Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity

<p>Abstract</p> <p>Background</p> <p>The presence of cholesterol in the Human Immunodeficiency Virus (HIV) lipid envelop is important for viral function as cholesterol depleted viral particles show reduced infectivity. However, it is less well established whether other viral membrane lipids are also important for HIV infection.</p> <p>The ABCB4 protein is a phosphatidyl choline (PC) floppase that mediates transport of PC from the inner to the outer membrane leaflet. This property enabled us to modulate the lipid composition of HIV vectors and study the effects on membrane composition and infection efficiency.</p> <p>Results</p> <p>Virus generated in the presence of ABCB4 was enriched in PC and cholesterol but contained less sphingomyelin (SM). Viral titers were reduced 5.9 fold. These effects were not observed with an inactive ABCB4 mutant. The presence of the ABC transport inhibitor verapamil abolished the effect of ABCB4 expression on viral titers.</p> <p>The ABCB4 mediated reduction in infectivity was caused by changes in the viral particles and not by components co purified with the virus because virus made in the presence of ABCB4 did not inhibit virus made without ABCB4 in a competition assay.</p> <p>Incorporation of the envelope protein was not affected by the expression of ABCB4. The inhibitory effect of ABCB4 was independent of the viral envelope as the effect was observed with two different envelope proteins.</p> <p>Conclusion</p> <p>Our data indicate that increasing the PC content of HIV particles reduces infectivity.</p

Long-term correction of bilirubin UDPglucuronyltransferase deficiency in rats by in utero lentiviral gene transfer

Bilirubin is glucuronidated by bilirubin UDP-glucuronyltransferase (UGT1A1) before biliary excretion. Because bilirubin is toxic, patients with Crigler-Najjar type I (CN), who have no UGT1A1 activity, suffer severe brain damage early in childhood. The Gunn rat is the model for CN type 1. Gunn rat fetuses were injected with 10(7) transducing units of UGT1A1 lentiviral vector at the end of the third trimester on embryonic day 19. Serum bilirubin of injected Gunn rats was lowered by 45% compared to untreated controls. This decrease was highly significant (P < 10(6)) and was sustained for more than a year. In treated Gunn rats, bilirubin glucuronides were present in bile and UGT1A1 protein was detected in tissue. Liver, intestine, stomach, pancreas, and other organs were transduced and mostly contained 1% or less vector copies per genome. Tissue distribution was variable among experimental animals but high transduction levels were seen in pancreas and intestine in most animals. Immunohistochemistry of these organs revealed transduction of pancreatic acinar cells and intestinal epithelium. Injection of a lentiviral UGT1A1 vector into third-trimester Gunn rat fetuses corrects the metabolic deficiency and mediates a reduction of serum bilirubin levels that would be therapeutic in human

Kupffer cells and not liver sinusoidal endothelial cells prevent lentiviral transduction of hepatocytes

Lentiviral vectors can stably transduce dividing and nondividing cells in vivo and are best suited to long-term correction of inherited liver diseases. Intraportal administration of lentiviral vectors expressing green fluorescent protein (Lenti-GFP) in mice resulted in a higher transduction of nonparenchymal cells than hepatocytes (7.32 +/- 3.66% vs 0.22 +/- 0.08%, respectively). Therefore, various treatments were explored to increase lentiviral transduction of hepatocytes. Lenti-GFP was injected into the common bile duct, which led to transduction of biliary epithelium and hepatocytes at low efficiency. Transient removal of the sinusoidal endothelial cell layer by cyclophosphamide to increase accessibility to hepatocytes did not improve hepatocyte transduction (0.42 +/- 0.36%). Inhibition of Kupffer cell function by gadolinium chloride led to a significant decrease in GFP-positive nonparenchymal cells (2.15 +/- 3.14%) and a sevenfold increase in GFP-positive hepatocytes compared to nonpretreated mice (1.48 +/- 2.01%). These findings suggest that sinusoidal endothelial cells do not significantly limit lentiviral transduction of hepatocytes, while Kupffer cells sequester lentiviral particles thereby preventing hepatocyte transduction. Therefore, the use of agents that inhibit Kupffer cell function may be important for lentiviral vector treatment of liver diseas

Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity-0

E ABCB4, (B) mutant ABCB4. (C) ABCC1, (D) untransfected 293T cells. Nuclei were stained with DAPI. Strong immunoreactive staining was observed in cells expressing ABCB4, ABCB4mut and ABCC1.<p><b>Copyright information:</b></p><p>Taken from "Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity"</p><p>http://www.retrovirology.com/content/5/1/14</p><p>Retrovirology 2008;5():14-14.</p><p>Published online 1 Feb 2008</p><p>PMCID:PMC2265746.</p><p></p

Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity-2

N equal amounts of HIV p24 were loaded on the gel, the intensity of VSVg staining is also identical. With loading of equal amounts of transducing units (TU) the intensity of VSVg staining is much stronger in the virus produced with ABCB4. This indicates that ABCB4 expression during production of viral vectors does not compromise VSVg incorporation.<p><b>Copyright information:</b></p><p>Taken from "Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity"</p><p>http://www.retrovirology.com/content/5/1/14</p><p>Retrovirology 2008;5():14-14.</p><p>Published online 1 Feb 2008</p><p>PMCID:PMC2265746.</p><p></p

Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity-4

E ABCB4, (B) mutant ABCB4. (C) ABCC1, (D) untransfected 293T cells. Nuclei were stained with DAPI. Strong immunoreactive staining was observed in cells expressing ABCB4, ABCB4mut and ABCC1.<p><b>Copyright information:</b></p><p>Taken from "Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity"</p><p>http://www.retrovirology.com/content/5/1/14</p><p>Retrovirology 2008;5():14-14.</p><p>Published online 1 Feb 2008</p><p>PMCID:PMC2265746.</p><p></p

Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity-5

O western blotting as described. Lanes represent: (1) wild type ABCB4, (2) mutant ABCB4, (3) negative control and (4) ABCC1. Wild type and mutant ABCB4 migrated at an apparent molecular mass of 140 kDa. ABCC1 migrated at a molecular mass of 170 kD. The two bands in both ABCB4 and ABCC1 lanes most likely correspond to differently glycosylated forms. Wild type and mutant ABCB4 appear to be differently processed. Similar amounts of cellular protein were loaded as shown by β-actin antibody reactivity.<p><b>Copyright information:</b></p><p>Taken from "Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity"</p><p>http://www.retrovirology.com/content/5/1/14</p><p>Retrovirology 2008;5():14-14.</p><p>Published online 1 Feb 2008</p><p>PMCID:PMC2265746.</p><p></p