Polyunsaturated liposomes are antiviral against hepatitis B and C viruses and HIV by decreasing cholesterol levels in infected cells.

The pressing need for broad-spectrum antivirals could be met by targeting host rather than viral processes. Cholesterol biosynthesis within the infected cell is one promising target for a large number of viral systems, including hepatitis C virus (HCV), hepatitis B virus (HBV) and HIV. Liposomes developed for intracellular, endoplasmic reticulum (ER)-targeted in vivo drug delivery have been modified to include polyunsaturated fatty acids that exert an independent antiviral activity through the reduction of cellular cholesterol. These polyunsaturated ER liposomes (PERLs) have greater activity than lovastatin (Mevacor, Altoprev), which is clinically approved for lowering cholesterol and preventing cardiovascular disease. Treatment of HCV, HBV, and HIV infections with PERLs significantly decreased viral secretion and infectivity, and pretreatment of naïve cells reduced the ability of both HCV and HIV to establish infections because of the decreased levels of plasma membrane cholesterol. Direct competition for cellular receptors was an added effect of PERLs against HCV infections. The greatest antiviral activity in all three systems was the inhibition of viral infectivity through the reduction of virus-associated cholesterol. Our study demonstrates that PERLs are a broadly effective antiviral therapy and should be developed further in combination with encapsulated drug mixtures for enhanced in vivo efficacy.

Uptake and trafficking of liposomes to the endoplasmic reticulum.

Liposomes are vesicular structures consisting of an aqueous core surrounded by a lipid bilayer. Apart from the cytosol and lysosomes, no other intracellular compartment has been successfully targeted using liposomal delivery. Here, we report the development of liposomes capable of specific targeting to the endoplasmic reticulum (ER) and associated membranes. Using competition and inhibitor assays along with confocal microscopy, we have determined that ER liposomes utilize scavenger and low-density lipoprotein receptors for endocytosis and enter cells through a caveolin- and microtubule-dependent mechanism. They traffic intact to the ER, where fusion with the ER membrane occurs after 22-25 min, which was confirmed by fluorescence-dequenching assays. Once inside the ER, tagged lipids intercalate with the ER membrane and are subsequently incorporated into ER-assembling entities, such as the ER-budding viruses hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV), lipid droplets, and secreted lipoproteins. ER liposomes are superior to cytosolic liposome formulations for the intracellular delivery of aqueous cargo, such as HIV-1 antivirals, and are especially suited for the prolonged delivery of lipids and lipophilic drugs into human cells.

Hepatitis B virus requires intact caveolin-1 function for productive infection in HepaRG cells.

Investigation of the entry pathways of hepatitis B virus (HBV), a member of the family Hepadnaviridae, has been hampered by the lack of versatile in vitro infectivity models. Most concepts of hepadnaviral infection come from the more robust duck HBV system; however, whether the two viruses use the same mechanisms to invade target cells is still a matter of controversy. In this study, we investigate the role of an important plasma membrane component, caveolin-1 (Cav-1), in HBV infection. Caveolins are the main structural components of caveolae, plasma membrane microdomains enriched in cholesterol and sphingolipids, which are involved in the endocytosis of numerous ligands and complex signaling pathways within the cell. We used the HepaRG cell line permissive for HBV infection to stably express dominant-negative Cav-1 and dynamin-2, a GTPase involved in vesicle formation at the plasma membrane and other organelles. The endocytic properties of the newly established cell lines, designated HepaRG(Cav-1), HepaRG(Cav-1Delta1-81), HepaRG(Dyn-2), and HepaRG(Dyn-2K44A), were validated using specific markers for different entry routes. The cells maintained their properties during cell culture, supported differentiation, and were permissive for HBV infection. The levels of both HBV transcripts and antigens were significantly decreased in cells expressing the mutant proteins, while viral replication was not directly affected. Chemical inhibitors that specifically inhibit clathrin-mediated endocytosis had no effect on HBV infection. We concluded that HBV requires a Cav-1-mediated entry pathway to initiate productive infection in HepaRG cells.