470. ABCB4 P-Glycoprotein Reduces Infectivity of Lentiviral Particles by Increasing Their Phosphatidylcholine Content

Abstract

Human Immunodeficiency Virus-1 (HIV-1) particles are assembled and released from membrane microdomains (lipid rafts). The membrane composition of the viral particle, which is important for viral integrity and infectivity, is therefore determined by the raft lipid composition.ATP binding cassette (ABC) transporters are transmembrane proteins, which require ATP to transport a broad array of compounds through membranes. The ABC transporter ABCB4 (MDR3 P-glycoprotein) is a phospholipid floppase that translocates phosphatidylcholine (PC) from the inner leaflet to the outer leaflet of the cellular membrane. ABCB4 is located in the apical membrane of hepatocytes; translocation of PC by ABCB4 promotes PC release into bile, which neutralizes the toxic effect of bile salts. Lack of ABCB4 expression leads to the severe liver disease Progressive Familial Intrahepatic Cholestasis type 3 (PFIC3). In order to develop gene therapy for this disease, we created a third generation HIV-1 lentiviral vector with a cassette consisting of ABCB4 cDNA driven by a phosphoglycerokinase (PGK) promoter. Our attempts at producing high titer (>1 × 105 transducing units per mL) lentiviral ABCB4 expression vector were unsuccessful. Because during lentiviral vector production ABCB4 is also expressed, we proposed that the PC floppase activity of ABCB4 might have affected lentiviral vector production negatively.To test this, we co-expressed ABCB4 during GFP lentiviral vector production. We observed that co-expression of ABCB4 inhibited viral infectivity, but the production of viral particles was not affected. Co-expression of an inactive ABCB4 mutant did not reduce viral infectivity. In addition, the ratio of phosphatidylcholine to cholesterol of lentiviral particles increased significantly from 0.4 in producer cells expressing no ABCB4 or a mutant ABCB4 to 1.3 if the wildtype ABCB4 protein was co-expressed.We therefore conclude that the specific translocase activity of ABCB4 can affect viral infectivity negatively by changing the lipid membrane composition of the viral particles. This has implications for the strategy of development of a successful lentiviral gene therapy system for the treatment of PFIC3. Human Immunodeficiency Virus-1 (HIV-1) particles are assembled and released from membrane microdomains (lipid rafts). The membrane composition of the viral particle, which is important for viral integrity and infectivity, is therefore determined by the raft lipid composition. ATP binding cassette (ABC) transporters are transmembrane proteins, which require ATP to transport a broad array of compounds through membranes. The ABC transporter ABCB4 (MDR3 P-glycoprotein) is a phospholipid floppase that translocates phosphatidylcholine (PC) from the inner leaflet to the outer leaflet of the cellular membrane. ABCB4 is located in the apical membrane of hepatocytes; translocation of PC by ABCB4 promotes PC release into bile, which neutralizes the toxic effect of bile salts. Lack of ABCB4 expression leads to the severe liver disease Progressive Familial Intrahepatic Cholestasis type 3 (PFIC3). In order to develop gene therapy for this disease, we created a third generation HIV-1 lentiviral vector with a cassette consisting of ABCB4 cDNA driven by a phosphoglycerokinase (PGK) promoter. Our attempts at producing high titer (>1 × 105 transducing units per mL) lentiviral ABCB4 expression vector were unsuccessful. Because during lentiviral vector production ABCB4 is also expressed, we proposed that the PC floppase activity of ABCB4 might have affected lentiviral vector production negatively. To test this, we co-expressed ABCB4 during GFP lentiviral vector production. We observed that co-expression of ABCB4 inhibited viral infectivity, but the production of viral particles was not affected. Co-expression of an inactive ABCB4 mutant did not reduce viral infectivity. In addition, the ratio of phosphatidylcholine to cholesterol of lentiviral particles increased significantly from 0.4 in producer cells expressing no ABCB4 or a mutant ABCB4 to 1.3 if the wildtype ABCB4 protein was co-expressed. We therefore conclude that the specific translocase activity of ABCB4 can affect viral infectivity negatively by changing the lipid membrane composition of the viral particles. This has implications for the strategy of development of a successful lentiviral gene therapy system for the treatment of PFIC3.