sucrose solution and incubated overnight at 4°C, snap frozen in liquid nitrogen and stored at -80°C the following day.
Cryosections were made from both the left and medial lobes. The tissue was embedded in Tissue-Tek OCT (Bayer) and sections (6 μm) were applied to poly lysine coated glass slides and enclosed in Vectashield mounting media (Vector Laboratories).
Liver sections were prepared as described above and frozen without mounting media. Following thawing at room temperature sections were washed three times 5 minutes each in PBS and blocked with 10% Normal Goat Serum in PBS/0.05% Tween-20 for one hour. Kupffer cells were stained with a rat anti mouse F4/80 antigen (1:20 Serotec) for one hour. Slides were washed three times 5 minutes each in PBS/0.05% Tween-20 and incubated with a goat anti rat Texas Red conjugated antibody (1:500 Rockland Immunochemicals) for one hour. Following three washing steps of 5 minutes each, sections were embedded in Vectashield mounting media containing DAPI. Images were captured at (400×) magnification using a fluorescent microscope (Leica DMRA2).
Cell counting and statistics
GFP positive cells were counted in sections made from the left and median lobes using a fluorescent microscope (Leica DMRA2). All sections/slides were prepared and coded independent of the counter. Identification of parenchymal and nonparenchymal cells was made based on morphology as described before .
Per animal, one section from the left lobe and median lobe were counted for GFP positive hepatocytes at (200×). Three fields (200×) per section were counted for GFP positive nonparenchymal liver cells. Images of the counted sections were captured and the surface area of sections was calculated using Leica FW4000 software. The number of total hepatocytes per mm2 was estimated by counting amount of hepatocytes present in one field at (400×). Data are reported as number of GFP expressing cells per square millimeter.
Statistical analysis was performed using SPSS 11.0 software using the Mann-Whitney U test. Values were determined to be significantly different with p < 0.05.
Genomic DNA isolation and PCR
Genomic DNA was isolated from snap frozen liver and spleen tissue using Dneasy tissue kit (Qiagen) according to manufacturers instructions. The following primer pairs were used to generate a 274 bp product: HIV-U3 forward primer 5'-CTGGAAGGGCTAATTCACTC-3' and HIV PSI reverse primer 5'-GGTTTCCCTTTCGCTTTCAG-3'. This primer pair is designed to specifically amplify integrated provirus and thus reduce contamination from amplification of the transfer plasmid. Additionally primers directed against GAPDH were used as a template loading control GAPDH forward primer 5'-CAATCACCATCTTCCAGGAG-3' and GAPDH reverse primer 5'-TGCCCACAGCCTTGGCAGC-3'. 100 ng of total DNA was used per PCR reaction using the following conditions: 95°C for 5 minutes, followed by 33 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds with a fill in at 72°C for 10 minutes. Negative control samples were taken from animals that had not been injected with virus.