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The authors describe a novel lentiviral packaging system in which not only …


Biology Articles » Virology » Design of a trans protease lentiviral packaging system that produces high titer virus » Methods

Methods
- Design of a trans protease lentiviral packaging system that produces high titer virus

Plasmid construction of transfer vectors

All the lentiviral components used in plasmid construction with the exception of Vpr [27] and RRE [42], were derived from PLNENV-1, accession # M19921 [43]. Oligonucleotides used were purchased from Life Technologies and all PCR products were verified by sequencing. The basic vector design of the wt-LTR and SIN lentiviral vectors has been described previously [44]. Briefly, both of the wt-LTR and SIN vectors contain a packaging signal, central polypurine tract, RRE (Rev response element), and an Ef1α promoter [45] driving the expression of eGFP (Clontech), which replaces the β-Globin cassette located between the BamHI and Kpn I restriction sites. The SIN vector contains a 400 bp deletion between EcoRV and Pvu II sites in the U3 region of the 5' LTR as well as a complete deletion of the U5, which was replaced by an "ideal" termination/polyadenylation sequence (ATG TGT GTG TTG GTT TTT TGT GT). In addition the SIN vector also contains two stops in the packaging signal placed at the 1st and 35th amino acid of Gag to prevent translation of Gag.

Plasmid construction of Gag expression plasmids

The backbone used for all three Gag expression plasmids, Gag-Pol-Vif, Gag-PR, and Gag alone, was "pCI Vector" (Promega) in which Nhe I site was replaced by a BssHII linker, the CMV promoter from the Bgl II to the Sac I sites was replaced by a Nhe 1 to Sac I fragment from cRev plasmid [42], this inserted the SV40 origin next to CMV promoter, and finally a RRE obtained from the Bgl II to Hind III sites of pgTatCMV [42] was inserted by blunt ligation into the Xba I to Sal I sites of the pCI Vector backbone. The Gag-Pol-Vif plasmid was made from digesting the backbone with BssHII and EcoRI and then inserting a 5032 bp BssHII to EcoRI Gag-Pol-Vif fragment from PNLENV-1. The Gag-PR plasmid was made by digesting the Gag-Pol-Vif plasmid with Bgl II and EcoRI to remove Pol-Vif, and then PCR was used to create a 463 bp fragment containing PR with a stop and EcoRI site. The primers used for PCR are 5' GGG AAG ATC TGG CCT TCC CAC 3' and 5' CGG AAT TCG GAT CCT TAA AAA TTT AAA GTG CAG CCA ATC TGA CT 3'. The Gag plasmid was made by first replacing the fragment from Nsi I to Bgl II with a PCR version (845 bp), in which the frameshift had been altered. The primers used are 5' TAA ATG CAT GGG TAA AAG TAG TA 3' and 5' CCA GAT CTT CCC TAA GAA GTT AGC CTG TCT CTC AGT ACA ATC 3'. Next the Bgl II to EcoRI fragment was replaced by a 208 bp PCR product, which contained P6 with an EcoRI site placed after the stop (all Pol components were removed). The primers used for PCR are 5' GGG AAG ATC TGG CCT TCC CAC 3' and 5' CGG AAT TCG CTA GCT ATC TTT ATT GTG ACG AGG GGT C 3'. The optimized version of the Gag plasmid was created by altering the coding sequence for the CAp24 and the start of MAp17 (502 bp), from BssHII to Nsi I sites, using PCR to first anneal 26 overlapping 40 mer oligonucleotides (sequence available upon request) and then a second PCR to create the 502 bp BssHII to Nsi I fragment from the annealed oligonucleotides. This protocol was described previously in [46,47].

Plasmid construction of VPR fusion plasmids

The backbone used to construct the Vpr fusion plasmids was "pCI Vector" (Promega) in which the CMV promoter from Bgl II to Mlu I had been replaced with an Hpa I to Mlu I fragment containing the Ef1α promoter [45]. The Vpr-RT/IN-Vif fusion plasmid was made by first inserting the 1–88 truncated Vpr (276 bp) into the Xba I site of the pCI Vector backbone. The Bgl II site at the end of Vpr was ligated to a Bgl II to EcoRV 468 bp PCR fragment consisting of the start of RT, keeping RT in frame with Vpr and the PR cleavage site intact. The primers used for PCR were 5' GGA AGA TCTCTG TTG ACT CAG ATT G 3' and 5' GTA CTG ATA TCT AAT CCC TGG 3'. The EcoRV site from the PCR fragment was ligated to an EcoRV to Not I 3372 bp fragment from the Gag-Pol-Vif plasmid containing the rest of RT, IN, Vif, and the RRE. Finally, in the Vpr-RT/IN-Vif plasmid a splice donor site was placed before Vpr (to ensure proper expression of Vif) using annealed oligonucleotides spanning the Mlu I to Xba I sites. The annealed oligonucleotides used were 5' CGC GTG CTA GCG GCG ACT GGT GAG TAC GCC AT 3' and 5' CTA GAT GGC GTA CTC ACCAGT CGC CGC TAG CA 3'. The Vpr-PR plasmid was made from the Vpr-RT/IN-Vif plasmid by digesting this backbone with Bgl II and EcoRI and then ligating the annealed oligonucleotides from Bgl II to BspEI and a 231 bp BspEI to EcoRI PCR fragment generated from the Gag-PR plasmid. The annealed oligonucleotides consisted of 5' GAT CTG TAT CCT TTA GCT TCC CTC AGA TCA CTC TTT GGC AGC GA 3', 5' CCC CTC GTC ACA ATA AAG ATA GGG GGG CAA TTA AAG GAA GCT CTA TTA GAT T 3', 5' CCG GAA TCT AAT AGA GCT TCC TTT AAT TGC CCC CCT ATC TTT ATT GTG ACG A 3', 5' GGG GTC GCT GCC AAA GAG TGA TCT GAG GGA AGC TAA AGG ATA CA 3' and were used to fuse Vpr in frame to PR at the Bgl II site while maintaining the PR cleavage site and inserting a T26S mutation (ACA changed to TCC) thereby creating a BspEI site. The primers used to PCR the 5' portion of PR and to create a stop at the end of PR are 5' GAA GAT CTA CGC GTT CCG GAG CAG ATG ATA CAG TAT TAG AAG 3' and 5' CGG AAT TCG GAT CCT TAA AAA TTT AAA GTG CAG CCA ATC TGA GT 3'.

Cells

Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 8% bovine growth serum, 2% fetal calf serum and 100-units/ml penicillin-streptomycin. NIH 3T3 were maintained in DMEM media supplemented with 10% calf serum and 100-units/ml penicillin-streptomycin. Jurkat cells were maintained in RPMI media supplemented with 10% fetal calf serum and 100-units/ml penicillin-streptomycin.

Virus production and titers

Virus was produced by transient transfection of 293T cells (10 cm dish) using Fugene (Roche). Twenty-four hours prior to transfection 293T cells were split 1:4 and at two hours prior to transfection media was removed and fresh media was added. Transfections were done using 5, 6, or 7 plasmids depending on the packaging system used. Plasmids transfected for the 5 plasmid system consisted of 3.2 μg lentiviral vector (wt-LTR or SIN, expressing GFP), 4 μg Gag-Pol-Vif packaging plasmid, 0.4 μg of each Rev, Tat, and VSV-G expression plasmids, for the 6 plasmid system the Gag-Pol-Vif plasmid was replaced by two plasmids one expressing Gag-PR (5.5 μg) and the other Vpr-RT/IN-Vif (2.0 μg), and for the 7 plasmid system the Gag-Pol-Vif plasmid was replaced by three plasmids expressing Gag (4.0 μg), Vpr-PR (1.8 μg) and Vpr-RT/IN-Vif (1.8 μg). Supernatants were collected and filtered through a 0.45 μM filter 48 h after transfection. Titers were determined by infecting either NIH 3T3 cells (2 × 105 cells/6 well dish) or Jurkat cells (3 × 106 cells/6 well dish) with serial dilutions of viral supernatants in a total volume of 900 μl in the presence of polybrene (8 μg/ml) or protamine sulfate (6 μg/ml), respectively. Transduced NIH 3T3 and Jurkat cells were analyzed, three or more days after infection for the expression of GFP by FACS. Transducing units (TU) were determined by multiplying the number of total cells at the time of infection by the percentage GFP positive cells by the dilution factor.

Immunoprecipitation analysis

Forty-eight hours post-transfection, cells were washed with PBS and radiolabeled with 250 μCi of [35S] methionine per 10 cm dish (Trans35S-Label; ICN, Irvine, CA) for 12 h. Following labeling, cell culture supernatants were collected and centrifuged at 3000 rpm for 30 min to remove any remaining cells or cell debris. Labeled viral particles in supernatants were isolated by ultracentrifugation through a 20% sucrose cushion at 32,000 rpm for 2 h using a Beckman Ti 61 rotor. Pelleted viral particles were then lysed in RIPA buffer (10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.25% sodium deoxycholate and 0.2% phenyl-methylsulfonyl fluoride (PMSF) and immunoprecipitated with the anti-HIV-1 serum (162) as described previously (53). Immunocomplexes were separated by SDS-12.5% polyacrylamide gel electrophoresis and analyzed by autoradiography. Densitometric analysis of autoradiograms was performed with a Molecular Dynamics Personal densitometer using the ImageQuant™ software version 3.22.


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