Direct Infection and Replication of Naturally Occurring Hepatitis C Virus Genotypes 1, 2, 3 and 4 in Normal Human Hepatocyte Cultures

Hepatitis C virus (HCV) infection afflicts about 170 million individuals worldwide. However, the HCV life cycle is only partially understood because it has not been possible to infect normal human hepatocytes in culture. The current Huh-7 systems use cloned, synthetic HCV RNA expressed in hepatocellular carcinoma cells to produce virions, but these cells cannot be infected with naturally occurring HCV obtained from infected patients.Here, we describe a human hepatocyte culture permissible to the direct infection with naturally occurring HCV genotypes 1, 2, 3 and 4 in the blood of HCV-infected patients. The culture system mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect naïve human hepatocytes.This culture system should complement the existing systems, and may facilitate the understanding of the HCV life cycle, its effects in the natural host cell, the hepatocyte, as well as the development of novel therapeutics and vaccines

Infection and propagation of hepatitis C virus in human CD4⁺ and CD8⁺ T lymphocytes in vitro

Accumulated molecular and clinical evidence indicate that human immune cells can support replication of hepatitis C virus (HCV). The aim of this study was to investigate the ability of authentic, plasma-occurring HCV to infect human CD4⁺ and CD8⁺ T lymphocytes in vitro. For this purpose, we adopted the previously established in vitro HCV replication system in total T cells derived from cultured normal human PBMC by employing affinity-purified CD4⁺ and CD8⁺ T lymphocytes as targets. Using this system, we were able to demonstrate that molecularly intact HCV can infect and productively replicate in both CD4⁺ and CD8⁺ T lymphocytes, albeit at low levels, by documenting: (1) Presence of HCV positive and replicative (negative) strands in infected cells; (2) Intracytoplasmic localization of viral proteins; (3) Emergence of new HCV variants in the de novo infected cells, and (4) Release of HCV RNA-reactive particles from the infected cells with biophysical properties distinct from those of virions in inocula used to infect these cells

Recombinant Semliki Forest virus replicon approach in search of an improved vaccine for Hepatitis B virus: induction of neutralizing antibodies

Elektroniskā versija nesatur pielikumusAr Semliki meža vīrusa vektoriem ierosināta neitralizējošu antivielu veidošanās pret B hepatīta vīrusa (HBV) virsmas proteīniem, lai pētītu iespējamos risinājumus patlaban izmantotās HBV vakcīnas nepilnībām - tā sastāv no viena HBV S virsmas proteīna subtipa, kas ekspresēts raugā, un atbilst vienam vīrusa genotipam. Ierosinātas neitralizējošas antivielas pret S proteīna diviem subtipiem, kas atbilst diviem vīrusa genotipiem, kā arī, lai paplašinātu imūno atbildi, pret saīsinātu L proteīnu, kas ietver divus par HBV saistīšanos pie hepatocītiem atbildīgos rajonus. Antivielu saistīšanās pie rekombinantā raugā ekspresētā HBsAg bija atkarīga no tā apakštipa, kamēr saistīšanās pie HBsAg, kas iegūts no HBV pacientiem, nebija atkarīga no apakštipa. Izmantojot HBV infekcijas in vitro modeli, parādīts, ka ar rSFV ierosināto antivielu neitralizējošais potenciāls nebija atkarīgs no S apakštipa. Atslēgas vārdi: B hepatīta vīrusa virsmas proteīni; apakštips; Semliki meža vīrusa vektors; neitralizējošas antivielasSemliki Forest virus vector (rSFV) approach was exploited for generation of neutralizing antibodies to surface proteins of hepatitis B virus (HBV) to address potential problems associated with current HBV vaccine, which consists of yeastexpressed small (S) protein, subtype ayw2, virus genotype A. After rSFV mediated delivery neutralizing antibodies were induced in mice to two S subtypes, representing different virus genotypes, and to shortened large proteins comprised of both HBV attachment determinants. Antibodies demonstrated preference to homologous subtype determinant of yeast-derived recombinant HBsAg, while binding to patient-derived HBsAg was not subtype dependent. In vitro HBV infection model showed that neutralizing antibodies were S subtype independent. The findings show that subtype dependency may not be as pronounced with vector-type vaccines. Keywords: Hepatitis B virus surface proteins; subtype; recombinant Semliki Forest virus vector, neutralizing antibodies

Recombinant Semliki Forest virus replicon approach in search of an improved vaccine for Hepatitis B virus: induction of neutralizing antibodies

Elektroniskā versija nesatur pielikumusAr Semliki meža vīrusa vektoriem ierosināta neitralizējošu antivielu veidošanās pret B hepatīta vīrusa (HBV) virsmas proteīniem, lai pētītu iespējamos risinājumus patlaban izmantotās HBV vakcīnas nepilnībām - tā sastāv no viena HBV S virsmas proteīna subtipa, kas ekspresēts raugā, un atbilst vienam vīrusa genotipam. Ierosinātas neitralizējošas antivielas pret S proteīna diviem subtipiem, kas atbilst diviem vīrusa genotipiem, kā arī, lai paplašinātu imūno atbildi, pret saīsinātu L proteīnu, kas ietver divus par HBV saistīšanos pie hepatocītiem atbildīgos rajonus. Antivielu saistīšanās pie rekombinantā raugā ekspresētā HBsAg bija atkarīga no tā apakštipa, kamēr saistīšanās pie HBsAg, kas iegūts no HBV pacientiem, nebija atkarīga no apakštipa. Izmantojot HBV infekcijas in vitro modeli, parādīts, ka ar rSFV ierosināto antivielu neitralizējošais potenciāls nebija atkarīgs no S apakštipa. Atslēgas vārdi: B hepatīta vīrusa virsmas proteīni; apakštips; Semliki meža vīrusa vektors; neitralizējošas antivielasSemliki Forest virus vector (rSFV) approach was exploited for generation of neutralizing antibodies to surface proteins of hepatitis B virus (HBV) to address potential problems associated with current HBV vaccine, which consists of yeastexpressed small (S) protein, subtype ayw2, virus genotype A. After rSFV mediated delivery neutralizing antibodies were induced in mice to two S subtypes, representing different virus genotypes, and to shortened large proteins comprised of both HBV attachment determinants. Antibodies demonstrated preference to homologous subtype determinant of yeast-derived recombinant HBsAg, while binding to patient-derived HBsAg was not subtype dependent. In vitro HBV infection model showed that neutralizing antibodies were S subtype independent. The findings show that subtype dependency may not be as pronounced with vector-type vaccines. Keywords: Hepatitis B virus surface proteins; subtype; recombinant Semliki Forest virus vector, neutralizing antibodies

CHARACTERIZATION OF FACTORS INVOLVED IN HOST-VIRAL INTERACTIONS AND HOW THEY CONTRIBUTE TO HEPATITIS C VIRUS-INDUCED CELL DEATH

Ph.DDOCTOR OF PHILOSOPH

Exploiting and exploring the interactions between microRNA-122 and Hepatitis C virus

Hepatitis C virus (HCV) is a single-stranded plus-sense RNA virus that is transmitted by blood-to-blood contact, and infects the human liver. HCV has a unique dependence on the liver-specific microRNA miR-122, where miR-122 binds the 5´ un-translated region of the viral RNA at two tandem sites and increases viral RNA abundance. The mechanisms of augmentation are not yet fully understood, but the interaction is known to stabilize the viral RNA, increase translation from the viral internal ribosomal entry site (IRES), and result in increased viral yield. In an attempt to create a small animal model for HCV, we added miR-122 to mouse cell lines previously thought non-permissive to HCV, which rendered these cells permissive to the virus, additionally showing that miR-122 is one of the major determinants of HCV hepatotropism. We found that some wild-type and knockout mouse cell lines – NCoA6 and PKR knockout embryonic fibroblasts – could be rendered permissive to transient HCV sub-genomic, but not full-length, RNA replication upon addition of miR-122, and that other wild-type and knockout cell lines cannot be rendered permissive to HCV replication by addition of miR-122. These knockout cell lines demonstrated varying permissiveness phenotypes between passages and isolates and eventually completely lost permissiveness, and we were unable to achieve sub-genomic RNA replication in PKR knockout primary hepatocytes. Knockdown of NCoA6 and PKR in Huh7.5 cells did not substantially impact sub-genomic replication, leading us to conclude that there are additional factors within the cell lines that affect their permissiveness for HCV replication such as epigenetic regulation during passage or transformation and immortalization. We also added miR-122 to Hep3B cells, a human hepatoma cell line lacking expression of miR-122 and previously thought to be non-permissive to HCV replication. Added miR-122 rendered the cells as highly permissive to HCV replication as the Huh7-derived cell lines commonly used to study the virus. In these cells, we were also able to observe miR-122-independent replication of sub-genomic HCV RNA. This was verified by use of a miR-122 antagonist that had no impact on the putative miR-122-independent replication, and by mutating the miR-122 binding sites to make them dependent on a single nucleotide-substituted microRNA. This replication in the absence of miR-122 was not detected in full-length HCV RNA, but was detectable using a bi-cistronic full-length genomic replicon, suggesting that the addition of a second IRES in sub-genomic and full-genomic replicons altered replication dynamics enough to allow detectable RNA replication without miR-122 binding. Because miR-122 has been implicated in protecting the viral RNA from destabilization and degradation by Xrn1, the main cytoplasmic 5´ to 3´ RNA exonuclease, we employed our miR-122-independent system to test this miR-122-mediated protection. We verified that miR-122 functions to protect the viral RNA from Xrn1, but this was insufficient to account for the overall impact of miR-122 on replication, meaning that miR-122 has further functions in the virus’ life cycle. We showed that the effect of miR-122 on translation is due to stabilization of the RNA by protecting it from Xrn1, through binding at both sites. We further evaluated the role of each miR-122 binding site (S1 and S2) in the virus life cycle, and found that binding at each site contributes equally to increasing viral RNA replication, while binding at both sites exerts a co-operative effect. Finally, we determined that binding of miR-122 at site S2 is more important for protection from Xrn1, suggesting that miR-122 binding at S1 is more important for the additional functions of miR-122 in enhancing HCV RNA accumulation. Altogether, we have shown that miR-122 is partially responsible for the hepatotropic nature of Hepatitis C virus, and that supplementation with this microRNA can render non-permissive cells permissive to viral replication. We have also identified and confirmed replication of both sub-genomic and full-length HCV RNA in the absence of miR-122. Finally, we have characterized the impact of the host RNA exonuclease Xrn1 on the HCV life cycle, and determined the roles of each miR-122 binding site in shielding the viral RNA from this host restriction factor

Etude de l'impact de la variabilité génétique de la protéine NS5A du virus de l'hépatite C dans la pathogenèse et la réplication virale

Le virus de l Hépatite C (VHC), de la famille Flaviviridae, est à l origine d une pandémiemondiale. L infection par le VHC provoque le dévelopment d hépatites chroniques, decirrhoses et de carcinomes hépatocellulaires (CHC). Les fonctions de la majorité des protéinesvirales sont connues, mis à part pour NS5A dont la seule fonction directe attribuée à ce jour,équivaut à celle d un facteur d'activation transcriptionnelle. Notre laboratoire a montréprécédemment que les variants de quasiespèce de NS5A isolés à partir du sérum d un mêmepatient présentaient des différences significatives dans leurs propriétés intrinsèques detransactivation. Fort de ces résultats, nous avons analysé des variants de NS5A isolés à partirde tissu hépatique d un patient chroniquement infecté par le VHC de génotype 1b. Cesanalyses ont révélé une compartimentation génétique et fonctionnelle des variants de NS5Aentre le tissu tumoral et le tissu non-tumoral adjacent. Nous avons donc émis l hypothèse queles propriétés transactivatrices de NS5A pourraient jouer un rôle dans la pathogenèse ainsique dans la réplication virale, et que la variabilité naturelle de NS5A pourrait influencer sespropriétés transactivatrices. L objectif de ce travail de thèse était d analyser le rôle despropriétés transactivatrices de NS5A dans la pathogenèse hépatique viro-induite ainsi quedans la réplication virale. Pour étudier le rôle des propriétés de transactivation de NS5A dans la pathogenèse hépatique,nous avons développé des vecteurs lentiviraux pour exprimer dans les hépatocytes primaireshumains les variants choisis de NS5A portants différents potentiels de transactivation. Enutilisant la technologie RNA-Seq d Illumina, l analyse des transcriptomes d hépatocytestransduits exprimant les variants transactivateurs fort et faible de NS5A, sera utiliser pouridentifier les voies cellulaires ciblées par les propriétés transactivatrices de NS5A. Pour lesétudes in vivo, nous avons lancé le développement des souris transgénique permettantl activation conditionnelle de l expression des variants de NS5A avec fort et faible potentielde transactivation, spécifiquement dans le foie. Ces souris transgéniques seront utilisées pourétudier le rôle potentiel des propriétés transactivatrices dans la pathogenèse VHC induite etplus particulièrement dans le développement des cancers. Pour étudier le rôle des propriétés de transactivation de NS5A dans la réplication virale, nousavons utilisé le système de réplicon subgénomique de VHC exprimant les variants de NS5Aprécédemment caractérisés. Pour exercer ses propriétés transactivatrices, NS5A doit êtrelocalisée au moins partiellement dans le noyau. Nous avons démontré qu une partie de NS5Ase retrouve dans noyau et est recruté sur des promoteurs cellulaires, modulant ainsidirectement l expression de gènes cellulaires essentiels pour la réplication de l ARN viral.Nous avons observé que les variants de NS5A avec différents potentiels de transactivation,confèrent différentes capacités de réplication au réplicon subgénomique, et corrèlent avec lepotentiel de transactivation de variant correspondant. En accord avec ces observations,l inhibition de translocation nucléaire de NS5A entraine une inhibition de la réplication virale,suggerant un rôle potentiel des propriétés transactivatrices de NS5A dans la réplication l ARNvirale. En conclusion, nous avons démontré que l activation transcriptionnelle des gènes cellulairespar la NS5A est essentielle pour la réplication de l ARN du VHC. Cette modulation des gènescellulaires pourrait également être impliquée dans les mécanismes de la pathogenèse viroinduite.Nous confirmerons cette hypothèse grâce aux souris NS5A. Par ailleurs, ces résultatspourraient contribuer au développement de nouvelles thérapies anti-VHC, basées surl inhibition de translocation nucléaire de NS5AHepatitis C virus (HCV) causes a chronic infection in the majority of infected patients,ultimately leading to liver cirrhosis and hepatocellular carcinoma (HCC). Although the rolesof the HCV proteins in the viral life cycle are increasingly understood, the precise function ofthe HCV NS5A protein has yet to be elucidated. To date, the only putative direct functionattributed to NS5A is its transcriptional transactivation properties. Our group has previouslyshown that quasispecies variants of NS5A isolated from the serum samples of the samepatient bear different transactivating properties according to their amino acid sequence. Basedon these observations, we performed preliminary phylogenetic and functional analysis ofNS5A variants isolated from liver tissue of individuals infected with HCV of genotype 1b.This analysis revealed genetic and functional compartmentation of NS5A variants in tumoraland adjacent non-tumoral tissue. We hypothesized that the natural variability of NS5A mayimpact its proposed transactivation properties. We also hypothesized that NS5A s putativetransactivation properties could play a role in HCV replication and in liver pathogenesis. Theaim of the study presented in this thesis was to investigate the role of NS5A transactivationproperties in the development of HCV-induced liver pathogenesis as well as in viralreplication. To study the role of NS5A transcriptional activation properties in liver pathogenesis, wedeveloped lentiviral vectors for the expression of selected NS5A variants bearing differenttransactivation potentials in cultured primary human hepatocytes. We now intend to extendthese preparations using RNAseq technology to analyse the, transcriptome of primaryhepatocytes transduced with lentiviral vectors encoding strongly and weakly transactivatingNS5A variants to identify the cellular pathways targeted by NS5A, allowing us to decipherthe role of NS5A mediated host gene regulation in development of HCV inducedpathogenesis. For in vivo studies, we have begun the development of transgenic mice allowingliver-specific conditional expression of NS5A variants with high and low transactivationpotentials. These transgenic mice will be used to study the possible role of NS5Atransactivation properties in development of HCC. To study the role of NS5A transcriptional activation properties in HCV RNA replication, weused the sub-genomic replicon system expressing previously characterized NS5A sequences..Using this system, we have demonstrated that a subset of NS5A protein can translocate to thenucleus and is recruited to cellular promoters of host cell genes known to be required forefficient replication of HCV replicon RNA as well as those implicated in pathogenesis.Moreover, we have shown that NS5A directly regulate the expression of these genes.Consequently, it was observed that replicons encoding NS5A variants with differenttransactivation potentials exhibited different replication capacities, and that this correlatedwith the transactivation potential of the corresponding NS5A variant. In agreement with theseobservations, inhibition of nuclear translocation of NS5A resulted in the inhibition ofreplication of the HCV subgenomic replicon, further confirming the role of NS5Atransactivation properties in viral RNA replication. In conclusion, we have demonstrated that NS5A-mediated transcriptional regulation ofcellular genes is required for HCV replication. Such NS5A-mediated modulation of cellulargenes may also constitute one of the mechanisms involved in HCV-related liver pathogenesisand development of HCC, an aspect which is currently under investigation using the toolsdeveloped during this project. This study will contribute towards deciphering the role ofNS5A in viral replication as well as providing insight into its role in HCV-induced liverpathogenesis...PARIS-EST-Université (770839901) / SudocPARIS12-Bib. électronique (940280011) / SudocSudocFranceF

Protection from HCV infection – Identification of mechanisms of resistance to HCV infection in exposed uninfected injection drug users.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease. In the developed world, injection drug use (IDU) through sharing of infected needles and other paraphernalia remains the principal risk factor for HCV transmission. Effective but expensive treatment is now possible but there remains a pressing need for a vaccine. A proportion of people who inject drugs (PWIDs) remain uninfected despite HCV exposure from a long history of sharing needles and other paraphernalia. These cases are termed exposed but uninfected (EU) and test negative for both HCV antibodies and RNA and exhibit a phenotype of resistance to HCV infection. Improved understanding of the mechanisms that confer resistance in the EUs has the potential to aid development of an effective vaccine and novel therapeutic strategies. This thesis reports on the findings from 3 different strategies to identify characteristics of HCV resistance. I used urinary metabolomics, serum lipidomics and the study of adaptive and innate immune responses. Each of these methods has demonstrated clear differences between EU cases and healthy controls and/or spontaneous resolvers of HCV infection. Urinary metabolomics suggest a potential role of the gut microbiome, the serum lipidomics showed marked differences in lipid profiles in EU cases pointing towards a perturbed lipid/virus interaction, and the immune studies confirmed previous work identifying low level T cell responses in many EU cases but has also identified a marked upregulation of interferon alpha production to low dose viral RNA in EU cases utilising ELISA assay. In conclusion, this thesis reports data that identifies a number of new findings that provide insight into mechanisms of resistance to HCV infection. My findings suggest that the complex interplay between the virus and lipids together with an upregulated innate immune response may together help determine the outcome following HCV exposure. In summary, studies performed in this thesis have demonstrated that there are different pathways that define the EU phenotype. Despite being a heterogenous subgroup of PWIDs, the EUs are clearly distinct from a healthy control population.University of Plymout

Analysis of diversity of hepatitis C virus glycoproteins E1 and E2

Hepatitis C Virus (HCV) exists as a population of sequence variants that evolves during infection adapting to host pressures. The main targets for the immune response are the envelope glycoproteins E1 and E2, which also mediate viral cell entry. The first hypervariable region (HVR1) of E2, previously implicated in the outcome of acute infection, has been a focus of many studies. However more broadly neutralising antibodies tend to target epitopes outside this region, yet evolution of full length E1E2 heterodimer is poorly understood. The HCV transmission and window period as well as seroconversion are the evolutionary events shaping primary infection hence influencing outcome of acute infection. However, due to the asymptomatic character of the early phases of HCV infection, evolutionary data describing this interval is still lacking depth. Defining the genetic and phenotypic characteristics of HCV population of sequence variants that establish infection in a new host would aid vaccine and new therapy design. This study aimed to identify patterns of HCV envelope glycoprotein evolution upon transmission and during early phases of disease. We studied this in three settings: experimental transmission of immunocompromised mice by known inoculum; occurrence of horizontal transmission in a haemodialysis unit between hypothesised source and index case individuals; and unrelated cases of acutely infected HCV patients. The single genome amplification (SGA) approach was utilised, which allowed us to accurately assign linkage between substitutions and determine the frequency distribution of E1E2 variants in analysed viral populations. Data from the first experimental setting indicates that a selective sweep occurs upon HCV transmission, with selective amplification of envelope sequence variants that possess fitness advantage at entry level. Molecular determinants associated with this enhanced infectivity have also been identified. In further part of the project we confirmed a horizontal infection in haemodialysis unit with use of phylogenetic methods and suggested revision of current safety guidelines. Analysis of sequences from the last setting showed that indeed HVR1 might not be a good enough indicator of evolutionary events in the acute phase, as linked substitutions occur also outside this region. Seroconversion is associated with increasing population diversity indicating role of antibodies in driving HCV evolution, which is host specific