TMPRSS2-mediated SARS-CoV-2 uptake boosts innate immune activation, enhances cytopathology, and drives convergent virus evolution.

The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models.

Reconstruction of the historic time course of blood-borne virus contamination of clotting factor concentrates, 1974-1992.

Factor VIII and IX clotting factor concentrates manufactured from pooled plasma have been identified as potent sources of virus infection in persons with hemophilia (PWHs) in the 1970s and 1980s. To investigate the range and diversity of viruses over this period, we analysed 24 clotting factor concentrates for several blood-borne viruses. Nucleic acid was extracted from 14 commercially produced clotting factors and 10 from nonremunerated donors, preserved in lyophilized form (expiry dates: 1974-1992). Clotting factors were tested by commercial and in-house quantitative PCRs for blood-borne viruses hepatitis A, B, C and E viruses (HAV, HBV, HCV, HEV), HIV- types 1/2, parvoviruses B19V and PARV4, and human pegiviruses types 1 and 2 (HPgV-1,-2). HCV and HPgV-1 were the most frequently detected viruses (both 14/24 tested) primarily in commercial clotting factors, with frequently extremely high viral loads in the late 1970s-1985 and a diverse range of HCV genotypes. Detection frequencies sharply declined following introduction of virus inactivation. HIV-1, HBV, and HAV were less frequently detected (3/24, 1/24, and 1/24 respectively); none were positive for HEV. Contrastingly, B19V and PARV4 were detected throughout the study period, even after introduction of dry heat treatment, consistent with ongoing documented transmission to PWHs into the early 1990s. While hemophilia treatment is now largely based on recombinant factor VIII/IX in the UK and elsewhere, the comprehensive screen of historical plasma-derived clotting factors reveals extensive exposure of PWHs to blood-borne viruses throughout 1970s-early 1990s, and the epidemiological and manufacturing parameters that influenced clotting factor contamination.

An Antigenically Diverse, Representative Panel of Envelope Glycoproteins for Hepatitis C Virus Vaccine Development.

BACKGROUND & AIMS: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays. METHODS: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps. RESULTS: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones. CONCLUSIONS: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.

Scavenger receptor class B type I genetic variants associated with disease severity in chronic hepatitis C virus infection.

Analysis of host genetic polymorphisms is an increasingly important tool for understanding and predicting pathogenesis and treatment response of viral diseases. The gene locus of scavenger receptor class B type I (SR-BI), encoding a cell entry factor and receptor for hepatitis C virus (HCV), contains several genetic polymorphisms. We applied a probe extension assay to determine the frequency of six single nucleotide polymorphisms (SNPs) within the SR-BI gene locus in 374 individuals with history of HCV infection. In addition, SR-BI messenger RNA (mRNA) levels were analyzed in liver biopsy specimens of chronically infected HCV subjects. The rs5888 variant allele T was present at a higher frequency in subjects with advanced fibrosis (χ2 , p = 0.016) and after adjusting for age, duration of infection and alcohol intake as confounding factors. Haplotype analysis of SNP frequencies showed that a haplotype consisting of rs61932577 variant allele C and rs5888 variant allele T was associated with an increased risk of advanced liver fibrosis (defined by an Ishak score 4-6) (adjusted odds ratio 2.81; 95% confidence interval 1.06-7.46. p = 0.038). Carriers of the rs5888 variant allele T displayed reduced SR-BI mRNA expression in liver biopsy specimens. In conclusion the rs5888 polymorphism variant is associated with decreased SR-BI expression and an increased risk of development of advanced fibrosis in chronic HCV infection. These findings provide further evidence for a role of SR-BI in HCV pathogenesis and provides a genetic marker for prediction of those infected individuals at greater risk of developing severe disease.

Hepatitis C subtyping assay failure in UK patients born in sub-Saharan Africa: Implications for global treatment and elimination.

BACKGROUND AND AIMS: The newly developed direct-acting antivirals have revolutionized the treatment of chronic hepatitis C virus (HCV), with cure rates as high as 98% in some cohorts. Although genome sequencing has demonstrated that some subtypes of HCV naturally harbor drug resistance associated substitutions (RAS), these are often overlooked as "rarities." Furthermore, commercial subtyping assays and associated epidemiological findings are skewed towards Western cohorts and whole-genome sequencing can be problematic to deploy without significant infrastructure and training support. We thus aimed to develop a simple, robust and accurate HCV subtyping pipeline, to optimize and streamline molecular detection and sequence-based typing of diverse RAS-containing subtypes. METHODS: HCV serum derived from 146 individuals, whose likely source of infection was from sub-Saharan Africa (SSA) was investigated with a novel panel of single round polymerase chain reaction (PCR) assays targeting NS5B and NS5A genomic regions. Virus subtype assignments were determined by pairwise-distance analysis and compared to both diagnostic laboratory assignments and free-to-use online typing tools. RESULTS: Partial NS5A and NS5B sequences were respectively obtained from 131 to 135 HCV-positive patients born in 19 different countries from SSA but attending clinics in the UK. We determined that routine clinical diagnostic methods incorrectly subtyped 59.0% of samples, with a further 6.8% incorrectly genotyped. Of five commonly used online tools, Geno2Pheno performed most effectively in determining a subtype in agreement with pairwise distance analysis. CONCLUSION: This study provides a simple low-cost pathway to accurately subtype in SSA, guide regional therapeutic choice and assist global surveillance and elimination initiatives.

Increasing SARS-CoV-2 seroprevalence among UK pediatric patients on dialysis and kidney transplantation between January 2020 and August 2021.

BACKGROUND: Coronavirus disease 2019 (COVID-19) was officially declared a pandemic by the World Health Organisation (WHO) on 11 March 2020, as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread rapidly across the world. We investigated the seroprevalence of anti-SARS-CoV-2 antibodies in pediatric patients on dialysis or kidney transplantation in the UK. METHODS: Excess sera samples were obtained prospectively during outpatient visits or haemodialysis sessions and analysed using a custom immunoassay calibrated with population age-matched healthy controls. Two large pediatric centres contributed samples. RESULTS: In total, 520 sera from 145 patients (16 peritoneal dialysis, 16 haemodialysis, 113 transplantation) were analysed cross-sectionally from January 2020 until August 2021. No anti-SARS-CoV-2 antibody positive samples were detected in 2020 when lockdown and enhanced social distancing measures were enacted. Thereafter, the proportion of positive samples increased from 5% (January 2021) to 32% (August 2021) following the emergence of the Alpha variant. Taking all patients, 32/145 (22%) were seropositive, including 8/32 (25%) with prior laboratory-confirmed SARS-CoV-2 infection and 12/32 (38%) post-vaccination (one of whom was also infected after vaccination). The remaining 13 (41%) seropositive patients had no known stimulus, representing subclinical cases. Antibody binding signals were comparable across patient ages and dialysis versus transplantation and highest against full-length spike protein versus spike subunit-1 and nucleocapsid protein. CONCLUSIONS: Anti-SARS-CoV-2 seroprevalence was low in 2020 and increased in early 2021. Serological surveillance complements nucleic acid detection and antigen testing to build a greater picture of the epidemiology of COVID-19 and is therefore important to guide public health responses. A higher resolution version of the Graphical abstract is available as Supplementary information.

Serum Levels of Proinflammatory Lipid Mediators and Specialized Proresolving Molecules Are Increased in Patients With Severe Acute Respiratory Syndrome Coronavirus 2 and Correlate With Markers of the Adaptive Immune Response.

BACKGROUND: Specialized proresolution molecules (SPMs) halt the transition to chronic pathogenic inflammation. We aimed to quantify serum levels of pro- and anti-inflammatory bioactive lipids in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients, and to identify potential relationships with innate responses and clinical outcome. METHODS: Serum from 50 hospital admitted inpatients (22 female, 28 male) with confirmed symptomatic SARS-CoV-2 infection and 94 age- and sex-matched controls collected prior to the pandemic (SARS-CoV-2 negative), were processed for quantification of bioactive lipids and anti-nucleocapsid and anti-spike quantitative binding assays. RESULTS: SARS-CoV-2 serum had significantly higher concentrations of omega-6-derived proinflammatory lipids and omega-6- and omega-3-derived SPMs, compared to the age- and sex-matched SARS-CoV-2-negative group, which were not markedly altered by age or sex. There were significant positive correlations between SPMs, proinflammatory bioactive lipids, and anti-spike antibody binding. Levels of some SPMs were significantly higher in patients with an anti-spike antibody value >0.5. Levels of linoleic acid and 5,6-dihydroxy-8Z,11Z,14Z-eicosatrienoic acid were significantly lower in SARS-CoV-2 patients who died. CONCLUSIONS: SARS-CoV-2 infection was associated with increased levels of SPMs and other pro- and anti-inflammatory bioactive lipids, supporting the future investigation of the underlying enzymatic pathways, which may inform the development of novel treatments.

Sero-reactivity to three distinct regions within the hepatitis C virus alternative reading frame protein (ARFP/core+1) in patients with chronic HCV genotype-3 infection.

Hepatitis C virus (HCV) infection affects more than 71 million people worldwide. The disease slowly progresses to chronic, long-term liver injury which leads to hepatocellular carcinoma (HCC) in 5 % of infections. The alternative reading frame protein (ARFP/core+1) is encoded by a sequence overlapping the HCV core gene in the +1 reading frame. Its role in hepatitis C pathogenesis and the viral life cycle is unclear, although some observers have related its production to disease progression and the development of HCC. The aim of this study was to determine whether ARFP is immunogenic in patients with chronic HCV genotype 3 infection and to assess whether sero-reactivity is associated with disease progression, particularly to HCC. Immunogenic epitopes within the protein were predicted by a bioinformatics tool, and three -20 aa length-peptides (ARFP-P1, ARFP-P2 and ARFP-P3) were synthesized and used in an avidin-biotin ARFP/core+1 peptide ELISA. Serum samples from 50 patients with chronic HCV genotype 3 infection, 50 genotype-1 patients, 50 HBV patients and 110 healthy controls were tested. Sero-reactivity to the ARFP peptides was also tested and compared in 114 chronic HCV genotype-3 patients subdivided on the basis of disease severity into non-cirrhotic, cirrhotic and HCC groups. Chronic HCV genotype-3 patients showed noticeable rates of reactivity to ARFP and core peptides. Seropositivity rates were 58% for ARFP-P1, 47 % for ARFP-P2, 5.9 % for ARFP-P3 and 100 % for C22 peptides. There was no significant difference between these seroreactivities between HCV genotype-3 patients with HCC, and HCV genotype-3 patients with and without liver cirrhosis. Patients with chronic HCV genotype-3 infection frequently produce antibodies against ARFP/core+1 protein. ARFP peptide reactivity was not associated with disease severity in patients with HCV genotype-3. These results support the conclusion that ARFP/core+1 is produced during HCV infection, but they do not confirm that antibodies to ARFP can indicate HCV disease progression.

Optimization of the pseudoparticle system for standardized assessments of neutralizing antibodies against hepatitis C virus.

A better understanding of the antibody response during natural infection and the effect on disease progression and reinfection is necessary for the development of a protective hepatitis C virus (HCV) vaccine. The HCV pseudoparticle (HCVpp) system enables the study of viral entry and inhibition by antibody neutralization. A robust and comparable neutralization assay is crucial for the development and evaluation of experimental vaccines.With the aim of optimizing the HCVpp-murine leukaemia virus (MLV) system, we tested the neutralization of HCVpp-harbouring E1E2 from 21 HCV isolates representing 6 different genotypes by several monoclonal antibodies (mAbs). HCVpps are generated by expressing functional envelope glycoproteins (E1E2) onto pseudoparticles derived from env-deleted MLV. Adjustments of E1E2, gag-pol and luciferase plasmid ratios resulted in increased yields for most HCVpps and recovery of one non-infectious HCVpp. We simplified and improved the protocol to achieve higher signal/noise ratios and minimized the amount of HCVpps and mAbs needed for the detection of neutralization. Using our optimized protocol, we demonstrated comparable results to previously reported data with both diluted and freeze-thawed HCVpps.In conclusion, we successfully established a simplified and reproducible HCVpp neutralization protocol for studying a wide range of HCV variants. This simplified protocol provides highly consistent results and could be easily adopted by others to evaluate precious biological material. This will contribute to a better understanding of the antibody response during natural infection and help evaluate experimental HCV vaccines.

Interferon-Induced Transmembrane Proteins Mediate Viral Evasion in Acute and Chronic Hepatitis C Virus Infection.

Although adaptive immune responses against hepatitis C virus (HCV) infection have been studied in great detail, the role of innate immunity in protection against HCV infection and immune evasion is only partially understood. Interferon-induced transmembrane proteins (IFITMs) are innate effector proteins restricting host cell entry of many enveloped viruses, including HCV. However, the clinical impact of IFITMs on HCV immune escape remains to be determined. Here, we show that IFITMs promote viral escape from the neutralizing antibody (nAb) response in clinical cohorts of HCV-infected patients. Using pseudoparticles bearing HCV envelope proteins from acutely infected patients, we show that HCV variants isolated preseroconversion are more sensitive to the antiviral activity of IFITMs than variants from patients isolated during chronic infection postseroconversion. Furthermore, HCV variants escaping nAb responses during liver transplantation exhibited a significantly higher resistance to IFITMs than variants that were eliminated posttransplantation. Gain-of-function and mechanistic studies revealed that IFITMs markedly enhance the antiviral activity of nAbs and suggest a cooperative effect of human monoclonal antibodies and IFITMs for antibody-mediated neutralization driving the selection pressure in viral evasion. Perturbation studies with the IFITM antagonist amphotericin B revealed that modulation of membrane properties by IFITM proteins is responsible for the IFITM-mediated blockade of viral entry and enhancement of antibody-mediated neutralization. Conclusion: Our results indicate IFITM proteins as drivers of viral immune escape and antibody-mediated HCV neutralization in acute and chronic HCV infection. These findings are of clinical relevance for the design of urgently needed HCV B-cell vaccines and might help to increase the efficacy of future vaccine candidates.

Cross-genotype AR3-specific neutralizing antibodies confer long-term protection in injecting drug users after HCV clearance.

BACKGROUND & AIMS: In order to design an effective vaccine against hepatitis C virus (HCV) infection, it is necessary to understand immune protection. A number of broadly reactive neutralizing antibodies have been isolated from B cells of HCV-infected patients. However, it remains unclear whether B cells producing such antibodies contribute to HCV clearance and long-term immune protection against HCV. METHODS: We analysed the B cell repertoire of 13 injecting drug users from the Amsterdam Cohort Study, who were followed up for a median of 17.5 years after primary infection. Individuals were classified into 2 groups based on the outcome of HCV infection: 5 who became chronically infected either after primary infection or after reinfection, and 8 who were HCV RNA negative following spontaneous clearance of ≥1 HCV infection(s). From each individual, 10,000 CD27+IgG+B cells, collected 0.75 year after HCV infection, were cultured to characterize the antibody repertoire. RESULTS: Using a multiplex flow cytometry-based assay to study the antibody binding to E1E2 from genotype 1 to 6, we found that a high frequency of cross-genotype antibodies was associated with spontaneous clearance of 1 or multiple infections (p = 0.03). Epitope specificity of these cross-genotype antibodies was determined by alanine mutant scanning in 4 individuals who were HCV RNA negative following spontaneous clearance of 1 or multiple infections. Interestingly, the cross-genotype antibodies were mainly antigenic region 3 (AR3)-specific and showed cross-neutralizing activity against HCV. In addition to AR3 antibodies, 3 individuals developed antibodies recognizing antigenic region 4, of which 1 monoclonal antibody showed cross-neutralizing capacity. CONCLUSIONS: Together, these data suggest that a strong B cell response producing cross-genotype and neutralizing antibodies, especially targeting AR3, contributes to HCV clearance and long-term immune protection against HCV. LAY SUMMARY: Although effective treatments against hepatitis C virus (HCV) are available, 500,000 people die from liver disease caused by HCV each year and approximately 1.75 million people are newly infected. This could be prevented by a vaccine. To design a vaccine against HCV, more insight into the role of antibodies in the protection against HCV infection is needed. In a cohort of injecting drug users, we found that antibodies interfering with virus cell entry, and recognizing multiple HCV genotypes, conferred long-term protection against chronic HCV infection.

Functional and immunogenic characterization of diverse HCV glycoprotein E2 variants.

BACKGROUND & AIMS: Induction of cross-reactive antibodies targeting conserved epitopes of the envelope proteins E1E2 is a key requirement for an hepatitis C virus vaccine. Conserved epitopes like the viral CD81-binding site are targeted by rare broadly neutralizing antibodies. However, these viral segments are occluded by variable regions and glycans. We aimed to identify antigens exposing conserved epitopes and to characterize their immunogenicity. METHODS: We created hepatitis C virus variants with mutated glycosylation sites and/or hypervariable region 1 (HVR1). Exposure of the CD81 binding site and conserved epitopes was quantified by soluble CD81 and antibody interaction and neutralization assays. E2 or E1-E2 heterodimers with mutations causing epitope exposure were used to immunize mice. Vaccine-induced antibodies were examined and compared with patient-derived antibodies. RESULTS: Mutant viruses bound soluble CD81 and antibodies targeting the CD81 binding site with enhanced efficacy. Mice immunized with E2 or E1E2 heterodimers incorporating these modifications mounted strong, cross-binding, and non-interfering antibodies. E2-induced antibodies neutralized the autologous virus but they were not cross-neutralizing. CONCLUSIONS: Viruses lacking the HVR1 and selected glycosylation sites expose the CD81 binding site and cross-neutralization antibody epitopes. Recombinant E2 proteins carrying these modifications induce strong cross-binding but not cross-neutralizing antibodies. LAY SUMMARY: Conserved viral epitopes can be made considerably more accessible for binding of potently neutralizing antibodies by deletion of hypervariable region 1 and selected glycosylation sites. Recombinant E2 proteins carrying these mutations are unable to elicit cross-neutralizing antibodies suggesting that exposure of conserved epitopes is not sufficient to focus antibody responses on production of cross-neutralizing antibodies.

High resolution sequencing of hepatitis C virus reveals limited intra-hepatic compartmentalization in end-stage liver disease.

BACKGROUND & AIMS: The high replication and mutation rate of hepatitis C virus (HCV) results in a heterogeneous population of viral sequences in vivo. HCV replicates in the liver and infected hepatocytes occur as foci surrounded by uninfected cells that may promote compartmentalization of viral variants. Given recent reports showing interferon stimulated gene (ISG) expression in chronic hepatitis C, we hypothesized that local interferon responses may limit HCV replication and evolution. METHODS: To investigate the spatial influence of liver architecture on viral replication we measured HCV RNA and ISG mRNA from each of the 8 Couinaud segments of the liver from 21 patients undergoing liver transplant. RESULTS: HCV RNA and ISG mRNA levels were comparable across all sites from an individual liver but showed up to 500-fold difference between patients. Importantly, there was no association between ISG and HCV RNA expression across all sites in the liver or plasma. Deep sequencing of HCV RNA isolated from the 8 hepatic sites from two subjects showed a similar distribution of viral quasispecies across the liver and uniform sequence diversity. Single genome amplification of HCV E1E2-envelope clones from 6 selected patients at 2 hepatic sites supported these data and showed no evidence for HCV compartmentalization. CONCLUSIONS: We found no differences between the hepatic and plasma viral quasispecies in all patients sampled. We conclude that in end-stage liver disease HCV RNA levels and the genetic pool of HCV envelope sequences are indistinguishable between distant sites in the liver and plasma, arguing against viral compartmentalization. LAY SUMMARY: HCV is an RNA virus that exists as a quasispecies of closely related genomes that are under continuous selection by host innate and adaptive immune responses and antiviral drug therapy. The primary site of HCV replication is the liver and yet our understanding of the spatial distribution of viral variants within the liver is limited. High resolution sequencing of HCV and monitoring of innate immune responses at multiple sites across the liver identified a uniform pattern of diversity and argues against viral compartmentalization.

Novel functional hepatitis C virus glycoprotein isolates identified using an optimized viral pseudotype entry assay.

Retrovirus pseudotypes are a highly tractable model used to study the entry pathways of enveloped viruses. This model has been extensively applied to the study of the hepatitis C virus (HCV) entry pathway, preclinical screening of antiviral antibodies and for assessing the phenotype of patient-derived viruses using HCV pseudoparticles (HCVpp) possessing the HCV E1 and E2 glycoproteins. However, not all patient-isolated clones produce particles that are infectious in this model. This study investigated factors that might limit phenotyping of patient-isolated HCV glycoproteins. Genetically related HCV glycoproteins from quasispecies in individual patients were discovered to behave very differently in this entry model. Empirical optimization of the ratio of packaging construct and glycoprotein-encoding plasmid was required for successful HCVpp genesis for different clones. The selection of retroviral packaging construct also influenced the function of HCV pseudoparticles. Some glycoprotein constructs tolerated a wide range of assay parameters, while others were much more sensitive to alterations. Furthermore, glycoproteins previously characterized as unable to mediate entry were found to be functional. These findings were validated using chimeric cell-cultured HCV bearing these glycoproteins. Using the same empirical approach we demonstrated that generation of infectious ebolavirus pseudoviruses (EBOVpv) was also sensitive to the amount and ratio of plasmids used, and that protocols for optimal production of these pseudoviruses are dependent on the exact virus glycoprotein construct. These findings demonstrate that it is crucial for studies utilizing pseudoviruses to conduct empirical optimization of pseudotype production for each specific glycoprotein sequence to achieve optimal titres and facilitate accurate phenotyping.

Flexible and rapid construction of viral chimeras applied to hepatitis C virus.

A novel and broadly applicable strategy combining site-directed mutagenesis and DNA assembly for constructing seamless viral chimeras is described using hepatitis C virus (HCV) as an exemplar. Full-length HCV genomic cloning cassettes, which contained flexibly situated restriction endonuclease sites, were prepared via a single, site-directed mutagenesis reaction and digested to receive PCR-amplified virus envelope genes by In-Fusion cloning. Using this method, we were able to construct gene-shuttle cassettes for generation of cell culture-infectious JFH-1-based chimeras containing genotype 1-3 E1E2 genes. Importantly, using this method we also show that E1E2 clones that were not able to support cell entry in the HCV pseudoparticle assay did confer entry when shuttled into the chimeric cell culture chimera system. This method can be easily applied to other genes of study and other viruses and, as such, will greatly simplify reverse genetics studies of variable viruses.

A Diverse Panel of Hepatitis C Virus Glycoproteins for Use in Vaccine Research Reveals Extremes of Monoclonal Antibody Neutralization Resistance.

UNLABELLED: Despite significant advances in the treatment of hepatitis C virus (HCV) infection, the need to develop preventative vaccines remains. Identification of the best vaccine candidates and evaluation of their performance in preclinical and clinical development will require appropriate neutralization assays utilizing diverse HCV isolates. We aimed to generate and characterize a panel of HCV E1E2 glycoproteins suitable for subsequent use in vaccine and therapeutic antibody testing. Full-length E1E2 clones were PCR amplified from patient-derived serum samples, cloned into an expression vector, and used to generate viral pseudoparticles (HCVpp). In addition, some of these clones were used to generate cell culture infectious (HCVcc) clones. The infectivity and neutralization sensitivity of these viruses were then determined. Bioinformatic and HCVpp infectivity screening of approximately 900 E1E2 clones resulted in the assembly of a panel of 78 functional E1E2 proteins representing distinct HCV genotypes and different stages of infection. These HCV glycoproteins differed markedly in their sensitivity to neutralizing antibodies. We used this panel to predict antibody efficacy against circulating HCV strains, highlighting the likely reason why some monoclonal antibodies failed in previous clinical trials. This study provides the first objective categorization of cross-genotype patient-derived HCV E1E2 clones according to their sensitivity to antibody neutralization. It has shown that HCV isolates have clearly distinguishable neutralization-sensitive, -resistant, or -intermediate phenotypes, which are independent of genotype. The panel provides a systematic means for characterization of the neutralizing response elicited by candidate vaccines and for defining the therapeutic potential of monoclonal antibodies. IMPORTANCE: Hepatitis C virus (HCV) has a global burden of more than 170 million people, many of whom cannot attain the new, expensive, direct-acting antiviral therapies. A safe and effective vaccine that generates both T cell responses and neutralizing antibodies is required to eradicate the disease. Regions within the HCV surface glycoproteins E1 and E2 are essential for virus entry and are targets for neutralizing antibodies. Screening of vaccine candidates requires suitable panels of glycoproteins that represent the breadth of neutralization resistance. Use of a standard reference panel for vaccine studies will ensure comparability of data sets, as has become routine for HIV-1. Here, we describe a large panel of patient-derived HCV glycoproteins with an assessment of their neutralization sensitivity to defined monoclonal antibodies, which has enabled us to predict their likely efficacy in the wider HCV-infected population. The panel could also be important for future selection of additional therapeutic antibodies and for vaccine design.

Structural flexibility of a conserved antigenic region in hepatitis C virus glycoprotein E2 recognized by broadly neutralizing antibodies.

UNLABELLED: Neutralizing antibodies (NAbs) targeting glycoprotein E2 are important for the control of hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412 to 423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a ß-hairpin in complex with three independent NAbs. Our structure of the same peptide in complex with NAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412 to 423 are essential for virus entry but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab fragment, this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and ß-hairpin conformations, respectively) display similar neutralizing activities despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as an immune evasion strategy, contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and nonenveloped viruses. IMPORTANCE: Approximately 180 million people worldwide are infected with hepatitis C virus (HCV), and neutralizing antibodies play an important role in controlling the replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412 to 423), which is disordered in the recently reported crystal structure of an E2 core fragment, can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activities in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than those targeting other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic region contributes to the evasion of the humoral host immune response, facilitating chronicity and the viral spread of HCV within an infected individual.

The P body protein LSm1 contributes to stimulation of hepatitis C virus translation, but not replication, by microRNA-122.

The P body protein LSm1 stimulates translation and replication of hepatitis C virus (HCV). As the liver-specific microRNA-122 (miR-122) is required for HCV replication and is associated with P bodies, we investigated whether regulation of HCV by LSm1 involves miR-122. Here, we demonstrate that LSm1 contributes to activation of HCV internal ribosome entry site (IRES)-driven translation by miR-122. This role for LSm1 is specialized for miR-122 translation activation, as LSm1 depletion does not affect the repressive function of miR-122 at 3' untranslated region (UTR) sites, or miR-122-mediated cleavage at a perfectly complementary site. We find that LSm1 does not influence recruitment of the microRNA (miRNA)-induced silencing complex to the HCV 5'UTR, implying that it regulates miR-122 function subsequent to target binding. In contrast to the interplay between miR-122 and LSm1 in translation, we find that LSm1 is not required for miR-122 to stimulate HCV replication, suggesting that miR-122 regulation of HCV translation and replication have different requirements. For the first time, we have identified a protein factor that specifically contributes to activation of HCV IRES-driven translation by miR-122, but not to other activities of the miRNA. Our results enhance understanding of the mechanisms by which miR-122 and LSm1 regulate HCV.

Dramatic potentiation of the antiviral activity of HIV antibodies by cholesterol conjugation.

The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane-proximal external region of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization; first, they bind and preconcentrate at the viral membrane through their long, hydrophobic CDRH3 loops, and second, they form a high affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol-conjugated antibodies bind to lipid raft domains on the membrane, and because of this enrichment, they show increased antiviral potency. In particular, we find that cholesterol conjugation (i) rescues the antiviral activity of CDRH3-mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane-binding HIV antibody D5 10-100-fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears to be a general strategy to boost the potency of antiviral antibodies, and, because membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies.

Colicin import into E. coli cells: a model system for insights into the import mechanisms of bacteriocins.

Bacteriocins are a diverse group of ribosomally synthesized protein antibiotics produced by most bacteria. They range from small lanthipeptides produced by lactic acid bacteria to much larger multi domain proteins of Gram negative bacteria such as the colicins from Escherichia coli. For activity bacteriocins must be released from the producing cell and then bind to the surface of a sensitive cell to instigate the import process leading to cell death. For over 50years, colicins have provided a working platform for elucidating the structure/function studies of bacteriocin import and modes of action. An understanding of the processes that contribute to the delivery of a colicin molecule across two lipid membranes of the cell envelope has advanced our knowledge of protein-protein interactions (PPI), protein-lipid interactions and the role of order-disorder transitions of protein domains pertinent to protein transport. In this review, we provide an overview of the arrangement of genes that controls the synthesis and release of the mature protein. We examine the uptake processes of colicins from initial binding and sequestration of binding partners to crossing of the outer membrane, and then discuss the translocation of colicins through the cell periplasm and across the inner membrane to their cytotoxic site of action. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.