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RO7119929 is being developed as an orally administered prodrug of the TLR7-specific agonist and active drug, RO7117418, for the treatment of patients with solid tumours.In this publication, we present a case study wherein the human pharmacokinetics and pharmacological active dose were prospectively predicted following oral administration of the prodrug.A simple translational pharmacokinetic-pharmacodynamic strategy was applied to predict the pharmacological active dose of the prodrug in human. In vivo studies in monkey showed that an unbound plasma exposure of active drug of 1.5 ng/mL elicited secretion of key serum pharmacodynamic cytokine and chemokine biomarkers in monkey. This threshold of 1.5 ng/mL was close to the minimum effective concentration of active drug required to induce cytokine secretion in human peripheral blood mononuclear cells (3 ng/mL).Measured in vitro physicochemical and biochemical properties of the prodrug and active drug were applied as input parameters in physiologically based pharmacokinetic models to predict the pharmacokinetics of active drug after oral dosing of the prodrug in humans. Then, using the PBPK model, a dose which delivered an unbound plasma Cmax in line with the target pharmacodynamic threshold of 1.5 ng/mL was found. This defined the lowest pharmacologically active dose as 3 mg.The prodrug entered the clinic in 2020 in patients with primary or secondary liver cancers. Clear pharmacodynamic, transient, and dose-dependent cytokine induction was observed at prodrug doses > 1 mg. RO7119929 is being developed as an orally administered prodrug of the TLR7-specific agonist and active drug, RO7117418, for the treatment of patients with solid tumours. In this publication, we present a case study wherein the human pharmacokinetics and pharmacological active dose were prospectively predicted following oral administration of the prodrug. A simple translational pharmacokinetic-pharmacodynamic strategy was applied to predict the pharmacological active dose of the prodrug in human. In vivo studies in monkey showed that an unbound plasma exposure of active drug of 1.5 ng/mL elicited secretion of key serum pharmacodynamic cytokine and chemokine biomarkers in monkey. This threshold of 1.5 ng/mL was close to the minimum effective concentration of active drug required to induce cytokine secretion in human peripheral blood mononuclear cells (3 ng/mL). Measured in vitro physicochemical and biochemical properties of the prodrug and active drug were applied as input parameters in physiologically based pharmacokinetic models to predict the pharmacokinetics of active drug after oral dosing of the prodrug in humans. Then, using the PBPK model, a dose which delivered an unbound plasma Cmax in line with the target pharmacodynamic threshold of 1.5 ng/mL was found. This defined the lowest pharmacologically active dose as 3 mg. The prodrug entered the clinic in 2020 in patients with primary or secondary liver cancers. Clear pharmacodynamic, transient, and dose-dependent cytokine induction was observed at prodrug doses > 1 mg.

The paramyxo- and pneumovirus family includes a wide range of viruses that can cause respiratory and/or systemic infections in humans and animals. The significant disease burden of these viruses is further exacerbated by the limited therapeutics that are currently available. Host cellular proteins that can antagonize or limit virus replication are therefore a promising area of research to identify candidate molecules with the potential for host-targeted therapies. Host proteins known as host cell restriction factors are constitutively expressed and/or induced in response to virus infection and include proteins from interferon-stimulated genes (ISGs). Many ISG proteins have been identified but relatively few have been characterized in detail and most studies have focused on studying their antiviral activities against particular viruses, such as influenza A viruses and human immunodeficiency virus (HIV)-1. This review summarizes current literature regarding host cell restriction factors against paramyxo- and pneumoviruses, on which there is more limited data. Alongside discussion of known restriction factors, this review also considers viral countermeasures in overcoming host restriction, the strengths and limitations in different experimental approaches in studies reported to date, and the challenges in reconciling differences between in vitro and in vivo data. Furthermore, this review provides an outlook regarding the landscape of emerging technologies and tools available to study host cell restriction factors, as well as the suitability of these proteins as targets for broad-spectrum antiviral therapeutics.

International audience; Rare individuals can naturally clear chronic hepatitis B virus (HBV) infection and acquire protection from reinfection as conferred by vaccination. To examine the protective humoral response against HBV, we cloned and characterized human antibodies specific to the viral surface glycoproteins (HBsAg) from memory B cells of HBV vaccinees and controllers. We found that human HBV antibodies are encoded by a diverse set of immunoglobulin genes and recognize various conformational HBsAg epitopes. Strikingly, HBsAg-specific memory B cells from natural controllers mainly produced neutralizing antibodies able to cross-react with several viral genotypes. Furthermore, monotherapy with the potent broadly neutralizing antibody Bc1.187 suppressed viremia in vivo in HBV mouse models and led to post-therapy control of the infection in a fraction of animals. Thus, human neutralizing HBsAg antibodies appear to play a key role in the spontaneous control of HBV and represent promising immunotherapeutic tools for achieving HBV functional cure in chronically infected humans.

TLR7 agonists are of high interest for the treatment of cancer, auto-immunity and chronic viral infections. They are known to activate plasmacytoid dendritic cells (pDCs) to produce high amounts of Type I Interferon (IFN) and to facilitate T and B cell responses, the latter with the help of maturation markers such as CD40, CD80 and CD86. The TLR7 single nucleotide polymorphism (SNP) rs179008 (GLn11Leu), sex and chronic viral infection have all been reported to influence pDC IFN production. It is unknown, however, whether these factors also influence pDC phenotypic maturation and thereby IFN-independent pDC functions. Furthermore, it is unclear whether SNP rs179008 influences HBV susceptibility and/or clearance. Here we investigated whether the SNP rs179008, sex and HBV infection affected phenotypic maturation of pDCs from 38 healthy individuals and 28 chronic HBV patients. In addition, we assessed SNP prevalence in a large cohort of healthy individuals (n = 231) and chronic HBV patients (n = 1054). Consistent with previous reports, the rs179008 variant allele was largely absent in Asians and more prevalent in Caucasians. Among Caucasians, the SNP was equally prevalent in healthy and chronically infected males. The SNP was, however, significantly more prevalent in healthy females than in those with chronic HBV infection (42 versus 28%), suggesting that in females it may offer protection from chronic infection. Ex vivo experiments demonstrated that induction of the co-stimulatory molecules CD40 and CD86 by TLR7 ligands, but not TLR9 ligands, was augmented in pDCs from healthy SNP-carrying females. Furthermore, CD80 and CD86 upregulation was more pronounced in females independent of the SNP. Lastly, our data suggested that chronic HBV infection impairs pDC maturation. These findings provide insight into factors determining TLR7 responses, which is important for further clinical development of TLR7-based therapies.

International audience; We previously reported that Toll-like receptor 9 (TLR9)-CpG oligonucleo-tides could inhibit the establishment of hepatitis B virus (HBV) infections in hepato-cytes. Our aim was to uncover the underlying mechanisms of this inhibition. HepaRG cells, RPMI-B lymphoblastoma cells, and primary plasmacytoid dendritic cells (pDCs) exposed to HBV and TLR9 ligands/agonists in various configurations were used. We observed an inhibition of HBV infection upon TLR9 stimulations only when agonist was applied during inoculation. This inhibition was independent of interleukin-6 (IL-6)/interferon-inducible protein 10 (IP-10) production as well as of TLR9 expression in hepatocytes. We further demonstrated an entry inhibition mechanism by showing a noncovalent binding of TLR9 agonist to HBV particles. Besides inhibiting HBV entry into hepatocytes, this biophysical interaction between HBV virions and TLR9 agonist was responsible for a reduction of alpha interferon (IFN-) expression by pDCs. Interestingly , subviral particles composed of only HBsAg were able to genuinely inhibit the TLR9 pathway, without titrating TLR9 ligands. To conclude, our data suggest that synthetic TLR9-CpG oligonucleotides can strongly inhibit HBV entry by " coating " HBV virions and thereby preventing their interaction with cellular receptor. This titration effect of TLR9 agonist is also artifactually responsible for the inhibition of TLR9 engagement in pDCs, whereas a genuine inhibition of this innate pathway was confirmed with HBsAg subviral particles. H epatitis B virus (HBV) chronically infects 240 million people worldwide and represents one of the major etiologies for cirrhosis and hepatocellular carcinoma (1, 2). The progression rate of chronicity is tightly linked with the maturity of the immune system, since 90 to 95% of infected newborns become chronic carriers, whereas only 5 to 10% of adults do (3). As HBV long-term persistence is characterized by subversion of both innate and adaptive immunity (4, 5), a better understanding of the underlying cellular and molecular mechanisms of this escape should help in defining new therapeutic strategies to reactivate the host immune system and control viral replication. Current front-line therapies include the use of nucleoside analogues to specifically inhibit viral reverse transcription and/or pegylated interferon alpha 2a or 2b (Peg-IFN

Fibrinogen-beta (FBG-beta), an important acute-phase protein (APP), is generated by the liver as a target for inflammatory mediators. Here we identified FBG-beta as a hepatitis C virus (HCV) core interacting protein by screening a human liver complementary DNA (cDNA) library using mammalian two-hybrid analysis. An association between FBG-beta and HCV core protein was verified by confocal microscopy and coimmunoprecipitation from the transfected human hepatocyte (Huh-7) cell line. HCV core or genomic RNA transfected Huh-7 cells modestly increased FBG-beta protein expression when compared to the basal level in control hepatocytes. Transfection of HCV core or full-length (FL) gene into Huh-7 cells up-regulated basal FBG-beta promoter activity. Exogenous addition of IL-6 stimulates FBG-beta promoter activity in hepatocytes. However, ectopic expression of HCV core or FL in hepatocytes inhibited IL-6-stimulated FBG-beta promoter activation. Inhibition of endogenous FBG-beta expression following introduction of small interfering RNA (siRNA) into cells displayed a gain of function of promoter regulation by HCV core protein. Further studies suggested that HCV core gene expression in stable transfectants of Huh-7 cells resulted in a basal up-regulation of FBG-beta and other APPs. However, treatment with cytokines, interleukin-6 (IL-6), or tumor necrosis factor-alpha repressed FBG-beta and other acute-phase response (APR) genes.Our results reveal that the core/FBG-beta interaction may act as a regulatory feedback, allowing repression of IL-6-stimulated APR genes. Together, these data suggested a network of interactions between HCV core and the hepatic APR genes, and may contribute to impaired innate immunity for viral persistence.

Hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease worldwide. In the early phase of acute infection, HCV continues to replicate in the liver, overcoming innate and acquired immunity in a majority of infected humans. Sustained, vigorous, and multiepitope-specific CD4-positive (CD4+) and CD8+ T-cell responses are essential for spontaneous HCV clearance (26). Infected cells display peptides derived from viral antigens bound to major histocompatibility complex (MHC)/human leukocyte antigen (HLA) receptors at the cell surface and are recognized by T cells. Generation of HCV-specific CD4+ T cells occurs after stimulation by MHC class II/viral peptide complexes and facilitates the induction of HCV-specific CD8+ T-cell responses and optimal antibody production. CD8+ T cells recognize and attack infected cells expressing viral peptides in the context of MHC class I surface molecules. Dendritic cells (DCs) are important for the initiation of T-cell responses to foreign antigens. The antigen uptake and presentation capacities of DCs enable them to prime and activate naive T cells. Immature DCs are optimally phagocytic and pinocytose antigens. However, DCs must be activated to mature before serving as efficient antigen-presenting cells (APCs). Chronic HCV infection has been shown to affect the allostimulatory function of DCs (3). Monocyte-derived DCs from patients with chronic HCV infection do not respond to maturation stimuli, maintaining their immature phenotype (1). The presence of HCV genomic sequences in DCs could be documented for HCV carriers (4), although DCs may not support HCV replication. DC-SIGN and DC-SIGNR are two closely related membrane-associated C-type lectins. DC-SIGN is expressed on some DCs, while DC-SIGNR is localized to certain endothelial cell populations, including hepatic sinusoidal endothelial cells. Interactions of HCV envelope glycoprotein E2 with DC-SIGN and DC-SIGNR may contribute to the capture and delivery of virus to the liver by DCs (29, 39). Several HLA polymorphisms are associated with viral clearance or persistence (21, 34, 35). The HLA-DR molecules are pivotal for the adaptive immune system, as they guide the development and activation of CD4+ T helper cells (41, 42). This complex gene expression profile is controlled almost exclusively by the CIITA, a global regulator for the expression of genes involved in antigen presentation (10, 30). Differential activation of independent promoters that drive expression of the gene encoding CIITA ultimately determines the exquisitely regulated pattern of MHC gene expression. Transcription of CIITA is driven by a large regulatory region that contains four distinct promoters known as pI, pII, pIII, and pIV (36). Cytokine regulation by CIITA is complex and may reflect both direct and indirect mechanisms of T-cell development and differentiation (37). The promoter pIV is induced by gamma interferon (IFN-γ), and a conserved 300-bp promoter-proximal region is sufficient for the activation. This region contains three regulatory elements, GAS, an E box, and an IFN-regulatory factor element (IRF-E), all of which are required for the induction of pIV. The main function of HLA-DR molecules encoded by one of the three MHC class II alleles is to present processed antigens which are derived primarily from exogenous sources to CD4+ T lymphocytes (22). Therefore, HLA-DR molecules are critical for the initiation of the antigen-specific immune response. Constitutive expression of MHC class II molecules is confined to professional APCs of the immune system, while MHC class II molecule expression can be induced by a variety of immune regulators in nonprofessional APCs. MHC class II proteins are important for the initiation of immune responses and are essential for specific recognition of foreign antigens by the immune system. Thus, an interaction of DCs with HCV particles may play an important role in immunopathogenesis. In this study, we have examined the role of HCV proteins and infectious HCV in suppression of APC function/T-cell activation. IFN-γ is the most potent inducer of MHC class II expression in many cell types (8). Our results indicate that cells endogenously expressing HCV proteins from an HCV1-2962 (FL) construct or those exposed to cell culture-grown HCV (HCVcc) perturb HLA-DR cell surface expression. The inhibition of MHC class II cell surface expression was evident on IFN-γ-induced HT1080 cells and on professional antigen-presenting monocyte-derived DCs. A deficiency in expression of HLA-DR molecules was further observed to be associated with the negative regulation of CIITA, GAS, and the HLA-DR promoters at the transcriptional level, induction of interleukin-10 (IL-10) in DCs, and inhibition of DC-mediated antigen-specific T-cell activation.

Hepatitis C virus (HCV) causes a spectrum of disease ranging from an asymptomatic carrier state to progressive liver disease, which includes diabetes, cirrhosis and hepatocellular carcinoma (17, 18, 29, 33). Patients with chronic HCV infection have a significantly increased prevalence of type 2 diabetes mellitus compared to controls or HBV-infected patients. Insulin resistance is a critical component of type 2 diabetes mellitus pathogenesis. Several mechanisms are likely to be involved in the pathogenesis of HCV-related insulin resistance (2). Both insulin resistance and diabetes can adversely affect the course of chronic hepatitis C and lead to enhanced steatosis, steatohepatitis, and liver fibrosis (1, 14). Although several hypotheses have been made, the link between insulin resistance and steatosis is complex, and the exact sequence of events is unclear (16). In chronic hepatitis C patients, the prevalence of steatosis ranges from 40 to 86%. Hepatic steatosis can develop secondary to obesity, diabetes mellitus, and chronic HCV infection (6). Studies have also suggested that NF-κB activation is involved in the induction of downstream cytokine (interleukin-6 [IL-6]) production, leading to insulin resistance (5, 13). Several cellular lesions have been associated with insulin resistance, but the precise mechanism whereby HCV induces insulin resistance remains elusive. This knowledge may allow for the development of intervention strategies directed toward treating the pathogenesis related to chronic HCV infection. Insulin generally carries out its biological effects through the phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-2 (55, 61, 62). Thus, research has focused on IRS-1 and IRS-2 as a locus for insulin resistance. Impairment of IRS-1 and IRS-2 expression has been observed in the liver of patients with chronic hepatitis C, as well as in HCV core transgenic mice. HCV mediates dysfunction of the insulin signaling pathways by upregulating the expression of suppressors of cytokine signaling 3 expression (31) and increased TNF-α secretion (51). Ser/Thr phosphorylation of IRS-1 inhibits its association with the insulin receptor, which in turn inhibits tyrosine phosphorylation of IRS-1, and promotes degradation. On the other hand, increased Ser phosphorylation of IRS-1 is a key negative feedback mechanism under physiological conditions to terminate the action of insulin. In an insulin-resistant state, an imbalance occurs between positive IRS-1 Tyr phosphorylation and negative Ser phosphorylation of IRS-1 (59). Tumor necrosis factor alpha (TNF-α), IL-6, free fatty acids, or cellular stress can induce insulin resistance by activating Ser phosphorylation of IRS-1, thereby inhibiting its function. However, since there are several Ser sites involved in the phosphorylation of IRS-1, the mechanism by which Ser phosphorylation inhibits insulin signaling is difficult to establish. c-Jun N-terminal kinase (JNK) is especially important for IRS-1 function because it associates with IRS-1 and phosphorylates Ser312 (3, 4). The Akt/protein kinase B (PKB) signal transduction pathway, one of those associated with insulin receptor signaling, is responsible for passing insulin receptor instructions from the plasma membrane to the metabolic, transcription, and translation machinery within the cell (12). Akt is activated as part of the downstream pathway of multiple classes of growth factor receptors, from receptor tyrosine kinases to cytokine receptors and integrins (42). In most cell types, activation of the Akt pathway by cell surface receptors dictates changes in cellular metabolism, coordinated with alterations in cell growth, mitogenesis, and susceptibility to apoptosis. Activation of Akt in response to growth factors or oncogenes is sufficient to cause increased transcription and plasma membrane localization of the glucose transporter expressed in most cell types (7). Akt activation requires phosphatidylinositol triphosphate generated by phosphatidylinositol 3-kinase (PI-3K) and phosphorylation on separate sites by the upstream kinases phosphatidylinositol-dependent kinase-1 (PDK1) and the mammalian target of rapamycin (mTOR)-rictor complex (TORc2) (47). Direct effects of Akt on glycolysis and mitochondrial function can be considered “immediate-early” metabolic responses. However, Akt also signals long-term alterations in cellular metabolism that can have profound effects on the homeostatic regulation of circulating glucose and overall organism longevity. These effects are mediated in part through the regulation of Forkhead Box subclass O transcription factors (42). Multiple oncoproteins and tumor suppressors connected with cell signaling and metabolic regulation intersect the Akt signal transduction pathway and are activated or inactivated, respectively, in a wide array of cancers (56). To understand the molecular mechanisms involved in HCV core-mediated insulin resistance, IRS-1 phosphorylation was studied in stable transfectants of hepatocytes expressing core protein in the presence or absence of TNF-α or IL-6. Activation of Akt in the presence of cell culture-grown HCV was also examined separately. Our results suggested that HCV core protein expression in hepatocytes increases IRS-1 phosphorylation at Ser312, impairs activation of Akt at the Thr308 phosphorylation site, and inhibits insulin stimulated glucose uptake.

Hepatitis C virus (HCV) often causes a persistent infection associated with hypergammaglobulinemia, high levels of antiviral antibody and circulating immune complexes, and immune complex disease. We previously reported that only a limited neutralizing activity to vesicular stomatitis virus or HCV pseudotype is generated in animals immunized with recombinant HCV envelope proteins and chronically infected HCV patient sera. Interestingly, when some of these neutralizing sera were diluted into a range of concentrations below those that reduced virus plaque number, an increase in pseudotype plaque formation was observed. Purified HCV E2-specific human monoclonal antibodies were used to further verify the specificity of this enhancement, and one- to twofold increases were apparent on permissive Huh-7 cells. The enhancement of HCV pseudotype titer could be inhibited by the addition of a Fc-specific anti-human immunoglobulin G Fab fragment to the virus-antibody mixture prior to infection. Treatment of cells with antibody to Fc receptor I (FcRI) or FcRII, but not FcRIII, also led to an inhibition of pseudotype titer enhancement in an additive manner. Human lymphoblastoid cell line (Raji), a poor host for HCV pseudotype infection, exhibited a four- to sixfold enhancement of pseudotype-mediated cell death upon incubation with antibody at nonneutralizing concentrations. A similar enhancement of cell culture-grown HCV infectivity by a human monoclonal antibody was also observed. Taken together, antibodies to viral epitopes enhancing HCV infection need to be taken into consideration for pathogenesis and in the development of an effective vaccine.

We have previously reported that immortalized human hepatocytes (IHH) support the generation of infectious hepatitis C virus (HCV) genotype 1a (clone H77). In the present study, we have investigated the growth of HCV genotype 1a (clone H77) through serial passages and accompanying changes in IHH in response to infection. Eleven serial passages of HCV genotype 1a (clone H77) in IHH were completed. Virus replication was ascertained from the presence of HCV-specific sequences, the detection of core antigen, the virus genome copy number, and the virus titer in IHH culture fluid. Electron microscopy suggested that HCV infection induces autophagic vacuole formation in IHH. Fluorescence microscopy displayed localization of autophagic markers, microtubule-associated protein-1 light chain-3 and Apg5, on the vacuoles of HCV-infected hepatocytes. Taken together, our results suggested that HCV genotype 1a (clone H77) can be serially passaged in IHH and that HCV infection induces an autophagic response in hepatocytes.

International audience; Background & Aims: The outcome of hepatitis B virus (HBV) infection may be influenced by early interactions between the virus and hepatocyte innate immune responses. To date, the study of such interactions during the very early step of infection has not been adequately investigated.Methods: We used the HepaRG cell line, as well as primary human hepatocytes to analyze, within 24 h of exposure to HBV, either delivered by a physiologic route or baculovirus vector (Bac-HBV), the early modulation of the expression of selected antiviral/pro-inflammatory cytokines and interferon stimulated genes. Experiments were also performed in the presence or absence of innate receptor agonists to investigate early HBV-induced blockade of innate responses.Results: We show that hepatocytes themselves could detect HBV, and express innate genes when exposed to either HBV virions or Bac-HBV. Whereas Bac-HBV triggered a strong antiviral cytokine secretion followed by the clearance of replicative intermediates, a physiologic HBV exposure led to an abortive response. The early inhibition of innate response by HBV was mainly evidenced on Toll-like receptor 3 and RIG-I/MDA5 signaling pathways upon engagement with exogenous agonist, leading to a decreased expression of several pro-inflammatory and antiviral cytokine genes. Finally, we demonstrate that this early inhibition of dsRNA-mediated response is due to factor(s) present in the HBV inoculum, but not being HBsAg or HBeAg themselves, and does not require de novo viral protein synthesis and replication.Conclusions: Our data provide strong evidence that HBV viral particles themselves can readily inhibit host innate immune responses upon virion/cell interactions, and may explain, at least partially, the “stealthy” character of HBV.

International audience; Background & Aims: HepaRG cells are considered as the best surrogate model to primary human hepatocyte (PHH) culture to investigate host-pathogen interactions. Yet their innate immune functions remain unknown. In this study, we explored the expression and functionality of Toll-like (TLR) and retinoic acid-inducible gene-1 (RIG-I)-like receptors (RLR) in these cells.Methods: Gene and protein expression levels of TLR-1 to 9 and RLR in HepaRG were mainly compared to PHH, by RT-qPCR, FACS, and Western blotting. Their functionality was assessed, by measuring the induction of toll/rig-like themselves and several target innate gene expressions, as well as the secretion of IL-6, IP-10, and type I interferon (IFN), upon agonist stimulation. Their functionality was also shown by measuring the antiviral activity of some TLR/RLR agonists against hepatitis B virus (HBV) infection.Results: The basal gene and protein expression profile of TLR/RLR in HepaRG cells was similar to PHH. Most receptors, except for TLR-7 and 9, were expressed as proteins and functionally active as shown by the induction of some innate genes, as well as by the secretion of IL-6 and IP-10, upon agonist stimulation. The highest levels of IL-6 and IP-10 secretion were obtained by TLR-2 and TLR-3 agonist stimulation respectively. The highest preventive anti-HBV activity was obtained following TLR-2, TLR-4 or RIG-I/MDA-5 stimulations, which correlated with their high capacity to produce both cytokines.Conclusions: Our results indicate that HepaRG cells express a similar pattern of functional TLR/RLR as compared to PHH, thus qualifying HepaRG cells as a surrogate model to study pathogen interactions within a hepatocyte innate system

Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self-assembles into HCV-like particles (HCV-LPs) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV particle morphogenesis by electron microscopy. Various mutated HCV core proteins with engineered internal deletions were expressed with this system, to identify core domains required or dispensable for HCV-LP assembly. The HCV core protein sequence was compared with its counterpart in GB virus B (GBV-B), the virus most closely related to HCV, to identify conserved domains. GBV-B and HCV display similar tropism for liver hepatocytes and their core proteins are organized similarly into three main domains (I, II and III), although GBV-B core is smaller and lacks approximately 35 amino acids (aa) in domain I. The deletion of short hydrophobic domains (aa 133-152 and 153-167 in HCV core) that appear highly conserved in domain II of both GBV-B and HCV core proteins resulted in loss of HCV core ER anchoring and self-assembly into HCV-LPs. The deletion of short domains found within domain I of HCV core protein but not in the corresponding domain of GBV-B core according to sequence alignment had contrasting effects. Amino acids 15-28 and 60-66 were shown to be dispensable for HCV-LP assembly and morphogenesis, whereas aa 88-106 were required for this process. The production of GBV-B core protein from a recombinant SFV vector was associated with specific ER ultrastructural changes, but did not lead to the morphogenesis of GBV-B-LPs, suggesting that different budding mechanisms occur in members of the Flaviviridae family.

Hepatitis C virus (HCV) core protein, expressed with a Semliki Forest virus replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. This model was used to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. Several mutants were designed as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electron-dense ER membrane, with no evidence of HCV-LP assembly. These data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly.

Details of the ultrastructure of hepatitis C virus (HCV) virion remain unclear because it has proved extremely difficult to visualise virus particles from infected serum and tissues directly. In addition, although much is known about the viral genome, first cloned in 1989, little is known about HCV morphogenesis, due to the lack of an efficient in vitro culture system for HCV propagation. Virus-like particles (VLPs) obtained by expressing genes encoding the HCV structural proteins in mammalian cells can be used as an alternative model for studying HCV morphogenesis. In particular, this HCV-LP model has made it possible to demonstrate that HCV budding occurs at the ER membrane and that the core protein drives this process. The HCV-LP model opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions, which may make it possible to establish the long-awaited in vitro culture system for HCV.

In the absence of a hepatitis C virus (HCV) culture system, the use of a Semliki Forest virus replicon expressing genes encoding HCV structural proteins that assemble into HCV-like particles provides an opportunity to study HCV morphogenesis. Using this system, we showed that the HCV core protein constitutes the budding apparatus of the virus and that its targeting to the endoplasmic reticulum by means of the signal sequence of E1 protein is essential for budding. In addition, the aspartic acid at position 111 in the HCV core protein sequence was found to be crucial for virus assembly, demonstrating the usefulness of this system for mapping amino acids critical to HCV morphogenesis.