GB Virus B and Hepatitis C Virus, Distantly Related Hepaciviruses, Share an Entry Factor, Claudin-1.

Due to increased and broadened screening efforts, the last decade has seen a rapid expansion in the number of viral species classified into the Hepacivirus genus. Conserved genetic features of hepaciviruses suggest that they have undergone specific adaptation and have evolved to hijack similar host proteins for efficient propagation in the liver. Here, we developed pseudotyped viruses to elucidate the entry factors of GB virus B (GBV-B), the first hepacivirus described in an animal after hepatitis C virus (HCV). GBV-B-pseudotyped viral particles (GBVBpp) were shown to be uniquely sensitive to the sera of tamarins infected with GBV-B, validating their usefulness as a surrogate for GBV-B entry studies. We screened GBVBpp infection of human hepatoma cell lines that were CRISPR/Cas9 engineered to ablate the expression of individual HCV receptors/entry factors and found that claudin-1 is essential for GBV-B infection, indicating the GBV-B and HCV share an entry factor. Our data suggest that claudin-1 facilitates HCV and GBV-B entry through distinct mechanisms since the former requires the first extracellular loop and the latter is reliant on a C-terminal region containing the second extracellular loop. The observation that claudin-1 is an entry factor shared between these two hepaciviruses suggests that the tight junction protein is of fundamental mechanistic importance during cell entry. IMPORTANCE Hepatitis C virus (HCV) is a major public health burden; approximately 58 million individuals have chronic HCV infection and are at risk of developing cirrhosis and liver cancer. To achieve the World Health Organization's target of eliminating hepatitis by 2030, new therapeutics and vaccines are needed. Understanding how HCV enters cells can inform the design of new vaccines and treatments targeting the first stage of infection. However, the HCV cell entry mechanism is complex and has been sparsely described. Studying the entry of related hepaciviruses will increase the knowledge of the molecular mechanisms of the first stages of HCV infection, such as membrane fusion, and inform structure-guided HCV vaccine design; in this work, we have identified a protein, claudin-1, that facilitates the entry of an HCV-related hepacivirus but with a mechanism not described for HCV. Similar work on other hepaciviruses may unveil a commonality of entry factors and, possibly, new mechanisms.

A novel and robust method for counting components within bio-molecular complexes using fluorescence microscopy and statistical modelling.

Cellular biology occurs through myriad interactions between diverse molecular components, many of which assemble in to specific complexes. Various techniques can provide a qualitative survey of which components are found in a given complex. However, quantitative analysis of the absolute number of molecules within a complex (known as stoichiometry) remains challenging. Here we provide a novel method that combines fluorescence microscopy and statistical modelling to derive accurate molecular counts. We have devised a system in which batches of a given biomolecule are differentially labelled with spectrally distinct fluorescent dyes (label A or B), and mixed such that B-labelled molecules are vastly outnumbered by those with label A. Complexes, containing this component, are then simply scored as either being positive or negative for label B. The frequency of positive complexes is directly related to the stoichiometry of interaction and molecular counts can be inferred by statistical modelling. We demonstrate this method using complexes of Adenovirus particles and monoclonal antibodies, achieving counts that are in excellent agreement with previous estimates. Beyond virology, this approach is readily transferable to other experimental systems and, therefore, provides a powerful tool for quantitative molecular biology.

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway.

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant.

An entropic safety catch controls hepatitis C virus entry and antibody resistance.

E1 and E2 (E1E2), the fusion proteins of Hepatitis C Virus (HCV), are unlike that of any other virus yet described, and the detailed molecular mechanisms of HCV entry/fusion remain unknown. Hypervariable region-1 (HVR-1) of E2 is a putative intrinsically disordered protein tail. Here, we demonstrate that HVR-1 has an autoinhibitory function that suppresses the activity of E1E2 on free virions; this is dependent on its conformational entropy. Thus, HVR-1 is akin to a safety catch that prevents premature triggering of E1E2 activity. Crucially, this mechanism is turned off by host receptor interactions at the cell surface to allow entry. Mutations that reduce conformational entropy in HVR-1, or genetic deletion of HVR-1, turn off the safety catch to generate hyper-reactive HCV that exhibits enhanced virus entry but is thermally unstable and acutely sensitive to neutralising antibodies. Therefore, the HVR-1 safety catch controls the efficiency of virus entry and maintains resistance to neutralising antibodies. This discovery provides an explanation for the ability of HCV to persist in the face of continual immune assault and represents a novel regulatory mechanism that is likely to be found in other viral fusion machinery.

Targeting human Acyl-CoA:cholesterol acyltransferase as a dual viral and T cell metabolic checkpoint.

Determining divergent metabolic requirements of T cells, and the viruses and tumours they fail to combat, could provide new therapeutic checkpoints. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) has direct anti-carcinogenic activity. Here, we show that ACAT inhibition has antiviral activity against hepatitis B (HBV), as well as boosting protective anti-HBV and anti-hepatocellular carcinoma (HCC) T cells. ACAT inhibition reduces CD8+ T cell neutral lipid droplets and promotes lipid microdomains, enhancing TCR signalling and TCR-independent bioenergetics. Dysfunctional HBV- and HCC-specific T cells are rescued by ACAT inhibitors directly ex vivo from human liver and tumour tissue respectively, including tissue-resident responses. ACAT inhibition enhances in vitro responsiveness of HBV-specific CD8+ T cells to PD-1 blockade and increases the functional avidity of TCR-gene-modified T cells. Finally, ACAT regulates HBV particle genesis in vitro, with inhibitors reducing both virions and subviral particles. Thus, ACAT inhibition provides a paradigm of a metabolic checkpoint able to constrain tumours and viruses but rescue exhausted T cells, rendering it an attractive therapeutic target for the functional cure of HBV and HBV-related HCC.

Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins.

Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.

Flexibility and intrinsic disorder are conserved features of hepatitis C virus E2 glycoprotein.

The glycoproteins of hepatitis C virus, E1E2, are unlike any other viral fusion machinery yet described, and are the current focus of immunogen design in HCV vaccine development; thus, making E1E2 both scientifically and medically important. We used pre-existing, but fragmentary, structures to model a complete ectodomain of the major glycoprotein E2 from three strains of HCV. We then performed molecular dynamic simulations to explore the conformational landscape of E2, revealing a number of important features. Despite high sequence divergence, and subtle differences in the models, E2 from different strains behave similarly, possessing a stable core flanked by highly flexible regions, some of which perform essential functions such as receptor binding. Comparison with sequence data suggest that this consistent behaviour is conferred by a network of conserved residues that act as hinge and anchor points throughout E2. The variable regions (HVR-1, HVR-2 and VR-3) exhibit particularly high flexibility, and bioinformatic analysis suggests that HVR-1 is a putative intrinsically disordered protein region. Dynamic cross-correlation analyses demonstrate intramolecular communication and suggest that specific regions, such as HVR-1, can exert influence throughout E2. To support our computational approach we performed small-angle X-ray scattering with purified E2 ectodomain; this data was consistent with our MD experiments, suggesting a compact globular core with peripheral flexible regions. This work captures the dynamic behaviour of E2 and has direct relevance to the interaction of HCV with cell-surface receptors and neutralising antibodies.

Building a mechanistic mathematical model of hepatitis C virus entry.

The mechanism by which hepatitis C virus (HCV) gains entry into cells is a complex one, involving a broad range of host proteins. Entry is a critical phase of the viral lifecycle, and a potential target for therapeutic or vaccine-mediated intervention. However, the mechanics of HCV entry remain poorly understood. Here we describe a novel computational model of viral entry, encompassing the relationship between HCV and the key host receptors CD81 and SR-B1. We conduct experiments to thoroughly quantify the influence of an increase or decrease in receptor availability upon the extent of viral entry. We use these data to build and parameterise a mathematical model, which we then validate by further experiments. Our results are consistent with sequential HCV-receptor interactions, whereby initial interaction between the HCV E2 glycoprotein and SR-B1 facilitates the accumulation CD81 receptors, leading to viral entry. However, we also demonstrate that a small minority of viruses can achieve entry in the absence of SR-B1. Our model estimates the impact of the different obstacles that viruses must surmount to achieve entry; among virus particles attaching to the cell surface, around one third of viruses accumulate sufficient CD81 receptors, of which 4-8% then complete the subsequent steps to achieve productive infection. Furthermore, we make estimates of receptor stoichiometry; in excess of 10 receptors are likely to be required to achieve viral entry. Our model provides a tool to investigate the entry characteristics of HCV variants and outlines a framework for future quantitative studies of the multi-receptor dynamics of HCV entry.

Small molecule scavenger receptor BI antagonists are potent HCV entry inhibitors.

BACKGROUND AND AIMS: ITX 5061 is a clinical stage small molecule compound that promotes high-density lipoprotein (HDL) levels in animals and patients by targeting the scavenger receptor BI protein pathway. Since SR-BI is a known co-receptor for HCV infection, we evaluated these compounds for their effects on HCV entry. METHODS: We obtained ITX 5061 and related compounds to characterize their interaction with SR-BI and effects on HCV entry and infection. RESULTS: We confirmed that a tritium-labeled compound analog (ITX 7650) binds cells expressing SR-BI, and both ITX 5061 and ITX 7650 compete for HDL-mediated lipid transfer in an SR-BI dependent manner. Both molecules inhibit HCVcc and HCVpp infection of primary human hepatocytes and/or human hepatoma cell lines and have minimal effects on HCV RNA replication. Kinetic studies suggest that the compounds act at an early post-binding step. CONCLUSIONS: These results suggest that the ITX compounds inhibit HCV infection with a mechanism of action distinct from other HCV therapies under development. Since ITX 5061 has already been evaluated in over 280 patients with good pharmacokinetic and safety profiles, it warrants proof-of-concept clinical studies in HCV infected patients.

Neutralizing antibody-resistant hepatitis C virus cell-to-cell transmission.

Hepatitis C virus (HCV) can initiate infection by cell-free particle and cell-cell contact-dependent transmission. In this study we use a novel infectious coculture system to examine these alternative modes of infection. Cell-to-cell transmission is relatively resistant to anti-HCV glycoprotein monoclonal antibodies and polyclonal immunoglobulin isolated from infected individuals, providing an effective strategy for escaping host humoral immune responses. Chimeric viruses expressing the structural proteins representing the seven major HCV genotypes demonstrate neutralizing antibody-resistant cell-to-cell transmission. HCV entry is a multistep process involving numerous receptors. In this study we demonstrate that, in contrast to earlier reports, CD81 and the tight-junction components claudin-1 and occludin are all essential for both cell-free and cell-to-cell viral transmission. However, scavenger receptor BI (SR-BI) has a more prominent role in cell-to-cell transmission of the virus, with SR-BI-specific antibodies and small-molecule inhibitors showing preferential inhibition of this infection route. These observations highlight the importance of targeting host cell receptors, in particular SR-BI, to control viral infection and spread in the liver.

Multiple effects of silymarin on the hepatitis C virus lifecycle.

UNLABELLED: Silymarin, an extract from milk thistle (Silybum marianum), and its purified flavonolignans have been recently shown to inhibit hepatitis C virus (HCV) infection, both in vitro and in vivo. In the current study, we further characterized silymarin's antiviral actions. Silymarin had antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin but not silibinin inhibited genotype 2a NS5B RNA-dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti-HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b BK and four 1b RDRPs derived from HCV-infected patients. Moreover, silymarin did not inhibit HCV replication in five independent genotype 1a, 1b, and 2a replicon cell lines that did not produce infectious virus. Silymarin inhibited microsomal triglyceride transfer protein activity, apolipoprotein B secretion, and infectious virion production into culture supernatants. Silymarin also blocked cell-to-cell spread of virus. CONCLUSION: Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarin's antiviral action appear to include blocking of virus entry and transmission, possibly by targeting the host cell.

Hepatoma cell density promotes claudin-1 and scavenger receptor BI expression and hepatitis C virus internalization.

Hepatitis C virus (HCV) entry occurs via a pH- and clathrin-dependent endocytic pathway and requires a number of cellular factors, including CD81, the tight-junction proteins claudin 1 (CLDN1) and occludin, and scavenger receptor class B member I (SR-BI). HCV tropism is restricted to the liver, where hepatocytes are tightly packed. Here, we demonstrate that SR-BI and CLDN1 expression is modulated in confluent human hepatoma cells, with both receptors being enriched at cell-cell junctions. Cellular contact increased HCV pseudoparticle (HCVpp) and HCV particle (HCVcc) infection and accelerated the internalization of cell-bound HCVcc, suggesting that the cell contact modulation of receptor levels may facilitate the assembly of receptor complexes required for virus internalization. CLDN1 overexpression in subconfluent cells was unable to recapitulate this effect, whereas increased SR-BI expression enhanced HCVpp entry and HCVcc internalization, demonstrating a rate-limiting role for SR-BI in HCV internalization.

Identification of a residue in hepatitis C virus E2 glycoprotein that determines scavenger receptor BI and CD81 receptor dependency and sensitivity to neutralizing antibodies.

Hepatitis C virus (HCV) infection is dependent on at least three coreceptors: CD81, scavenger receptor BI (SR-BI), and claudin-1. The mechanism of how these molecules coordinate HCV entry is unknown. In this study we demonstrate that a cell culture-adapted JFH-1 mutant, with an amino acid change in E2 at position 451 (G451R), has a reduced dependency on SR-BI. This altered receptor dependency is accompanied by an increased sensitivity to neutralization by soluble CD81 and enhanced binding of recombinant E2 to cell surface-expressed and soluble CD81. Fractionation of HCV by density gradient centrifugation allows the analysis of particle-lipoprotein associations. The cell culture-adapted mutation alters the relationship between particle density and infectivity, with the peak infectivity occurring at higher density than the parental virus. No association was observed between particle density and SR-BI or CD81 coreceptor dependence. JFH-1 G451R is highly sensitive to neutralization by gp-specific antibodies, suggesting increased epitope exposure at the virion surface. Finally, an association was observed between JFH-1 particle density and sensitivity to neutralizing antibodies (NAbs), suggesting that lipoprotein association reduces the sensitivity of particles to NAbs. In summary, mutation of E2 at position 451 alters the relationship between particle density and infectivity, disrupts coreceptor dependence, and increases virion sensitivity to receptor mimics and NAbs. Our data suggest that a balanced interplay between HCV particles, lipoprotein components, and viral receptors allows the evasion of host immune responses.

Effect of cell polarization on hepatitis C virus entry.

The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.

Hepatitis C virus receptor expression in normal and diseased liver tissue.

UNLABELLED: The principal site of hepatitis C virus (HCV) replication is the liver. HCV pseudoparticles infect human liver derived cell lines and this suggests that liver-specific receptors contribute to defining HCV hepatotropism. At least three host cell molecules have been reported to be important for HCV entry: the tetraspanin CD81, scavenger receptor class B member I (SR-BI), and the tight junction (TJ) protein Claudin 1 (CLDN1). Hepatocytes in liver tissue coexpress CD81, SR-BI, and CLDN1, consistent with their ability to support HCV entry. CLDN1 localized at the apical-canalicular TJ region and at basolateral-sinusoidal hepatocyte surfaces in normal tissue and colocalized with CD81 at both sites. In contrast, CLDN1 appeared to colocalize with SR-BI at the basolateral-sinusoidal surface. CLDN1 expression was increased on basolateral hepatocyte membranes in HCV-infected and other chronically inflamed liver tissue compared with normal liver. In contrast, CLDN4 hepatocellular staining was comparable in normal and diseased liver tissue. CONCLUSION: HCV infection of Huh-7.5 hepatoma cells in vitro significantly increased CLDN1 expression levels, consistent with a direct modulation of CLDN1 by virus infection. In HCV infected livers, immunohistochemical studies revealed focal patterns of CLDN1 staining, suggesting localized areas of increased CLDN1 expression in vivo which may potentiate local viral spread within the liver.

Scavenger receptor BI and BII expression levels modulate hepatitis C virus infectivity.

Hepatitis C virus (HCV) enters cells via a pH- and clathrin-dependent endocytic pathway. Scavenger receptor BI (SR-BI) and CD81 are important entry factors for HCV internalization into target cells. The SR-BI gene gives rise to at least two mRNA splice variants, SR-BI and SR-BII, which differ in their C termini. SR-BI internalization remains poorly understood, but SR-BII is reported to endocytose via a clathrin-dependent pathway, making it an attractive target for HCV internalization. We demonstrate that HCV soluble E2 can interact with human SR-BI and SR-BII. Increased expression of SR-BI and SR-BII in the Huh-7.5 hepatoma cell line enhanced HCV strain J6/JFH and JFH infectivity, suggesting that endogenous levels of these receptors limit infection. Elevated expression of SR-BI, but not SR-BII, increased the rate of J6/JFH infection, which may reflect altered intracellular trafficking of the splice variants. In human plasma, HCV particles have been reported to be complexed with lipoproteins, suggesting an indirect interaction of the virus with SR-BI and other lipoprotein receptors. Plasma from J6/JFH-infected uPA-SCID mice transplanted with human hepatocytes demonstrates an increased infectivity for SR-BI/II-overexpressing Huh-7.5 cells. Plasma-derived J6/JFH infectivity was inhibited by an anti-E2 monoclonal antibody, suggesting that plasma virus interaction with SR-BI was glycoprotein dependent. Finally, anti-SR-BI antibodies inhibited the infectivity of cell culture- and plasma-derived J6/JFH, suggesting a critical role for SR-BI/II in HCV infection.