Cholesterol Supplementation During Production Increases the Infectivity of Retroviral and Lentiviral Vectors Pseudotyped with the Vesicular Stomatitis Virus Glycoprotein (VSV-G)

Abstract

Cholesterol, a major component of plasma membrane lipid rafts, is important for assembly and budding of enveloped viruses, including influenza and HIV-1. Cholesterol depletion impairs virus assembly and infectivity. This study examined the effects of exogenous cholesterol addition (delivered as a complex with methyl beta cyclodextrin) on the production of Molony murine leukemia virus retroviral vector and HIV-1-based lentiviral vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Cholesterol supplementation before and during vector production enhanced the infectivity of retroviral and lentiviral vectors up to 4-fold and 6-fold, respectively. In contrast, the amount of retroviral vector produced was unchanged, and that of lentiviral vector was increased less than two-fold. Both free cholesterol and cholesterol ester content in 293-gag-pol producer cells increased with cholesterol addition. In contrast, the phospholipids headgroup composition was essentially unchanged by cholesterol supplementation in 293-gag-pol packaging cells. Based on these results, it is proposed that cholesterol supplementation increases the infectivity of VSV-G-pseudotyped retroviral and lentiviral vectors, possibly by altering the composition of the producer cell membrane where the viral vectors are assembled and bud, and/or by changing the lipid composition of the viral vectors.

Fig. 1

Cholesterol supplementation schemes for both retroviral MoMuLV vector (top) and lentiviral vector (bottom) production. Cholesterol was added to culture at periods P (24 h prior to transfection or induction), I (0–24 h post-transfection or induction), and/or II (24–48 h post-transfection or induction).

Fig. 2

MoMuLV Gag protein is associated with detergent-resistant membrane of untransfected 293-gag-pol packaging cells. Cells treated without (A) or with (B) 0.03 mM cholesterol for 48 h were homogenized and sonicated. Postnuclear supernatants were treated with (w) or without (w/o) 0.5% Triton X-100 (TX100) on ice or at 37 °C before equilibrium flotation centrifugation. Fractions containing MoMuLV Gag protein were detected by immunoblotting. Fractions (1–9) represent the sucrose gradient (10–85.5%). Fraction 1 corresponds to the top and fraction 9 corresponds to the bottom.

Fig. 3

Effects of cholesterol supplementation on transduction efficiency (TE) of NIH3T3 cells using either FBS (A) or FP (B), each at a serum concentration of 5% or 10%. 0.03 mM cholesterol was added at periods P + I + II (Fig. 1). A range of TE values was obtained by using different volumes of viral vector to transduce the target cells. All values were normalized to those under control condition at each collection, in which the same volume of viral vector produced without cholesterol supplementation was used to transduce the cells.

Fig. 4

Cholesterol supplementation after vector production had no effect on transduction efficiency using vectors from the 2nd collection. For supplementation after vector production, 0.03 mM cholesterol was added at 37 °C 1 h before transduction to viral vector produced under control conditions. For supplementation during transduction, cholesterol was added only during transduction. For supplementation during production, cholesterol was added to producer cells before and after transfection. All values were normalized to those under control condition at the 2nd collection, in which the same volume of viral vector produced without cholesterol supplementation was used to transduce the cells. Data points represent the mean ± S.D. (n = 2).

Fig. 5

Effects of adding MbCD alone during vector production on transduction efficiency using vectors from the 2nd collection. MbCD with the same concentration (0.25 mM) as that used for cholesterol supplementation was added either alone (0.25 mM MbCD) or complexed with cholesterol (0.03 mM cholesterol) to 293-gag-pol cells during vector production. The insert figure represents the results when 5 mM unloaded MbCD was added to 293-gag-pol cells 9 h before the 2nd collection. All values were normalized to those under control condition at the 2nd collection, in which the same volume of viral vector produced without cholesterol supplementation was used to transduce the cells. Data points represent the mean ± S.D. (n = 2). Results are shown separately for cultures with low (20–40%) and high (>40%) transductions efficiency under control conditions.

Fig. 6

Gag protein amounts in viral vectors determined by immunoblot for both control conditions and cholesterol supplementation. A typical immunoblot result for p30^gag in viral vectors from the 1st and 2nd collections with or without 0.03 mM cholesterol supplementation is shown in (A). For culture with cholesterol, cholesterol was added at periods P + I + II (Fig. 1). In (B) the amount of p30^gag (determined from densitometry analysis of immunoblots) in viral vectors with or without cholesterol supplementation for each collection was normalized to that of the 1st collection under control conditions. Data points represent the mean ± S.D. (n = 3). p = 0.26 for the 1st collection and p = 0.35 for the 2nd collection with or without cholesterol supplementation.

Fig. 7

Comparison of the stability of retroviral vector produced with or without cholesterol supplementation. MoMuLV vectors were incubated for different periods of time at 37 °C and then used for transduction. The x-axis represents the transduction efficiency for vectors without incubation at 37 °C and the y-axis represents the vector decay rate obtained from the experiment.

Fig. 8

Phospholipid composition in untransfected 293-gag-pol cells with or without cholesterol supplementation. Packaging cell line 293-gag-pol (without transfection) was incubated with 0.03 mM cholesterol for 3, 9, 23, or 46 h, respectively. Alternatively, cholesterol-free methyl-beta-cyclodextrin (10 mM) was added to 293-gag-pol cells and incubated for 30 min at 37 °C to deplete cholesterol (depletion). LPC: lysophosphatidylcholine; SM: sphingomyelin; PC: phosphatidylcholine; PI: phosphatidylinositol; PS: phsphatidylserine; PE: phosphatidylethanolamine; PA: phosphatidic acid. Data points represent the mean ± S.D. (n = 2).

Fig. 9

Total cholesterol (TC) and free cholesterol (FC) content in transfected 293-gag-pol cells with or without 0.03 mM cholesterol supplementation during vector production. Cholesterol was added at periods P + I + II (Fig. 1). All values were normalized to the total cholesterol amount under control conditions prior to transfection. Data points represent the mean ± S.D. (n = 3–7).