CD81 Is a Central Regulator of Cellular Events Required for Hepatitis C Virus Infection of Human Hepatocytes

Abstract

Infection with hepatitis C virus (HCV) is still a major public health problem, and the events leading to hepatocyte infection are not yet fully understood. Combining confocal microscopy with biochemical analysis and studies of infection requirements using pharmacological inhibitors and small interfering RNAs, we show here that engagement of CD81 activates the Rho GTPase family members Rac, Rho, and Cdc42 and that the block of these signaling pathways drastically reduces HCV infectivity. Activation of Rho GTPases mediates actin-dependent relocalization of the HCV E2/CD81 complex to cell-cell contact areas where CD81 comes into contact with the tight-junction proteins occludin, ZO-1, and claudin-1, which was recently described as an HCV coreceptor. Finally, we show that CD81 engagement activates the Raf/MEK/ERK signaling cascade and that this pathway affects postentry events of the virus life cycle. In conclusion, we describe a range of cellular events that are manipulated by HCV to coordinate interactions with its multiple coreceptors and to establish productive infections and find that CD81 is a central regulator of these events.Hepatitis C virus (HCV), the causal agent of hepatitis C, is a positive-strand RNA virus that belongs to the Flaviviridae family (11). The genome encodes a single polyprotein that is processed by a combination of host and viral peptidases into at least 10 different structural and nonstructural proteins. The HCV envelope is formed by the two heavily N-glycosylated type I transmembrane proteins E1 and E2, which are present on the virus as heterodimers (44,48) anchored to a host cellderived double-layer lipid membrane.HCV infects mainly hepatocytes, but the precise mechanisms of early infection are largely unknown. As for other flaviviruses, it is generally accepted that HCV glycoproteins E1 and E2 play a major role in virus binding and entry into target cells (58).Several putative HCV receptors have been identified so far. Some of these, like the low-density lipoprotein (LDL) receptor (LDLR) (1) and other cell surface proteins involved in serum lipoprotein binding and metabolism, seem to act as relatively nonspecific attachment factors to allow virus concentration on the cell surface, as in the infected host, HCV can be associated with LDL and very low-density lipoprotein (VLDL). The tetraspanin molecule CD81 (49) and the scavenger receptor class B type I (SR-BI) (54) were both identified as putative receptors based on their interaction with recombinant soluble E2 (rE2) protein, and their role has been confirmed by means of HCV pseudotyped retroviral particles (2,14,29) and by the recently established in vitro infectious HCV system (HCVcc) (21,(30)(31)(32)59). In addition, CD81 has been shown to play a role in the infection of primary human hepatocytes by serumderived HCV (45). CD81 is an integral membrane protein that shows the structure common to all tetraspanins, with four transmembrane domains delimiting a large extracellular loop and a small extracellular loop and three short intracel...

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Despite these difficulties, related viruses, easier to cultivate, for instance bovine viral diarrhoea virus, have been used as surrogate models [38]. Secondly, recombinant forms of the envelope glycoproteins were used to search for viral receptors [33,39] but also as probes for antibody screening or neutralization of binding assays [40,41], to study HCV-induced receptor signalling during entry [42], to delineate E2-receptor interacting surfaces [43,44], or to draw a theoretical model for E2 structure [45]. Most frequently a soluble form of E2 produced in mammalian or insect cells and capable of inhibiting HCV entry [46] is used.…”

“…How the virus triggers a cellular response leading to its internalization and delivery to a compartment favourable for fusion remains incompletely understood and requires systematic studies of the signalling cascades regulated after virus attachment onto the cell surface. Several groups attempted to tackle this issue, by screening libraries of kinases for those that are important for HCV entry [141] or by targeting specific signalling molecules using genetic or pharmaceutical approaches [42,173]. cells that express all HCV receptors (e.g.…”

Characterization of Hepatitis C Virus Particle Subpopulations Reveals Multiple Usage of the Scavenger Receptor BI for Entry Steps

“…Cholesterol transfer from SR-BI-bound lipo-viro-particles or lipoproteins could be important to facilitate post-attachment events at the site of entry into cells. Local enhanced membrane fluidity due to an elevated cholesterol/lipid content could favor crucial HCV cell entry events such as receptor movements (93) and interactions (68,94), particle internalization (61,95), and finally fusion with the host membrane (23,47,96). Furthermore, acceleration or facilitation of these crucial entry steps might help the virus to escape the host humoral immune response (60,61) and to establish persistence (26).…”

Impact of Intra- and Interspecies Variation of Occludin on Its Function as Coreceptor for Authentic Hepatitis C Virus Particles

“…These include the tetraspanin CD81 (30), scavenger receptor class B type I (SR-BI) (32), and more recently described, the tight junction components claudin 1 (CLDN1) (13) and occludin (OCLN) (27,31). It is thought that the viral envelope glycoproteins E1 and E2 interact with these four (co)receptors sequentially and that these interactions are orchestrated by cell signaling processes (6,14,28). SR-BI may act early and the tight junction components may act later in this process (11,13,21,24,42).…”