Figure 1.
Schematic representation of lentiviral plasmids used in this study.
A) The numbers in parentheses at the top designated as pLenti/EGFP indicate each region of viral sequence elements residing in the 5′ UTR of HIV-1 according to the genome sequence of HXB2 isolate [19]. Shown below is an enlargement of the 5′ UTR region and its nucleotide sequence. Black triangles around the packaging sequence Psi indicate nucleotide positions in which restriction enzyme sites were created to generate lentiviral transgene vectors used in this study. Open triangles indicate each nucleotide positions to generate serial deletion mutants of the Psi tested in this study, which are also schematically shown in Figure 6A. B) Nucleotide positions and sequence changes introduced in the transgene vector constructs are indicated.
Figure 2.
Transduction efficiency and RNA packaging of various lentiviral vectors.
A) One milliliter of lentiviral supernatants harvested from transiently-transfected 293FT cells was used to transduce 1×105 MT4 cells. Expression levels of the EGFP transgene were observed by fluorescence microscopy in 293FT cells and MT4 cells 30 hr post-transfection and 72 hr post-transduction, respectively. B) Amounts of virus particles in p24 in harvested viral supernatants were measured by ELISA. (error bars: ±S.E.M.) C) Percentage of EGFP positive MT4 cells after transduction was determined by FACS as described in Materials and Methods. D) The transducibility of each virus was calculated by normalization with p24 ELISA data, and each graph represents five different experiments performed independently. E) One microliter of viral supernatant was subjected to direct virus lysis and qRT-PCR to measure packaged viral RNA, and the results were normalized with the ELISA data. Statistically different results were tagged with two (p<0.05) or three (p<0.0001) asterisks (One-way ANOVA).
Figure 3.
Transduction efficiency and RNA packaging of the PMM.psi transgene vectors.
A) Lentiviruses harvested from transfected 293FT cells with pLenti/PMM1.psi or PMM2.psi were used for MT4 transduction, and the transduced cells were observed at 72 hr post-transduction. B) Amounts of virus particles in p24 in harvested viral supernatants were measured by ELISA, and C) Percentage of EGFP positive MT4 cells transduced was determined by FACS. D) Lentiviral transduction ratio was calculated from FACS and p24 ELISA results. Four independent experiments were performed. E) qRT-PCR was performed in triplicate as described in the legend of Figure 2, and the data were presented as p24 normalized ratios. All data were compared to Lenti/EGFP control in One-way ANOVA (F-test), and data showing significant differences (p<0.001) were marked with three asterisks.
Figure 4.
Titration of lentiviral titers using HT1080 cells.
Harvested lentiviral supernatants were subjected to 10-fold serial dilution for 5 times, and then used to transduce HT1080 cells. These cells were cultivated under Blasticidin selection (5 µg/ml) for 14 days, and stained with crystal violet. The stained colonies in plates were counted and used to calculate the titers of lentiviral vectors, which were presented collectively in Table 1.
Table 1.
Determination of lentiviral titers and transduction efficiency.
Figure 5.
Transduction efficiency and RNA packaging by alternative Psi packaging sequence in various transgene vectors.
A) Various transgene plasmids harboring SW8.4 or SW8.4.Mut sequence substituted in the SL3 position of HIV-1 Psi are schematically illustrated. B) Each mutant plasmid was transfected into 293FT cells with packaging plasmids, and the harvested supernatants were determined for viral p24 values and used to transduce MT4 cells. C) Percentage of EGFP-positive MT4 cells from FACS analysis are shown in parenthesis in the figures and the numbers in each figure indicate the relative transducibility of each vector compared to that of pLenti/EGFP transgene vector. D) The viral RNA packaging efficiency was measured by qRT-PCR and normalized to p24 ELISA data. All data are presented in comparison to the EGFP control in One-way ANOVA, and data showing significant differences (p<0.001) are marked with three asterisks.
Figure 6.
The effect of serial deletions of the Psi sequence on gene transduction efficiency and RNA packaging.
A) Psi sequence of PMM.psi vector was deleted from 3′ end serially. The nucleotide position numbers were also shown in parentheses at the end of each mutant constructs. B) Lentiviruses produced from PMM1.psi deletion mutants were transduced to MT4 cells for 72 hr, and expression of EGFP was visualized by fluorescence microscopy and percentage of EGFP positive cells was quantified with FACS and indicated on each histogram. C) Transduction efficiency of each vector in MT4 cells was calculated from FACS data normalized with p24 ELISA. D) Viral RNA packaging efficiency was determined from qRT-PCR and p24 ELISA. All data are presented in comparison to EGFP control in One-way ANOVA, and data showing significant differences (p<0.001) are marked with three asterisks.