Genetic recombination is an important evolutionary mechanism for RNA viruses and can facilitate escape from immune and drug pressure. Recombinant hepatitis C virus (HCV) variants have rarely been detected in patients, suggesting that HCV has intrinsic low recombination rate. Recombination of HCV has been demonstrated in vitro between non-functional genomes, but its frequency and relevance for viral evolution and life cycle has not been clarified. We developed a cell-based assay to detect and quantify recombination between fully viable HCV genomes, using the reconstitution of green fluorescent protein (GFP) as a surrogate marker for recombination. Here, two GFP-expressing HCV genomes carrying different inactivating GFP mutations can produce a virus carrying a functional GFP by recombining within the GFP region. Generated constructs allowed quantification of recombination rates between markers spaced 603 and 553 nucleotides apart by flow cytometry and next-generation sequencing (NGS). Viral constructs showed comparable spread kinetics and reached similar infectivity titers in Huh7.5 cells, allowing their use in co-transfections and co-infections. Single-cycle co-transfection experiments, performed in CD81-deficient S29 cells, showed GFP expression in double-infected cells, demonstrating genome mixing and occurrence of recombination. Quantification of recombinant genomes by NGS revealed an average rate of 6.1 per cent, corresponding to 49 per cent of maximum detectable recombination (MDR). Experiments examining recombination during the full replication cycle of HCV, performed in Huh7.5 cells, demonstrated average recombination rates of 5.0 per cent (40.0 per cent MDR) and 3.6 per cent (28.8 per cent MDR) for markers spaced by 603 and 553 nucleotides, respectively, supporting a linear relationship between marker distance and recombination rates. First passage infections using recombinant virus supernatant resulted in comparable recombination rates of 5.9 per cent (47.2 per cent MDR) and 3.5 per cent (28.0 per cent MDR), respectively, for markers spaced by 603 and 553 nucleotides. We developed a functional cell-based assay that, to the best of our knowledge, allows for the first time detailed quantification of recombination rates using fully viable HCV constructs. Our data indicate that HCV recombines at high frequency between highly similar genomes and that the frequency of recombination increases with the distance between marker sites. These results have implication for our understanding of HCV evolution and emphasize the importance of recombination in the reassortment of mutations in the HCV genome.

• COVID-19
• Challenges
• Hepatitis C virus
• Nucleic acid-based vaccines
• Recombinant vector-based vaccines
• Vaccine candidates

• B-cell vaccine
• antibody escape
• hepatitis C virus
• virus neutralization

• FLAG tag
• computational modeling
• epitope imprinting
• hierarchically imprinted polymer
• silane grafting

• Epitopes/chemistry
• Molecular Imprinting
• Molecular Structure
• Oligopeptides/analysis
• Particle Size
• Polymers/chemistry
• Silicon Dioxide/chemistry
• Surface Properties

• Antibodies, Neutralizing/immunology
• Antibodies, Viral/immunology
• Computer Simulation
• Epitopes/immunology
• Glycosylation
• HEK293 Cells
• Hepatitis C/virology
• Humans
• Molecular Dynamics Simulation
• Protein Binding
• Protein Domains
• Scattering, Radiation
• Viral Envelope Proteins/chemistry
• Virus Internalization
• X-Rays

• Wellcome Trust
• 107653/Z/15/Z Wellcome Trust
• 109162/Z/15/Z Wellcome Trust

• Amino Acid Sequence
• Base Sequence
• Carrier Proteins/genetics
• Carrier Proteins/metabolism
• Cell Line
• Hepacivirus/genetics
• Hepacivirus/metabolism
• Hepacivirus/physiology
• Hepatitis C, Chronic/virology
• Humans
• Intracellular Signaling Peptides and Proteins
• Mutation
• Protein Domains
• RNA, Viral
• Viral Envelope Proteins/genetics
• Viral Envelope Proteins/metabolism
• Viral Nonstructural Proteins/genetics
• Viral Nonstructural Proteins/metabolism
• Virion/metabolism
• Virion/physiology
• Virus Assembly
• Virus Replication

• R01 AI125416 NIAID NIH HHS
• T32 CA009111 NCI NIH HHS
• R21 AI129851 NIAID NIH HHS
• P30 AI064518 NIAID NIH HHS
• F31 AI131477 NIAID NIH HHS

• Antibodies
• HCV
• Hepatitis C virus
• Immunostaining
• Neutralization
• Vaccine

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

• Cell culture
• Chimeric HCV
• DAA
• Direct acting antivirals
• Full-length HCV
• HCV
• HCV treatment in vitro
• HCV vaccine
• HCVcc
• Hepatitis C virus
• In vitro
• Neutralizing antibodies
• Pseudo-particle
• Replicon

Hepatitis C virus (HCV) is a globally disseminated human pathogen for which no vaccine is currently available. HCV is highly diverse genetically and can be classified into 7 genotypes and multiple sub-types. Due to this antigenic variation, the induction of cross-reactive and at the same time neutralizing antibodies is a challenge in vaccine production. Here we report the analysis of immunogenicity of recombinant HCV envelope glycoproteins from genotypes 1a, 1b and 2a, with a Flag tag inserted in the hypervariable region 1 of E2. This modification did not affect protein expression or conformation or its capacity to bind the crucial virus entry factor, CD81. Importantly, in immunogenicity studies on mice, the purified E2-Flag mutants elicited high-titer, cross-reactive antibodies that were able to neutralize HCV infectious particles from two genotypes tested (1a and 2a). These findings indicate that E1E2-Flag envelope glycoproteins could be important immunogen candidates for vaccine aiming to induce broad HCV-neutralizing responses.

• E1E2
• Envelope glycoproteins
• Flag tag
• Hepatitis C virus
• Neutralization
• Vaccine

• Flaviviridae
• antiviral screening
• in vivo dynamics
• reporter virus

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

• envelope glycoproteins
• epitopes
• hepatitis C virus
• neutralizing antibodies
• vaccines

Evidence is accumulating that retroviruses can produce microRNAs (miRNAs). To prevent cleavage of their RNA genome, retroviruses have to use an alternative RNA source as miRNA precursor. The transacting responsive (TAR) hairpin structure in HIV-1 RNA has been suggested as source for miRNAs, but how these small RNAs are produced without impeding virus replication remained unclear. We used deep sequencing analysis of AGO2-bound HIV-1 RNAs to demonstrate that the 3′ side of the TAR hairpin is processed into a miRNA-like small RNA. This ∼21 nt RNA product is able to repress the expression of mRNAs bearing a complementary target sequence. Analysis of the small RNAs produced by wild-type and mutant HIV-1 variants revealed that non-processive transcription from the HIV-1 LTR promoter results in the production of short TAR RNAs that serve as precursor. These TAR RNAs are cleaved by Dicer and processing is stimulated by the viral Tat protein. This biogenesis pathway differs from the canonical miRNA pathway and allows HIV-1 to produce the TAR-encoded miRNA-like molecule without cleavage of the RNA genome.

• 3A
• Foot-and-mouth disease virus
• Foreign epitope

• Amino Acid Sequence
• Animals
• Cattle
• Cell Line
• Epitopes/chemistry
• Epitopes/genetics
• Epitopes/immunology
• Foot-and-Mouth Disease/immunology
• Foot-and-Mouth Disease/virology
• Foot-and-Mouth Disease Virus/genetics
• Foot-and-Mouth Disease Virus/immunology
• Gene Expression
• Genetic Engineering
• Genome, Viral
• Molecular Sequence Data
• Viral Nonstructural Proteins/chemistry
• Viral Nonstructural Proteins/genetics
• Viral Nonstructural Proteins/immunology
• Viral Plaque Assay
• Virus Replication

Pairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains.

Our insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability. We have also generated complementary resources, including a website (http://hangfei.bol.ucla.edu) and a panel of epitope-tagged mutant viruses that should enhance the research capabilities of investigators studying HCV. Researchers can now detect epitope-tagged viral proteins by established antibodies, which will allow biochemical studies of HCV proteins for which antibodies are not readily available. Furthermore, researchers can now quickly look up genotype-phenotype relationships and base further mechanistic studies on the residue-by-residue information from the functional profile. More broadly, this approach offers a general strategy for the systematic functional characterization of viruses on the genome scale.

• Animals
• Antibodies, Viral
• Blotting, Western
• Capsid Proteins/metabolism
• Enzyme-Linked Immunosorbent Assay
• Epitopes
• Foot-and-Mouth Disease/prevention & control
• Foot-and-Mouth Disease/virology
• Foot-and-Mouth Disease Virus/classification
• Models, Molecular
• Protein Conformation
• RNA, Viral
• Staining and Labeling
• Vaccines, DNA
• Vaccines, Inactivated
• Viral Vaccines/immunology

• BBS/E/I/00001442 Biotechnology and Biological Sciences Research Council
• G1100525 Medical Research Council
• 089755 Wellcome Trust
• 090532 Wellcome Trust
• Wellcome Trust
• G1000099 Medical Research Council

Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447_Δc), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447_Δc. Upon infection of the natural host, Vp447_Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general.

Virus particles of members of the Flaviviridae consist of an inner complex of viral RNA genome and core protein that together form the nucleocapsid, and an outer lipid layer containing the viral glycoproteins. Functional analyses of core protein of the classical swine fever virus (CSFV), a pestivirus related to hepatitis C virus (HCV), led to the observation that crippling mutations or even complete deletion of the core gene were compensated by single amino acid substitutions in the helicase domain of non-structural protein 3 (NS3). NS3 is well conserved among the Flaviviridae and acts as protease and helicase. In addition to its essential role in RNA replication, NS3 apparently organizes the incorporation of RNA into budding virus particles. Characterization of core deficient CSFV particles (Vp447_Δc) revealed that the lack of core had no effect with regard to thermostability, size, density, and morphology. Vp447_Δc was fully attenuated in the natural host. Our results provide evidence that core protein is not essential for virus assembly. Hence, Vp447_Δc might help to explain the enigmatic existence of GB viruses -A and -C, close relatives of HCV that do not encode an apparent core protein.

• Animals
• Cell Line
• Classical Swine Fever/blood
• Classical Swine Fever/virology
• Classical Swine Fever Virus/pathogenicity
• Classical Swine Fever Virus/physiology
• Disease Models, Animal
• Host-Pathogen Interactions
• Swine
• Viral Core Proteins/physiology
• Viral Nonstructural Proteins/physiology
• Virulence
• Virus Replication