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How does interferon inhibit HCV cell entry?
Commentaries
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Gut May 2008;57:573-574
Meleri Jones1, James S Owen2
1 Department of Infection, Royal Free and University College Medical School, London, UK
2 Centre for Hepatology, Royal Free and University College Medical School, London, UK
Hepatitis C virus (HCV), the sole member of the Hepacivirus genus of the Flaviviridae family, is an enveloped virus with a positive-sense single-stranded RNA genome of 9600 nucleotides. HCV virions mainly infect hepatocytes, entering via specific receptors and clathrin-mediated endocytosis to release their genome from acidified endosomes.1 Over 170 million people worldwide are infected with HCV, and 80-85% of these develop chronic hepatitis C, which in a sizeable portion (up to 20%) leads to cirrhosis, hepatic failure or hepatocellular carcinoma. Current HCV treatment is based on the antiviral action of interferon (IFN) in combination with ribavirin, although this clears the virus in only 40-55% of patients.
IFN plays a central role in the innate immune system by inducing an antiviral state in target cells,2 most notably for HCV by inhibiting viral replication.3 However, it is less clear whether IFN initiates specific actions to limit HCV infections, such as restricting virion attachment and/or entry into hepatocytes. In this edition of Gut (see page 10.1136/gut.2006.111443), Murao et al4 demonstrate in vitro that IFN decreases cell surface expression of scavenger receptor class B type I (SR-BI), a pivotal molecule in early stages of the HCV life cycle. Before discussing their findings, however, we first describe the molecular ensemble of host cellular factors which orchestrate HCV entry.
Until the last decade, HCV research was hampered by lack of infectious clones and an inability to propagate the virus in cultured liver cells. Introduction of the HCV replicon system (a self-replicating subgenomic construct)5 allowed studies of viral replication, but unravelling molecular details of HCV attachment and entry into hepatocytes has required the development of additional tools. These encompass reconstituted HCV envelope glycoproteins, E1 and E2,6 7 HCV pseudoparticles (HCVpp) in which E1 and E2 are displayed on retroviral core particles8 and, more recently, HCVcc, infectious viral particles released in cell culture following transfection with a full-length HCV genome.3 9 10 These systems identified several cell surface molecules which interact with HCV, including glycosaminoglycans (GAGs), CD81, claudin-1 and SR-BI.1 11 12 Notwithstanding, these putative receptors are by themselves insufficient for HCV binding and cell entry; current studies focus on their interplay. Initial attachment via GAGs is likely, as recombinant E2 binds strongly to heparan sulphates,13 but these have wide tissue expression and thus cannot confer HCV hepatotropism; an additional hepatocyte-specific surface molecule(s) is required.
One vital protein is CD81, a tetraspanin with two extracellular domains: it binds glycoprotein E2;14 infection of Huh7.5 hepatoma cells is inhibited by CD81 antibodies or by silencing CD81 gene expression; and HepG2 cells which resist HCVpp infection become permissive if transfected to express CD81.15 Significantly, however, expressing CD81 in non-hepatic cells does not allow HCVpp entry, suggesting that a co-receptor is involved\strong experimental evidence implicates SR-BI, though reports that CD81 has a suppressive partner EW1-2wint,1 and that the tight junction protein claudin-1, and also claudins 6 and 9,16 act late in the HCV entry pathway, emphasise that the overall process is complex and multistep.
SR-BI, an 82 kDa glycoprotein with a large extracellular loop and a membrane-spanning cytoplasmic N-terminus and C-terminus, is highly expressed in hepatocytes, and steroidogenic cells, where it facilitates bidirectional cholesterol transport. Though able to bind oxidised or chemically modified low-density lipoprotein (LDL), SR-BI is a physiological receptor for high-density lipoproteins (HDLs) and functions to extract cholesteryl esters selectively.17 18 Intriguingly, SR-BI also binds apolipoprotein E,19 a 34 kDa polymorphic constituent of various plasma lipoproteins which has multiple roles in HCV virion assembly and infectivity,20 including effects on the persistence of infection.21 As with HCV-CD81 interactions, SR-BI binds the HCV envelope glycoprotein E2,22 and the infectivity of HCVpp and HCVcc is stimulated by SR-BI overexpression in Huh7.5 cells or, conversely, is decreased by SR-BI gene silencing or by addition of anti-SR-BI antibodies.23 Noteworthy, but still ill-understood,19 is the association of HCV in human plasma with lipoprotein particles and, while oxidised LDL inhibits HCV cell entry in vitro,24 HDL stimulates infectivity and protects HCVpps from neutralising antibodies.25 26 Thus, although the finer details of HCV cell binding and entry remain elusive, the HCVpp and HCVcc models verify the interplay of CD81 and SR-BI for productive infection and establish that HCV-SR-BI interactions are enhanced by HDLs.
How then might IFN disrupt these processes? As SR-BI appears pivotal, since HCV virions first engage this receptor before interacting with CD81,17 Murao and colleagues4 studied its expression and activity in human liver cell lines (HepG2 and Huh7) and primary rat hepatocytes following incubations with IFN. They found SR-BI to be downregulated in a time- and dose-dependent manner at the levels of both mRNA (by quantitative real-time PCR) and protein (by western blotting and also flow cytometry, which measured cell surface protein).
Next, by coupling the SR-BI promoter to a reporter gene, they showed inhibition of gene transcription and implicated signal transducer and activator of transcription 1 (STAT1)/STAT2 heterodimers as key mediators. Thus, when cells were depleted of these transcription factors, the ability of IFN to downregulate SR-BI was lost; conversely, STAT1/STAT2 were rapidly phosphorylated (activated) in response to IFN. Verification was obtained by selective blocking of STAT1/STAT2 binding to the SR-BI promoter, including mutagenesis of the response motif they had identified within the promoter. Finally, following IFN pretreatment of liver cells, Murao et al4 demonstrated certain functional consequences of SR-BI downregulation: a decrease in HDL-cell association and transfer of HDL cholesteryl esters; and a reduction in specific binding of synthetic E1 and E2 peptides.
This report in Gut is the first to link the antiviral action of IFN to the inhibition of HCV cell entry. Earlier studies of subgenomic replicons27 or of HCVcc3 had verified that HCV replication declined during IFN treatment, but until recently the complexity of probing HCV cell entry had precluded insightful investigations. Predictably, the finding of Murao and colleagues4 that SR-BI is a downstream target of IFN signalling raises questions but, importantly, also has therapeutic implications. One uncertainty is the effect of IFN on levels of SR-BII protein. This isoform arises as a splice variant of the SR-B gene; it differs from SR-BI only in the cytoplasmic C-terminus28 and binds E2 peptide as effectively as SR-BI.29 About 70% of SR-B protein in HepG2 cells is SR-BI, and conceivably IFN could reduce its content indirectly by regulating alternative splicing, as occurs with oestrogen.30 If so, this mechanism of decreasing SR-BI in favour of SR-BII would contribute to the IFN-mediated reductions of cell surface HDL association and E2 binding noted by Murao et al4; SR-BII, unlike SR-BI, is predominantly located in intracellular compartments and so has limited cell surface binding activity. Another query concerns possible HCV retaliation against IFN-mediated suppression of its entry into hepatocytes. Thus, synthesis of HCV proteins during intracellular virus replication promotes the degradation of STAT131 and, as noted by Murao et al4 using gene silencing, such a depletion will prevent the downregulation of SR-BI by IFN.
In the absence of effective vaccination, pegylated IFN in combination with ribavirin remains the best treatment for chronic HCV infection. Protease inhibitors and replicase inhibitors of HCV are under development, but work primarily by inhibiting viral replication. Thus, the discovery that IFN has additional anti-HCV effects at the point of virion entry endorses this process as a valuable new target for drug therapy; indeed, endogenous SR-BI is suggested to limit HCV infection,29 while broad-spectrum antivirals, such as arbidol and cyanoviridin-N, restrict HCV entry.32 33 The goal is clear\to refine the selectivity and potency of antiviral drugs to block HCV cell attachment and entry.
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