Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis

Abstract

Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell endomembrane system to produce a membranous replication organelle (RO). The underlying mechanisms are far from being elucidated fully. In this report, we provide evidence that HCV RNA replication depends on functional lipid transport along the endosomal-lysosomal pathway that is mediated by several lipid transfer proteins, such as the Niemann-Pick type C1 (NPC1) protein. Pharmacological inhibition of NPC1 function reduced viral replication, impaired the transport of cholesterol to the viral replication organelle, and altered organelle morphology. Besides NPC1, our study reports the importance of additional endosomal and lysosomal lipid transfer proteins required for viral replication, thus contributing to our understanding of how HCV manipulates their function in order to generate a membranous replication organelle. These results might have implications for the biogenesis of replication organelles of other positive-strand RNA viruses.

Gene Expression Profiling of Different Huh7 Variants Reveals Novel Hepatitis C Virus Host Factors

“…On the one hand, it is well established that NR0B2 inhibits bile acid synthesis by downregulating the expression of the crucial cholesterol hydroxylase/monooxygenase CYP7A1 [79,80]. On the other hand, over the last ten years it became increasingly evident that HCV vitally depends on proper distribution of cholesterol in the cell and particularly in the ER-membrane, from which HCV derives its replicative membrane system ("membranous web") [21,105,106]. In fact, we found NR0B2 overexpression to strongly redistribute cellular cholesterol away from ER-like perinuclear structures towards the plasma membrane.…”

“…Infectious full-length viral particles (JcR2a [38], Jc1 [50]) were prepared as described in [21]. In brief, in vitro-transcribed RNA was electroporated into Huh7.5 cells [51] and cell culture supernatants were harvested and sterile-filtered after 24, 48, and 72 h. Jc1 supernatants were further concentrated by incubation with 8% PEG-8000 overnight at 4 • C and centrifugation at 4000× g, 30 min, 4 • C. Supernatants were discarded and the pellet was resuspended in complete medium, aliquoted and stored at −80 • C. Virus titers were determined on Huh7.5 cells with an end-point dilution assay (TCID 50 [52]) using the mouse monoclonal α-NS5A antibody 9E10 (a kind gift from Dr. Charles Rice, The Rockefeller University, New York, NY, USA) in a 1:15,000 dilution.…”

“…In support of this notion, we found less than 50% of total bile acids in cell lysates from Huh7-Lunet cells overexpressing HA-NR0B2 compared to the HA-eGFP control ( Figure S8); however, absolute levels of bile acids were at the lower limit of detection even for the control cells. On the other hand, NR0B2 regulates bile acid anabolism by transcriptional inhibition of the cholesterol-hydroxylase CYP7A1 and cholesterol is known to play a crucial role in the HCV life cycle as well [21]. Thus, we also examined the distribution of free cholesterol in the cell using Filipin-III [81].…”

“…Replication takes place in the cytoplasm at specialized, ER-derived mono-, double-, or multi-membrane vesicles, designated the "membranous web" [14] and is tightly linked to lipid droplets [15]. HCV hijacks many cellular pathways to establish and maintain a productive infection, e.g., autophagy [16] as well as glucose [17][18][19][20] and cholesterol metabolism [21,22]. Recent evidence implies that nuclear receptors contribute to mediating these changes and are, thus, important players during HCV infection [18,23,24].…”

Lipid transfer proteins: the lipid commute via shuttles, bridges and tubes

“…Accordingly speed of the counter-transport is determined by the rates of PI4P generation and degradation. Despite neither of these yet being established, there is strong evidence for the counter-transport system from the hijacking of the entire system by viruses to drive cholesterol transport to virally determined membranes (see section "LTPs and disease") 100 . Specificity for "lipid B" varies between ORPs, and phosphoinositides other than PI4P ("lipid A") might also drive countertransport 44,45 .…”