Cholesterol Depletion of Hepatoma Cells Impairs Hepatitis B Virus Envelopment by Altering the Topology of the Large Envelope Protein

Abstract

Previous reports have shown that cholesterol depletion of the membrane envelope of the hepatitis B virus (HBV) impairs viral infection of target cells.A potential function of this lipid in later steps of the viral life cycle remained controversial, with secretion of virions and subviral particles (SVP) being either inhibited or not affected, depending on the experimental approach employed to decrease the intracellular cholesterol level. This work addressed the role of host cell cholesterol on HBV replication, assembly, and secretion, using an alternative method to inhibition of the enzymes involved in the biosynthesis pathway. Growing HBV-producing cells with lipoprotein-depleted serum (LPDS) resulted in an important reduction of the amount of cholesterol within 24 h of treatment (about 40%). Cell exposure to chlorpromazine, an inhibitor of the clathrin-mediated pathway used by the low-density lipoprotein receptor for endocytosis, also impacted the cholesterol level; however, this level of inhibition was not achievable when the synthesis inhibitor lovastatin was used. HBV secretion was significantly inhibited in cholesterol-depleted cells (by ϳ80%), while SVP release remained unaffected. The viral DNA genome accumulated in LPDS-treated cells in a time-dependent manner. Specific immunoprecipitation of nucleocapsids and mature virions revealed an increased amount of naked nucleocapsids, while synthesis of the envelope proteins occurred as normally. Following analysis of the large envelope protein conformation in purified microsomes, we concluded that cholesterol is important in maintaining the dual topology of this polypeptide, which is critical for viral envelopment.A large variety of viruses, of which many are important human pathogens, depend on lipid and cholesterol metabolism in host cells during at least one step of their life cycle. Hepatitis C virus, for instance, relies on lipids for entry into target cells (21), RNA replication (22), viral assembly (45), as well as infectivity (1, 37). Cholesterol-rich plasma membrane domains (lipid rafts) are important for HIV entry, assembly, and infectivity (2). In the case of hepatitis B virus (HBV), efficient infection of hepatocytes was shown to be dependent on the cholesterol content of the viral envelope (6, 37); more recently, a role for caveolin-1, a structural protein of lipid rafts, was suggested in HBV entry (30).HBV is an enveloped member of the Hepadnaviridae family bearing an unusual feature among animal viruses, in that multiple types of virus-related particles are assembled in infected cells. The infectious virions, also called "Dane" particles, are sphere-shaped, 42-nm-diameter particles containing the nucleocapsid surrounded by an envelope composed of cellular lipids and three structural viral proteins. These are designated the large (L), middle (M), and small (S) proteins and derive from the same open reading frame, sharing a common S domain (43). In addition to mature virions, coreless, noninfectious lipoprotein particles occurring in two morphologi...

Statin use and risk of hepatocellular carcinoma in a U.S. population

“…In persons infected with HCV, statins have also been demonstrated to inhibit viral replication via depletion of mevalonate which leads to low levels of geranylgeranyl phosphate which is needed for HCV replication (30). In persons infected with HBV, a possible mechanism of action is simply the lowering of cholesterol levels as cholesterol depletion impairs the ability of HBV to infect target cells (31). …”

Lipid-dependent Generation of Dual Topology for a Membrane Protein

“…2 and 38) and small proteins of less than 50 amino acids (36). The L envelope protein of hepatitis B virus (48) exists in two functionally distinct topological isoforms whose topological distribution varies with the endoplasmic reticulum cholesterol content (49), which reciprocally affects a translocation of positively charged domains and marginally hydrophobic segments (50). It was suggested that cholesterol can either trigger conformational changes in the translocon by affecting the fluidity of the lipid bilayer or may change the surface charge distribution of the membrane and modulate electrostatic lipid-protein interactions (50).…”

The Hepatitis B Virus Receptor

“…This work thus provides direct in vivo experimental evidence showing that HBV infection is intimately linked to bile acid and lipid metabolism. Moreover, cholesterol has been implicated in HBV morphogenesis and infectivity, and promotion of cholesterol synthesis and uptake may be important in HBV infection (Bremer et al 2009, Dorobantu et al 2011.…”