Minimal impact of ZAP on lentiviral vector production and transduction efficiency

Figures

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  Figure 1. Schematic of CpG dinucleotides in retroviral vector genomes DNA sequences were analyzed for CpG sites using Methyl Primer Express (Applied Biosystems). Genomes within each panel are to scale. (A) CpG dinucleotides annotated for wild-type HIV-1 and HIV-1env86−561CpG genomic RNAs. (B) CpG dinucleotides annotated for lentiviral vector genomes pLV-CMV-GFP, plentiCRISPRv.2-GFP, pHR’SIN-cPPT-SEW (pSEW), and pSEW-lowCpG genomic RNAs. (C) CpG dinucleotides annotated for MLV-CMV-GFP retroviral vector genomic RNA. (D) CpG dinucleotides annotated for the packaging vectors pCMVΔR8.91, pSYNGP, and pCMV-MLV-Gag-Pol.

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  Figure 2. ZAP restricts HIV-1 with CpG dinucleotides introduced in env nucleotides (nt) 86−561 (A) HEK293T CRISPR control, ZAP-ex4, and ZAP-ex6 cells were transfected with wild-type pHIV-1 or pHIV-1env86–561CpG. The culture supernatants were used to infect TZM-bl reporter cells to measure infectious virus production. The bar chart shows the average values of three independent experiments. Data are shown as mean ± SD, ∗p < 0.05, as determined by an unpaired t test. (B) Intracellular HIV-1 Gag and ZAP expression were determined by western blotting. Virus production was determined by western blot of the producer cell supernatant.

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  Figure 3. Endogenous ZAP in HEK293T cells does not restrict lentiviral vector titers HEK293T producer cells (CRISPR control, ZAP-ex4, or ZAP-ex6) were transfected with the indicated genome plasmid, packaging plasmid, and pVSV-G. (A, C, and E) Infectious titers were determined in transduced target cells (HEK293T CRISPR control, ZAP-ex4, or ZAP-ex6 cells) by flow cytometry for GFP-positive cells. The bar charts show the average values of three independent experiments. Data are shown as mean ± SD. (B, D, and F) Intracellular HIV-1 Gag expression from the packaging vector, GFP expression from the vector genome, and HSP90 and ZAP expression were determined by western blots of the producer cell lysates. Virus production was determined by western blots of the producer cell supernatant.

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  Figure 4. ZAP overexpression does not inhibit lentiviral vector titers HEK293T cells were transfected with the indicated genome plasmid and packaging vector plus pVSV-G and pcDNA4, pHA-ZAP-L, or pHA-ZAP-S in pcDNA4. The infectious vector titer was determined by flow cytometry for GFP-positive HEK293T target cells. (A) Infectious titers of lentiviral vectors made with pLV-CMV-GFP, pCMVΔR8.91, and pVSV-G. (B) Infectious titers of lentiviral vectors made with pLV-CMV-GFP, pSYNGP, and pVSV-G. (C) infectious titers of lentiviral vectors made with plentiCRISPRv.2-GFP, pCMVΔR8.91, and pVSV-G. (A−C) Intracellular HIV-1 Gag expression from the packaging vector, GFP expression from the vector genome, HSP90, and ZAP were determined by western blots of the producer cell lysates. Virus production was determined by western blots of the producer cell supernatant. The bar charts show the average values of four (A) or three (B and C) independent experiments. Data are shown as mean ± SD.

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  Figure 5. Removal of non-HIV-1-derived CpG dinucleotides from the lentiviral vector genome does not improve lentiviral vector titers (A−C) HEK293T cells were transfected with pSEW or pSEW-lowCpG plus pCMVΔR8.91 and pVSV-G. (A) Jurkat T cells were transduced with the lentiviral vector supernatant. The infectious titer was determined by flow cytometry of GFP-positive cells. The bar chart shows the average values of three independent experiments. Data are shown as mean ± SD, ∗p < 0.05, as determined by an unpaired t test. (B) The relative mean fluorescence intensity (MFI) of GFP-positive cells, indicating the GFP expression efficiency. The bar chart shows the average values of three independent experiments normalized to pSEW. Data are shown as mean ± SD, ∗p < 0.05, as determined by an unpaired t test. (C) Expression of intracellular HIV-1 Gag from the packaging plasmid, GFP from the vector genome, and HSP90 were determined by western blotting. The band intensity of GFP was quantified for relative GFP expression. (D and E) Relative genomic RNA abundance within the producer cell lysates and virions was quantified by qRT-PCR. (B−E) The bar charts show the average values of three independent…

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  Figure 6. ZAP does not substantially inhibit a gammaretroviral vector (A) HEK293T CRISPR control, ZAP-ex4 ,or ZAP-ex6 producer cells were transfected with pMLV-CMV-GFP, pCMV-MLV Gag-Pol, and pVSV-G. Infectious titers were determined in transduced target cells (HEK293T CRISPR control, ZAP-ex4, or ZAP-ex6 cells) by flow cytometry for GFP-positive cells. (B and D) Intracellular MLV Gag expression from the packaging plasmid, GFP expression from the vector genome, and HSP90 and ZAP expression were determined by western blots of the producer cell lysates. Virus production was determined by western blots of the producer cell supernatant. (C) HEK293T cells were transfected with the pMLV-CMV-GFP genome and pCMV-MLV Gag-Pol packaging vector plus pVSV-G and pcDNA4, pZAP-L, or pZAP-S. Infectious titers of the vectors were determined by flow cytometry for GFP-positive HEK293T target cells. (A and C) The bar charts show the average values of three independent experiments. Data are shown as mean ± SD.