The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has highlighted the need for broad-spectrum antivirals against coronaviruses (CoVs). Here, pheophorbide a (Pba) was identified as a highly active antiviral molecule against human CoV-229E after bioguided fractionation of plant extracts. The antiviral activity of Pba was subsequently shown for SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV), and its mechanism of action was further assessed, showing that Pba is an inhibitor of coronavirus entry by directly targeting the viral particle. Interestingly, the antiviral activity of Pba depends on light exposure, and Pba was shown to inhibit virus-cell fusion by stiffening the viral membrane, as demonstrated by cryoelectron microscopy. Moreover, Pba was shown to be broadly active against several other enveloped viruses and reduced SARS-CoV-2 and MERS-CoV replication in primary human bronchial epithelial cells. Pba is the first described natural antiviral against SARS-CoV-2 with direct photosensitive virucidal activity that holds potential for COVID-19 therapy or disinfection of SARS-CoV-2-contaminated surfaces.

The pestivirus classical swine fever virus (CSFV) represents one of the most important pathogens of swine. Its virulence is dependent on the RNase activity of the essential structural glycoprotein E^rns that uses an amphipathic helix as a membrane anchor and forms homodimers via disulfide bonds employing cysteine 171. Dimerization is not necessary for CSFV viability but for its virulence. Mutant E^rns proteins lacking cysteine 171 are still able to interact transiently as shown in crosslink experiments. Deletion analysis did not reveal the presence of a primary sequence-defined contact surface essential for dimerization, but indicated a general importance of an intact ectodomain for efficient establishment of dimers. Pseudoreverted viruses reisolated in earlier experiments from pigs with mutations Cys171Ser/Ser209Cys exhibited partially restored virulence and restoration of the ability to form E^rns homodimers. Dimer formation was also observed for experimentally mutated proteins, in which other amino acids at different positions of the membrane anchor region of E^rns were replaced by cysteine. However, with one exception of two very closely located residues, the formation of disulfide-linked dimers was only observed for cysteine residues located at the same position of the helix.

• RNA virus
• amphipathic helix
• dimerization of glycoprotein
• disulfide bond formation
• envelope protein
• flavivirus
• pestivirus
• virulence factor

• E2 glycoprotein
• HCV vaccine
• HCV-neutralizing antibodies
• N-glycosylation
• plant biopharmaceuticals

• Animals
• Antibodies, Neutralizing
• Hepacivirus
• Hepatitis C Antibodies
• Mice
• Nicotiana
• Viral Envelope Proteins/genetics
• Viral Hepatitis Vaccines

Pestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, E^rns, E1, and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface but localizes predominantly in the endoplasmic reticulum (ER). Using engineered chimeric transmembrane domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labeling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C terminus, whereas it adopts a hairpin-like structure with the C terminus located in the ER lumen when the precleavage situation is mimicked by blocking the cleavage site between E1 and E2.

IMPORTANCE The shortage of specific antibodies against E1, making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to E^rns and E2 with regard to biosynthesis, structure, and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the transmembrane domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy terminus located on the luminal side of the ER in the precleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.

• envelope protein
• glycoprotein surface expression
• intracellular retention signal
• membrane topology
• pestivirus
• viral polyprotein processing

• RNA virus envelope protein
• envelope protein heterodimer
• envelope protein homooligomer
• intracellular localization
• intracellular retention
• pestivirus

• Animals
• Cattle
• Cell Line
• Cricetinae
• Dimerization
• Mutation/genetics
• Pestivirus/genetics
• Rabbits
• Viral Envelope Proteins/genetics
• Virus Internalization

Chronic hepatitis C virus (HCV) infections continue to be a major contributor to liver disease worldwide. HCV treatment has become highly effective, yet there are still no vaccines or prophylactic strategies available to prevent infection and allow effective management of the global HCV burden. Glycan-dependent interactions are crucial to many aspects of the highly complex HCV entry process, and also modulate immune evasion. This review provides an overview of the roles of viral and cellular glycans in HCV infection and highlights glycan-focused advances in the development of entry inhibitors and vaccines to effectively prevent HCV infection.

• glycans
• hepatitis C virus
• prophylaxis
• vaccines
• viral entry

• Antiviral Therapy
• Entry Inhibitor
• Hepatitis C Virus
• High-Mannose Glycan
• Plant-Made Pharmaceutical

• Animals
• Antiviral Agents/pharmacology
• Antiviral Agents/therapeutic use
• Disease Models, Animal
• Female
• Hepacivirus/drug effects
• Hepacivirus/isolation & purification
• Hepatitis C, Chronic/drug therapy
• Hepatitis C, Chronic/pathology
• Hepatitis C, Chronic/virology
• Hepatocytes/transplantation
• Humans
• Immunoconjugates/genetics
• Immunoconjugates/pharmacology
• Immunoconjugates/therapeutic use
• Lectins/genetics
• Lectins/pharmacology
• Lectins/therapeutic use
• Liver/drug effects
• Liver/pathology
• Liver/virology
• Male
• Mice
• Polysaccharides/antagonists & inhibitors
• Recombinant Fusion Proteins/genetics
• Recombinant Fusion Proteins/pharmacology
• Recombinant Fusion Proteins/therapeutic use
• Transplantation Chimera
• Viral Envelope Proteins

• R33 AI088585 NIAID NIH HHS
• T32 ES011564 NIEHS NIH HHS
• U01 HL127518 NHLBI NIH HHS

• Hepatitis C virus
• NS2
• Rimantadine
• dFLEX
• p7

• HCV assembly
• Phosphatidylserine-specific phospholipase A1 (PLA1A)
• Viral RNA replication

• Cell Line, Tumor
• Hepacivirus/physiology
• Hepatitis C/virology
• Host-Pathogen Interactions
• Humans
• Liver/virology
• Phosphatidylserines/chemistry
• Phospholipases A1/chemistry
• Phospholipases A1/genetics
• RNA, Viral/metabolism
• Viral Envelope Proteins/chemistry
• Viral Envelope Proteins/genetics
• Viral Nonstructural Proteins/chemistry
• Viral Nonstructural Proteins/genetics
• Virus Assembly

Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae. HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV.

Hepatitis C virus (HCV) is an enveloped virus that infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV has two envelope proteins, E1 and E2, which form heterodimers on viral surface and are critical for HCV cell entry. However, current studies of HCV E1E2 are often limited by the poor quality of the in vitro expressed E1E2 heterodimers. Here we express the ectodomains of HCV E1E2 with different tags, and are able to isolate soluble E1E2 ectodomains suitable for structural and functional studies. Then we generate the 3D reconstruction of E1E2 heterodimer by electron microscopy and also model the E1E2 structure by the coevolution based strategy with Rosetta, showing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the HCV cellular receptors, neutralizing antibodies as well as the inhibition of HCV infection. These results suggest that the expressed E1E2 heterodimer would be a promising target for both viral studies and vaccination against HCV.

• Antibodies, Neutralizing/metabolism
• HEK293 Cells
• Hepacivirus/physiology
• Hepatitis C/genetics
• Hepatitis C/metabolism
• Hepatitis C/virology
• Humans
• Protein Conformation
• Protein Multimerization
• Receptors, Cell Surface/metabolism
• Recombinant Fusion Proteins/chemistry
• Recombinant Fusion Proteins/genetics
• Recombinant Fusion Proteins/metabolism
• Viral Envelope Proteins/chemistry
• Viral Envelope Proteins/genetics
• Viral Envelope Proteins/metabolism
• Virus Internalization

Hepatitis C virus (HCV) infects 3% of world population being responsible for nearly half a million deaths annually urging the need for a prophylactic vaccine. Retrovirus like particles are commonly used scaffolds for antigens presentation being the core of diverse vaccine candidates. The immunogenicity of host proteins naturally incorporated in retrovirus was hypothesized to impact the performance of retrovirus based vaccines. In this work, the capacity of engineered retrovirus like particles devoided of host protein CD81 to display HCV envelope antigens was compared to non-engineered particles. A persistent inability of CD81 negative VLPs to incorporate HCV E2 protein as a result from the inefficient transport of HCV E2 to the plasma membrane, was observed. This work enabled the identification of a CD81-mediated transport of HCV E2 while stressing the importance of host proteins for the development of recombinant vaccines.

• CD81
• E1
• E2
• HCV
• Retrovirus
• Transport

• HCVcc
• HCVpp
• Hepatitis C virus

Chronic hepatitis C virus (HCV) infection is the cause of about 400,000 annual liver disease-related deaths. The global spread of this important human pathogen can potentially be prevented through the development of a vaccine, but this challenge has proven difficult, and much remains unknown about the multitude of mechanisms by which this heterogeneous RNA virus evades inactivation by neutralizing antibodies (NAbs). The N-terminal motif of envelope protein 2 (E2), termed hypervariable region 1 (HVR1), changes rapidly in immunoglobulin-competent patients due to antibody-driven antigenic drift. HVR1 contains NAb epitopes and is directly involved in protecting diverse antibody-specific epitopes on E1, E2, and E1/E2 through incompletely understood mechanisms. The ability of HVR1 to protect HCV from NAbs appears linked with modulation of HCV entry co-receptor interactions. Thus, removal of HVR1 increases interaction with CD81, while altering interaction with scavenger receptor class B, type I (SR-BI) in a complex fashion, and decreasing interaction with low-density lipoprotein receptor. Despite intensive efforts this modulation of receptor interactions by HVR1 remains incompletely understood. SR-BI has received the most attention and it appears that HVR1 is involved in a multimodal HCV/SR-BI interaction involving high-density-lipoprotein associated ApoCI, which may prime the virus for later entry events by exposing conserved NAb epitopes, like those in the CD81 binding site. To fully elucidate the multifunctional role of HVR1 in HCV entry and NAb evasion, improved E1/E2 models and comparative studies with other NAb evasion strategies are needed. Derived knowledge may be instrumental in the development of a prophylactic HCV vaccine.

• hepatitis C virus (HCV)
• hypervariable region 1 (HVR1)
• neutralization
• vaccine design
• viral entry

• glycoproteins
• glycosylation
• hepatitis C virus
• humoral immune response
• neutralizing antibodies

• Amino Acid Sequence
• Animals
• Antibodies, Monoclonal
• Antibodies, Viral
• Antigens, Viral/genetics
• Epitope Mapping
• Epitopes/chemistry
• Epitopes/genetics
• Hepacivirus/genetics
• Hepacivirus/immunology
• Hepacivirus/physiology
• High-Throughput Screening Assays
• Humans
• Models, Molecular
• Mutagenesis
• Protein Engineering
• Protein Folding
• Protein Stability
• Recombinant Proteins/chemistry
• Recombinant Proteins/genetics
• Recombinant Proteins/immunology
• Tetraspanin 28/metabolism
• Viral Envelope Proteins/chemistry
• Viral Envelope Proteins/genetics
• Viral Envelope Proteins/immunology
• Viral Hepatitis Vaccines/genetics
• Viral Hepatitis Vaccines/immunology
• Virus Internalization

• T32 AI007606 NIAID NIH HHS
• R01 AI079031 NIAID NIH HHS
• HHSN272201400028C NIAID NIH HHS
• R56 AI106005 NIAID NIH HHS
• U19 AI123861 NIAID NIH HHS
• R01 AI106005 NIAID NIH HHS
• U54 GM094586 NIGMS NIH HHS
• R01 AI123365 NIAID NIH HHS
• HHSN272201400058C NIAID NIH HHS
• National Institutes of Health

Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3–4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare cross-genotype conserved epitope. By analyzing the genotype 1a envelope proteins (E1/E2) of recovered Core-NS2 recombinant H77/JFH1_ΔHVR1 and performing reverse genetic studies we found that resistance to AR5A was caused by substitution L665W, also conferring resistance to the parental H77/JFH1. The mutation did not induce viral fitness loss, but abrogated AR5A binding to HCV particles and intracellular E1/E2 complexes. Culturing J6/JFH1_ΔHVR1 (genotype 2a), for which fitness was decreased by L665W, with AR5A generated AR5A-resistant viruses with the substitutions I345V, L665S, and S680T, which we introduced into J6/JFH1 and J6/JFH1_ΔHVR1. I345V increased fitness but had no effect on AR5A resistance. L665S impaired fitness and decreased AR5A sensitivity, while S680T combined with L665S compensated for fitness loss and decreased AR5A sensitivity even further. Interestingly, S680T alone had no fitness effect but sensitized the virus to AR5A. Of note, H77/JFH1_L665S was non-viable. The resistance mutations did not affect cell-to-cell spread or E1/E2 interactions. Finally, introducing L665W, identified in genotype 1, into genotypes 2–6 parental and HVR1-deleted variants (not available for genotype 4a) we observed diverse effects on viral fitness and a universally pronounced reduction in AR5A sensitivity. Thus, we were able to take advantage of the neutralization-sensitive HVR1-deleted viruses to rapidly generate escape viruses aiding our understanding of the divergent escape pathways used by HCV to evade AR5A.

Worldwide hepatitis C virus (HCV) is one of the leading causes of chronic liver diseases, including cirrhosis and cancer. Treatment accessibility is limited and development of a preventive vaccine has proven difficult, partly due to the high mutation rate of the virus. Recent studies of HCV antibody neutralization resistance have revealed important information about escape pathways and barriers to escape for several clinically promising human monoclonal antibodies. However, due to the varying levels of antibody shielding between HCV isolates these studies have been mostly limited to a few neutralization-sensitive HCV isolates. Here, we took advantage of the fact that deletion of the hypervariable region 1 (HVR1) increased antibody sensitivity of HCV isolates by increasing the exposure of important epitopes, thus facilitating studies of antibody escape for neutralization resistant isolates. We identified escape mutations in the envelope glycoprotein E2, at amino acid position L665, which conferred antibody resistance in parental HCV viruses from genotypes 1–6. We found that antibody escape was associated with loss of binding to HCV particles and intracellular envelope protein complexes. We also identified escape substitutions at L665 that were isolate-specific. Thus, our data sheds new light on antibody resistance mechanisms across diverse HCV isolates.

• Antibodies, Monoclonal/immunology
• Antibodies, Neutralizing/immunology
• Antibodies, Viral/immunology
• Cell Line
• Epitopes, B-Lymphocyte/immunology
• Hepacivirus/immunology
• Hepatitis C/immunology
• Humans
• Immune Evasion/immunology
• Immunoprecipitation
• Mutation
• Viral Envelope Proteins/genetics
• Viral Envelope Proteins/immunology
• Viral Proteins/genetics
• Viral Proteins/immunology

• R01 AI079031 NIAID NIH HHS
• R01 AI106005 NIAID NIH HHS
• R01 AI123365 NIAID NIH HHS
• U19 AI123861 NIAID NIH HHS
• Faculty of Health and Medical Sciences, University of Copenhagen
• Danish Council of Independent Research
• Lundbeckfonden
• Novo Nordisk Foundation
• Danish Council for Independent Research
• National Institutes of Health

• Adult
• Antibodies, Monoclonal/isolation & purification
• Antibodies, Neutralizing/isolation & purification
• B-Lymphocytes/cytology
• B-Lymphocytes/immunology
• Epitopes/immunology
• Genotype
• Hepacivirus/genetics
• Hepacivirus/immunology
• Hepacivirus/metabolism
• Hepatitis C/blood
• Hepatitis C/therapy
• Hepatitis C Antibodies/isolation & purification
• Humans
• Male
• Substance Abuse, Intravenous/virology
• Viral Envelope Proteins/immunology
• Viral Hepatitis Vaccines/immunology

• Virgo consortium, funded by the Dutch government
• Center for Infectious Disease Control of the Netherlands National Institute for Public Health and the Environment.

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are the main inducers of a cross-neutralizing antibody response which plays an important role in the early phase of viral infection. Correctly folded and immunologically active E1E2 complex can be expressed in mammalian cells, though the production process might still prove restrictive, even if the immunological response of a vaccine candidate is positive. Here, we report a characterization and immunogenicity study of a full-length (fE1E2) and soluble version of the E1E2 complex (tE1E2) from genotype 1a, successfully expressed in the cells of Leishmania tarentolae. In a functional study, we confirmed the binding of both Leishmania-derived E1E2 complexes to the CD-81 receptor and the presence of the major epitopes participating in a neutralizing antibody response. Both complexes were proved to be highly immunogenic in mice and elicited neutralizing antibody response. Moreover, cross-reactivity of the mouse sera was detected for all tested HCV genotypes with the highest signal intensity observed for genotypes 1a, 1b, 5 and 6. Since the development of a prophylactic vaccine against HCV is still needed to control the global infection, our Leishmania-derived E1E2 glycoproteins could be considered a potential cost-effective vaccine candidate.

Highly pathogenic human coronaviruses associated with a severe respiratory syndrome, including Middle East respiratory syndrome coronavirus (MERS-CoV), have recently emerged. The MERS-CoV epidemic started in 2012 and is still ongoing, with a mortality rate of approximately 35%. No vaccine is available against MERS-CoV and therapeutic options for MERS-CoV infections are limited to palliative and supportive care. A search for specific antiviral treatments is urgently needed. Coronaviruses are enveloped viruses, with the spike proteins present on their surface responsible for virus entry into the target cell. Lectins are attractive anti-coronavirus candidates because of the highly glycosylated nature of the spike protein. We tested the antiviral effect of griffithsin (GRFT), a lectin isolated from the red marine alga Griffithsia sp. against MERS-CoV infection. Our results demonstrate that while displaying no significant cytotoxicity, griffithsin is a potent inhibitor of MERS-CoV infection. Griffithsin also inhibits entry into host cells of particles pseudotyped with the MERS-CoV spike protein, suggesting that griffithsin inhibits spike protein function during entry. Spike proteins have a dual function during entry, they mediate binding to the host cell surface and also the fusion of the viral envelope with host cell membrane. Time course experiments show that griffithsin inhibits MERS-CoV infection at the binding step. In conclusion, we identify griffithsin as a potent inhibitor of MERS-CoV infection at the entry step.

•

We analyze the anti-MERS-CoV potential of the lectin griffithsin.

• Antiviral
• Lectin
• MERS-CoV
• Virus entry

• Hepatitis C virus
• Lipid-protein interaction
• Membrane fusion
• Protein spectroscopic properties
• Viral envelope proteins

• Carcinoma, Hepatocellular/immunology
• Carcinoma, Hepatocellular/metabolism
• Carcinoma, Hepatocellular/virology
• Cell Line, Tumor
• Cells, Cultured
• Claudins/genetics
• Claudins/immunology
• Claudins/metabolism
• Disease Resistance/genetics
• Disease Resistance/immunology
• Flow Cytometry
• HEK293 Cells
• Hep G2 Cells
• Hepacivirus/immunology
• Hepacivirus/physiology
• Hepatitis C/immunology
• Hepatitis C/metabolism
• Hepatitis C/virology
• Hepatocytes/immunology
• Hepatocytes/metabolism
• Hepatocytes/virology
• Host-Pathogen Interactions/immunology
• Humans
• Liver Neoplasms/immunology
• Liver Neoplasms/metabolism
• Liver Neoplasms/virology
• Microscopy, Fluorescence
• Mutation/immunology
• Occludin/genetics
• Occludin/immunology
• Occludin/metabolism
• Virion/immunology
• Virion/physiology

• Anthocyanins/administration & dosage
• Anthocyanins/pharmacology
• Antiviral Agents/administration & dosage
• Antiviral Agents/pharmacology
• Catechin/analogs & derivatives
• Catechin/pharmacology
• Cell Line
• Cryoelectron Microscopy
• Drug Evaluation, Preclinical
• HEK293 Cells
• Hepacivirus/drug effects
• Hepacivirus/physiology
• Hepacivirus/ultrastructure
• Hepatocytes/drug effects
• Hepatocytes/virology
• Humans
• Interferon-alpha/administration & dosage
• Polyphenols/administration & dosage
• Polyphenols/pharmacology
• Proline/administration & dosage
• Proline/analogs & derivatives
• Virus Internalization/drug effects

• HCV
• apolipoproteins
• cell polarity
• entry receptors
• glycoproteins
• lipoviralparticles
• membrane fusion
• tight junction
• very low-density lipoprotein particles
• viral entry

• Antiviral activity
• Claudin-1
• Hepatitis C Virus
• Sorafenib

• Baculovirus
• E1
• E2
• Envelope protein
• Glycosylation
• Hepatitis C virus

Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421–645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50’s ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.

• Amino Acid Sequence
• Antiviral Agents/chemistry
• Antiviral Agents/pharmacology
• Antiviral Agents/therapeutic use
• Binding Sites
• Cell Line, Tumor
• Cell Survival/drug effects
• Crystallography, X-Ray
• Genotype
• Hepacivirus/drug effects
• Hepacivirus/genetics
• Hepatitis C/drug therapy
• Hepatitis C/pathology
• Hepatitis C/virology
• Humans
• Kinetics
• Ligands
• Models, Molecular
• Molecular Sequence Data
• Protein Binding/drug effects
• Recombinant Proteins/metabolism
• Structural Homology, Protein
• Surface Plasmon Resonance
• Tetraspanin 28/metabolism
• Thermodynamics
• Viral Envelope Proteins/chemistry
• Viral Envelope Proteins/metabolism
• Virus Internalization/drug effects

• E2 ectodomain
• E2 glycoprotein
• Hepatitis C virus envelope proteins
• Protein structure

• Envelope glycoproteins
• Hepatitis C virus
• Membrane-associated proteins
• Nuclear magnetic resonance
• Protein structure
• Transmembrane helix

Hepatitis C virus (HCV) infection is a global health problem, with an estimated 170 million people being chronically infected. HCV cell entry is a complex multi-step process, involving several cellular factors that trigger virus uptake into the hepatocytes. The high- density lipoprotein receptor scavenger receptor class B type I, tetraspanin CD81, tight junction protein claudin-1, and occludin are the main receptors that mediate the initial step of HCV infection. In addition, the virus uses cell receptor tyrosine kinases as entry regulators, such as epidermal growth factor receptor and ephrin receptor A2. This review summarizes the current understanding about how cell surface molecules are involved in HCV attachment, internalization, and membrane fusion, and how host cell kinases regulate virus entry. The advances of the potential antiviral agents targeting this process are introduced.

• Hepatitis C virus
• Virus entry
• Hepatocytes
• Receptor
• Host kinase
• Antiviral target

• Animals
• Antiviral Agents/therapeutic use
• CD36 Antigens/metabolism
• Cell Membrane/virology
• Claudin-1/metabolism
• Endocytosis
• Endosomes
• Hepacivirus/physiology
• Hepatitis C/physiopathology
• Hepatocytes/virology
• Humans
• Liver/pathology
• Occludin/metabolism
• Phenylenediamines/pharmacology
• Receptor Protein-Tyrosine Kinases/metabolism
• Sulfonamides/pharmacology
• Tetraspanin 28/metabolism
• Virus Internalization

Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2. Their structure and mode of fusion remain unknown, and so does the virion architecture. The organization of the HCV envelope shell in particular is subject to discussion as it incorporates or associates with host-derived lipoproteins, to an extent that the biophysical properties of the virion resemble more very-low-density lipoproteins than of any virus known so far. The recent development of novel cell culture systems for HCV has provided new insights on the assembly of this atypical viral particle. Hence, the extensive E1E2 characterization accomplished for the last two decades in heterologous expression systems can now be brought into the context of a productive HCV infection. This review describes the biogenesis and maturation of HCV envelope glycoproteins, as well as the interplay between viral and host factors required for their incorporation in the viral envelope, in a way that allows efficient entry into target cells and evasion of the host immune response.

Envelope glycoproteins of Hepatitis C Virus (HCV) play an important role in the virus assembly and initial entry into host cells. Conserved charged residues of the E2 transmembrane (TM) domain were shown to be responsible for the heterodimerization with envelope glycoprotein E1. Despite intensive research on both envelope glycoproteins, the structural information is still not fully understood. Recent findings have revealed that the stem (ST) region of E2 also functions in the initial stage of the viral life cycle. We have previously shown the effect of the conserved charged residues on the TM helix monomer of E2. Here, we extended the model of the TM domain by adding the adjacent ST segment. Explicit molecular dynamics simulations were performed for the E2 amphiphilic segment of the ST region connected to the putative TM domain (residues 683-746). Structural conformation and behavior are studied and compared with the nuclear magnetic resonance (NMR)-derived segment of E2 ( 2KQZ.pdb). We observed that the central helix of the ST region (residues 689 - 703) remained stable as a helix in-plane to the lipid bilayer. Furthermore, the TM domain appeared to provide minimal contribution to the structural stability of the amphipathic region. This study also provides insight into the orientation and positional preferences of the ST segment with respect to the membrane lipid-water interface.

Hepatitis C Virus (HCV) infection is a global public health problem affecting over 160 million individuals worldwide. Its symptoms include chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped RNA virus mainly targeting liver cells and for which the initiation of infection occurs through a complex multistep process involving a series of specific cellular entry factors. This process is likely mediated through the formation of a tightly orchestrated complex of HCV entry factors at the plasma membrane. Among HCV entry factors, the tetraspanin CD81 is one of the best characterized and it is undoubtedly a key player in the HCV lifecycle. In this review, we detail the current knowledge on the involvement of CD81 in the HCV lifecycle, as well as in the immune response to HCV infection.

• Hepacivirus/physiology
• Host-Pathogen Interactions
• Humans
• Models, Biological
• Receptors, Virus/metabolism
• Tetraspanin 28/metabolism
• Virus Internalization

• hepatitis c
• antiviral lectin
• hcv entry inhibitor
• cyanovirin-n (cv-n)
• hcvcc
• biolayer interferometry

• Antiviral Agents/pharmacology
• Bacterial Proteins/pharmacology
• Carrier Proteins/pharmacology
• Cell Line, Tumor
• Cyanobacteria/metabolism
• Glycoproteins/metabolism
• Hepacivirus/drug effects
• Humans
• Mannose-Binding Lectins/pharmacology
• Microcystis/metabolism
• Plant Lectins/pharmacology
• Recombinant Proteins/pharmacology

• Z01 DK032103 Intramural NIH HHS
• ZIA DK032103 Intramural NIH HHS
• ZIA DK032103-13 Intramural NIH HHS

• hepacivirus
• hepatitis C virus
• neutralizing antibodies
• sequence variability
• viral glycoproteins

• Amino Acid Sequence
• Antibodies, Monoclonal/immunology
• Antibodies, Neutralizing/immunology
• Cell Line
• Epitopes/immunology
• Glycosylation
• Hepacivirus/genetics
• Hepacivirus/immunology
• Hepacivirus/metabolism
• Hepatitis C/immunology
• Hepatitis C/virology
• Hepatitis C Antibodies/immunology
• Humans
• Mutation
• Pakistan
• RNA, Viral/genetics
• Sequence Alignment
• Sequence Analysis, DNA
• Tetraspanin 28/immunology
• Viral Envelope Proteins/chemistry
• Viral Envelope Proteins/immunology
• Viral Envelope Proteins/metabolism

• 1,4-dithio-D-threitol
• 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol)
• 4,4-dimethyl-4-silapentane-1-sulfonic acid
• CD
• CPMG
• Carr–Purcell–Meiboom–Gil (pulse-train)
• DHPC
• DPC
• DSS
• DTT
• Envelope glycoproteins
• HCV
• HSQC
• Hepatitis C virus
• IPTG
• LPPG
• MBP
• MP
• Membrane-associated proteins
• NMR
• Nuclear magnetic resonance
• Protein structure
• SDS
• TEV
• TM
• Transmembrane helix
• circular dichroism
• dihexanoylphosphatidylcholine
• dodecyl-phosphocholine
• hepatitis C virus
• heteronuclear single-quantum coherence
• isopropyl β-D-1-thiogalactopyranoside
• maltose-binding protein
• membrane-embedded protein
• nuclear magnetic resonance
• sodium dodecylsulphate
• tobacco etch virus (protease)
• transmembrane domain

• Antigenicity
• CD81 binding
• Cysteine
• Disulfide bond
• E1E2 heterodimers
• Envelope protein 2
• HCVpp
• Hepatitis C virus

• Attachment
• Binding
• Cell culture
• Fetal bovine serum
• Hepatitis C virus
• Infection