Interaction of alpha-melanotropin with central dopamine systems: role of hormonal state and molecular structure

The tubero-infundibular and nigrostriatal DA neurone systems of rats respond to systemic (i.p.) injection of alpha-MSH (2-100 microgram/kg). The response of the tubero-infundibular (arcuate) DA neurones, an increase in cellular fluorescence intensity which can be interpreted as a sign of increased neuronal activity, is essentially the same in males, estrogen-progesterone-pretreated ovariectomized females and hypophysectomized males, whereas the type of response elicited by alpha-MSH in the nigral DA neurones depends upon the hormonal state of the animal. Differences between the two DA neurone groups exist also with regard to the effects of peptide fragments containing the two active sites of the alpha-MSH molecule. Results of lesion experiments in the lower brainstem (area postrema) and of blockade of muscarinic mechanisms by atropine further point to differences in the mechanisms underlying the peptide effects on the two neurone systems. The reaction of the tubero-infundibular DA system (which controls the pars intermedia of the pituitary) can be considered to reflect the activation of a feedback mechanism on MSH secretion, while the functional counterpart of the changes observed in the nigral system remains unknown at the present time.

Développement de la communication intentionnelle gestuelle à partir des usages culturels des objets dans l'interaction triadique enfant-objet-adulte

Pour que deux protagonistes parviennent à communiquer, ils doivent non seulement partager les signes verbaux et/ou non verbaux qu'ils utilisent, mais ils doivent également parvenir à accéder à l'intention communicative de chacun. Ce dernier aspect de la communication a été intimement lié aux significations et aux connaissances que les protagonistes partagent (angl. common ground). Malgré le fait que l'importance des significations partagées ait été soulignée dans la littérature concernant la communication entre adultes, cet aspect n'a pas été étudié en ce qui concerne le développement précoce de la communication. Dans le présent travail, nous suggérons d'investiguer le lien entre les significations partagées entre le jeune enfant et l'adulte et la capacité de l'enfant à communiquer de manière intentionnelle via des gestes. En nous appuyant sur l'approche de la Pragmatique de l'Objet (Moro & Rodríguez, 2005), nous suggérons que les connaissances concernant l'usage culturel de l'objet que construit progressivement l'enfant représentent un type de significations que l'enfant partage avec autrui. 12 dyades mère-enfant ont été enregistrées à l'aide d'une caméra lors d'interactions avec quatre objets. Les observations ont eu lieu au domicile des familles et ont été conduites chaque deuxième mois lorsque les enfants ont été âgés de 8 à 16 mois (groupe 1) et de 16 à 24 mois (groupe 2). Les observations ont été codées pour le niveau de maîtrise de l'usage culturel par l'enfant ainsi que pour la production de gestes par l'enfant et par l'adulte. Nous avons trouvé que plus l'enfant maîtrise les usages culturels des objets, plus il produit de gestes, et plus ces gestes remplissent une fonction communicative explicite. Concernant les gestes de l'adulte, les résultats dévoilent que plus l'enfant maîtrise les usages conventionnels des objets, plus l'adulte adresse des gestes communicatifs à l'enfant, plus ses gestes sont produits de manière complexe et plus l'intention des gestes communicatifs de l'adulte est complexe. Pris ensemble, ces résultats suggèrent que le partage de significations sur l'usage culturel des objets représente un type de connaissances communes qui permettent au bébé et à ses protagonistes de communiquer.

Interaction between widening of diameter of abdominal aorta and cardiovascular risk factors and atherosclerosis burden.

The aim of this study was to investigate influence of traditional cardiovascular risk factors (CVRF) and subclinical atherosclerosis (ATS) burden on early stages of abdominal aortic diameter (AAD) widening among adults. 2,052 consecutive patients (P) (39 % women), mean age 52 ± 13 years, were prospectively screened for CVRF, ATS, and AAD. B-mode ultrasound was used to evaluate the largest AAD and to detect carotid and femoral atherosclerotic plaques. Mean AAD was 15.2 ± 2.8 mm. Atherosclerotic plaques were detected in 71 % of patients. Significant univariate correlation between AAD, traditional CVRF, and ABS was found. However, multiple regression analysis showed that only seven of them were significantly and weakly correlated with AAD (R² = 0.27, p < 0.001). On the other hand, a multivariate logistic analysis was used to evaluate CVRF impact on enlarged AAD ≥25 mm (EAAD) as compared to those with AAD <25 mm. These factors did not account for more than 30 % of interaction (R² = 0.30, p = 0.001). Furthermore, despite a large proportion of patients with high number of CVRF, and subclinical ATS, rate of patients with AAD ≥25 mm was low (1 %) and scattered regardless their CHD risk score or ATS burden. In conclusion, these results suggest that although some traditional CVRF and presence of ATS are associated with early stages of EAAD, other determinants still need to be identified for a better understanding of abdominal aortic aneurysm pathogenesis.

Corticosterone mediates the condition-dependent component of melanin-based coloration

The handicap principle of sexual selection theory states that colourful phenotypic traits signal aspects of individual quality because only individuals in prime condition can afford to produce and bear conspicuous traits. Melanin-based pigments participate in the elaboration of many secondary sexual characters and, given their role in sexual selection, melanin-based coloration may therefore honestly reject individual quality. Although the expression of melanism is usually under genetic control, in some species it is condition dependent. However, the underlying physiological mechanism is yet unknown. Based on the negative feedback link between corticosterone and melanogenesis ( melanocortins, tyrosinase) in response to stressful environmental factors, we hypothesize that corticosterone mediates the condition-dependent component of melanism. This hypothesis predicts that stressful factors induce a rise in circulating corticosterone which inhibits the secretion of melanocortins and tyrosinase and in turn melanin production. We tested this prediction by manipulating the level of corticosterone at the time of melanin production in nestling barn owls, Tyto alba, a species showing heritable variation in the degree of phaeomelanism from reddish-brown to white. The finding that corticosterone-implanted nestlings produced feathers with less phaeomelanic coloration than placebo-implanted nestlings is consistent with the hypothesis that the environment-mediated reduction in the degree of melanism is, at least in part, caused by a rise in corticosterone. In species in which the expression of melanin-based coloration is condition dependent, we now need a test showing that individuals with less corticosterone and more melanin-based signals are individuals in better condition.

Shaping the interaction landscape of bioactive molecules.

MOTIVATION: Most bioactive molecules perform their action by interacting with proteins or other macromolecules. However, for a significant fraction of them, the primary target remains unknown. In addition, the majority of bioactive molecules have more than one target, many of which are poorly characterized. Computational predictions of bioactive molecule targets based on similarity with known ligands are powerful to narrow down the number of potential targets and to rationalize side effects of known molecules. RESULTS: Using a reference set of 224 412 molecules active on 1700 human proteins, we show that accurate target prediction can be achieved by combining different measures of chemical similarity based on both chemical structure and molecular shape. Our results indicate that the combined approach is especially efficient when no ligand with the same scaffold or from the same chemical series has yet been discovered. We also observe that different combinations of similarity measures are optimal for different molecular properties, such as the number of heavy atoms. This further highlights the importance of considering different classes of similarity measures between new molecules and known ligands to accurately predict their targets. CONTACT: olivier.michielin@unil.ch or vincent.zoete@unil.ch SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genetic and maternal contributions to offspring viability under different environmental conditions in various salmonids

Summary : Due to anthropogenic impacts and natural fluctuations, fish usually have to cope with constantly changing and often hostile environments. Whereas adult fish have various possibilities to counteract unfavourable environmental conditions, embryos have much fewer options. Besides by their developing immune system, they are protected by the egg envelopes and several immune substances provided by their mothers. In addition to this, they may also adjust their hatching timing in reaction to various risks. However, individuals may vary in their defensive potential. This variation may be either based on their genetics and/or on differential maternal investments and may be dependent on the experienced stress. Nevertheless, in fish, the impact of such parental contributions on embryo and/or juvenile viability is still poorly investigated. The main objective of this thesis was to investigate the importance of paternal (i.e. genetic) and maternal (i.e. genetic + egg investment) contributions to offspring viability under different environmental conditions and at different life stages. In order to investigate this, we used gametes of various salmonids for in vitro fertilisation experiments based on full-factorial breeding designs. The individual studies are summarised in the following chapters: In the first chapter, we tested the effectiveness of the embryonic immune system in Lake whitefish (Coregonus palaea). Namely, we investigated paternal and maternal contributions to the embryos' tolerance to different kinds of pathogen exposure. Additionally, we tested whether an early sub-léthal exposure has a positive or a negative effect on an embryo's susceptibility to later pathogen exposures with the same pathogen. We found that pre-challenged embryos were more susceptible to future challenges. Moreover, pathogen susceptibility was dependent on maternal investments and/or the embryos' own genetics, depending on the challenge level. Chapter 2 summarises a similar study with brown trout (Salmo trutta). In addition to the previously described investigations, we analysed if genetic effects on offspring viability are mediated either by parental MHC genotypes or relatedness based on neutral microsatellite markers, and we tested if males signal their genetic quality either by their body size or their melanin-based skin colouration. We found that embryo survival was lower at higher stress levels and dependent on the embryos' genetics. Addirionally, parents with similar and/or, very common MHC genotypes had higher offspring viabilities. Finally, darker males produced more viable offspring. In the first two chapters we investigated the embryos' defensive potential based on their immune system, i.e. their pathogen tolerance. In chapter 3 we investigate whether hatching timing of Lake whitefìsh (C. palaea) is dependent on parental contributions and/or on pathogen pressure, and whether there are parental-environmental interactions. We found that whitefish embryos hatch earlier under increasing pathogen pressure. Moreover, hatching timing was affected by embryo genetics and/or maternally provided resources, but the magnitude of the effect was dependent on the pathogen. pressure. We also found a significant paternal-environmental interaction, indicating that the hatching efficiency of a certain sib group is dependent on the pathogen environment. Chapter 4 describes an analogous study with brown trout (S. trutta), with similar findings. In the former chapters, we only looked at offspring performance during the embryonic period, and only under semi-natural conditions. In chapter 5 we now test the performance and viability of embryonic and juvenile brown trout (S. trutta) under natural conditions. To measure embryo viability, we put them in brood boxes, buried them in the gravel of a natural river, and analysed survival after several months. To investigate juvenile survival and performance, wé reared embryos under different stress levels in the laboratory and subsequently released the resulting hatchlings in to a closed river section. Juvenile size and survival was then determined one year later. Additionally, we investigated if sires differ in their genetic quality, determined by embryo and juvenile survival as well as juvenile size, and if they signal their quality by either body size or melanin-based body darkness. We found hat juvenile size was dependent on genetic effects and on maternal investment, whereas this was neither the case for embryo nor for juvenile survival. Additionally, we found that offspring of darker males grew larger, and larger juveniles had also an increased survival. Finally, we found acarry-over effect of the early non-lethal challenge: exposing embryos to higher stress levels resulted in smaller juveniles. To evaluate the long-term performance of differently treated groups, mark-recapture studies are inevitable. For this purpose, effective mass-marking techniques are essential. In chapter 6 we tested the suitability of the fluorescent pigment spray marking method for the mass marking of European graylings (Thymallus thymallus), with very promising results. Our in vitro fertilisation studies on whitefish may reveal new insights on potential genetic benefits of mate choice, but the mating system of whitefish under natural conditions is still poorly investigated. In order to study this, we installed underwater cameras at the spawning place of a Coregonus suidteri population, recorded the whole mating period and subsequently analysed the recordings. Confirmations of previous findings as well as exciting new observations are listed and discussed in chapter 7. Dus aux impacts anthropogéniques et aux fluctuations naturelles, les poissons doivent faire face à des environnements en perpétuel changement. Ces changements font que les poissons doivent s'adapter à de nouvelles situations, souvent hostiles pour eux. Les adultes ont différentes possibilités d'échapper à un environnement peu favorable, ce n'est par contre pas le cas des embryons. Les embryons sont protégés d'une part par leur système immunitaire en développement, d'autre part, par la coquille de l'eeuf et différentes substances immunitaires fournies par leur mère. De plus, ils sont capables d'influencer leur propre date d'éclosion en réponse à différents facteurs de stress. Malgré tout, les individus varient dans leur capacité à se défendre. Cette variation peut être basé sur des facteurs génétiques et/ou sur des facteurs maternels, et est dépendante du stress subi. Néanmoins, chez les poissons, l'impact de telles contributions parentales sur la survie d'embryons et/ou juvéniles est peu étudié. L'objectif principal de cette thèse a été d'approfondir les connaissances sur l'importance de la contribution paternelle (c.a.d. génétique) et maternelle (c.a.d. génétique + investissement dans l'oeuf) sur la survie des jeunes dans différentes conditions expérimentales et stades de vie. Pour faire ces analyses, nous avons utilisé des gamètes de divers salmonidés issus de croisements 'full-factorial'. Les différentes expériences sont résumées dans les chapitres suivants: Dans le premier chapitre, nous avons testé l'efficacité du système immunitaire des embryons chez les corégones (Coregonus palea). Plus précisément nous avons étudié la contribution paternelle et maternelle à la tolérance des embryons à différents niveaux de stress pathogène. Nous avons aussi testé, si une première exposition non létale à un pathogène avait un effet positif ou négatif sur la susceptibilité d'un embryon a une deuxième exposition au même pathogène. Nous avons trouvé que des embryons qui avaient été exposés une première fois étaient plus sensibles au pathogène par la suite. Mais aussi que la sensibilité au pathogène était dépendante de l'investissement de la mère et/ou des gènes de l'embryon, dépendamment du niveau de stress. Le deuxième chapitre résume une étude similaire avec des truites (Salmo truffa). Nous avons examiné, si la survie des jeunes variait sous différentes intensités de stress, et si la variance observée était due aux gènes des parents. Nous avons aussi analysé si les effets génétiques sur la survie des juvéniles étaient dus au MHC (Major Histocompatibility Complex) ou au degré de parenté des parents. De plus, nous avons analysé si les mâles signalaient leur qualité génétique par la taille du corps ou par leur coloration noire, due à la mélanine. On a trouvé que la survie des embryons était plus basse quand le niveau de stress était plus haut mais que la variation restait dépendante de la génétique des embryons. De plus, les parents avec des MHC similaires et/ou communs avaient des embryons avec une meilleure survie. Par contre, des parents avec un degré de parenté plus haut produisent des embryons avec une survie plus mauvaise. Finalement nous avons montré que les mâles plus foncés ont des embryons qui survivent mieux, mais que la taille des mâles n'a pas d'influence sur la survie de ces mêmes embryons. Dans les deux premiers chapitres, nous avons étudié le potentiel de défense des embryons basé sur leur système immunitaire, c.a.d. leur tolérance aux pathogènes. Dans le troisième chapitre, nous nous intéressons à la date d'éclosion des corégones (C. palea), pour voir si elle est influencée par les parents ou par la pression des pathogènes, et si il y a une interaction entre ces deux facteurs. Nous avons trouvé que les jeunes naissent plus rapidement lorsque la pression en pathogènes augmente. La date d'éclosion est influencée par la génétique des embryons et/ou l'investissement des parents, mais c'est la magnitude des effets qui est dépendante de la pression du pathogène. Nous avons aussi trouvé une interaction entre l'effet paternel et l'environnement, ce qui indique que la rapidité d'éclosion de certains croisements est dépendante des pathogènes dans l'environnement. Le chapitre 4 décrit une étude analogue avec de truites (S. truffa), avec des résultats sitzimilaires. Dans les précédents chapitres nous nous sommes uniquement concentrés sur les performances des jeunes durant leur stade embryonnaire, et seulement dans des conditions semi naturelles. Dans le chapitre 5 nous testons la performance et la viabilité des embryons et de juvéniles de truites (S. truffa) dans des conditions naturelles. Nous avons trouvé que la taille des juvéniles était dépendante d'effets génétiques et de l'investissement maternel, mais ceci n'était ni les cas pour les survie des embryons et des juvéniles. De plus, nous avons trouvé que les jeunes des mâles plus foncés devenaient plus grands et que les grands ont un meilleur taux de survie. Finalement nous avons trouvé un 'carry-over effect' d'une première exposition non létale à un pathogène: exposer des embryons à des plus hauts niveaux de stress donnait des juvéniles plus petits. Pour évaluer la performance à long terme de groupes traités dé manières différentes, une méthode de marquage-recapture est inévitable. Pour cette raison, des techniques de marquage en masse sont nécessaires. Dans le chapitre 6, nous avons testé l'efficacité de la technique `fluorescent pigment spray marking' pour le marquage en masse de l'Ombre commun (Thymallus thymallus), avec des résultats très prometteurs. Les études de fertilisations in vitro avec les corégones nous donnent une idée du potentiel bénéfice génétique que représente la sélection d'un bon partenaire, même si le système d'accouplement des corégones en milieu naturel reste peu connu. Pour combler cette lacune, nous avons installé des caméras sous-marines autour de la frayère d'une population de corégones (C. suidteri), nous avons enregistré toute la période de reproduction et nous avons analysé les données par la suite. Ainsi, nous avons été capables de confirmer bien des résultats trouvés précédemment, mais aussi de faire de nouvelles observations. Ces résultats sont reportés dans le septième chapitre, où elles sont comparées avec des observations antérieures.

Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex.

Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide-major histocompatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen.These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.

A Role for cis Interaction between the Inhibitory Ly49A receptor and MHC class I for natural killer cell education.

Natural killer (NK) cells show enhanced functional competence when they express inhibitory receptors specific for inherited major histocompatibility complex class I (MHC-I) molecules. Current models imply that NK cell education requires an interaction of inhibitory receptors with MHC-I expressed on other cells. However, the inhibitory Ly49A receptor can also bind MHC-I ligand on the NK cell itself (in cis). Here we describe a Ly49A variant, which can engage MHC-I expressed on other cells but not in cis. Even though this variant inhibited NK cell effector function, it failed to educate NK cells. The association with MHC-I in cis sequestered wild-type Ly49A, and this was found to relieve NK cells from a suppressive effect of unengaged Ly49A. These data explain how inhibitory MHC-I receptors can facilitate NK cell activation. They dissociate classical inhibitory from educating functions of Ly49A and suggest that cis interaction of Ly49A is necessary for NK cell education.

A protein interaction atlas for the nuclear receptors: properties and quality of a hub-based dimerisation network.

BACKGROUND: The nuclear receptors are a large family of eukaryotic transcription factors that constitute major pharmacological targets. They exert their combinatorial control through homotypic heterodimerisation. Elucidation of this dimerisation network is vital in order to understand the complex dynamics and potential cross-talk involved. RESULTS: Phylogeny, protein-protein interactions, protein-DNA interactions and gene expression data have been integrated to provide a comprehensive and up-to-date description of the topology and properties of the nuclear receptor interaction network in humans. We discriminate between DNA-binding and non-DNA-binding dimers, and provide a comprehensive interaction map, that identifies potential cross-talk between the various pathways of nuclear receptors. CONCLUSION: We infer that the topology of this network is hub-based, and much more connected than previously thought. The hub-based topology of the network and the wide tissue expression pattern of NRs create a highly competitive environment for the common heterodimerising partners. Furthermore, a significant number of negative feedback loops is present, with the hub protein SHP [NR0B2] playing a major role. We also compare the evolution, topology and properties of the nuclear receptor network with the hub-based dimerisation network of the bHLH transcription factors in order to identify both unique themes and ubiquitous properties in gene regulation. In terms of methodology, we conclude that such a comprehensive picture can only be assembled by semi-automated text-mining, manual curation and integration of data from various sources.

Melanin-based coloration covaries with ovary size in an age-specific manner in the barn owl.

While the adaptive function of black eumelanin-based coloration is relatively well known, the function of reddish-brown pheomelanin-based coloration is still unclear. Only a few studies have shown or suggested that the degree of reddish-brownness is associated with predator-prey relationships, reproductive parameters, growth rate and immunity. To gain insight into the physiological correlates of melanin-based coloration, I collected barn owl (Tyto alba) cadavers and examined the covariation between this colour trait and ovary size, an organ that increases in size before reproduction. A relationship is expected because melanin-based coloration often co-varies with sexual activity. The results showed that reddish-brown juveniles had larger ovaries than whiter juveniles particularly in individuals in poor condition and outside the breeding season, while in birds older than 2 years lightly coloured females had larger ovaries than reddish-brown conspecifics. As barn owls become less reddish-brown between the first and second year of age, the present study suggests that reddish-brown pheomelanic and whitish colorations are associated with juvenile- and adult-specific adaptations, respectively.

Phosphorylation modulates FOXC2 transcriptional program by controlling the high-order interaction with DNA.

Phosphorylation of transcription factors is a rapid and reversible process linking cell signaling and control of gene expression, therefore understanding how it controls the transcription factor functions is one of the challenges of functional genomics. We performed such analysis for the forkhead transcription factor FOXC2 mutated in human hereditary disease lymphedemadistichiasis and important for the development of venous and lymphatic valves and lymphatic collecting vessels. We found that FOXC2 is phosphorylated in a cell-cycle dependent manner on eight evolutionary conserved serine/threonine residues, seven of which are clustered within a 70 amino acid domain. Surprisingly, the mutation of phosphorylation sites or a complete deletion of the domain did not affect the transcriptional activity of FOXC2 in a synthetic reporter assay. However, overexpression of the wild type or phosphorylation-deficient mutant resulted in overlapping but distinct gene expression profiles suggesting that binding of FOXC2 to individual sites under physiological conditions is affected by phosphorylation. To gain a direct insight into the role of FOXC2 phosphorylation, we performed comparative genome-wide location analysis (ChIP-chip) of wild type and phosphorylation-deficient FOXC2 in primary lymphatic endothelial cells. The effect of loss of phosphorylation on FOXC2 binding to genomic sites ranged from no effect to nearly complete inhibition of binding, suggesting a mechanism for how FOXC2 transcriptional program can be differentially regulated depending on FOXC2 phosphorylation status. Based on these results, we propose an extension to the enhanceosome model, where a network of genomic context-dependent DNA-protein and protein-protein interactions not only distinguishes a functional site from a nonphysiological site, but also determines whether binding to the functional site can be regulated by phosphorylation. Moreover, our results indicate that FOXC2 may have different roles in quiescent versus proliferating lymphatic endothelial cells in vivo.

Tabagisme, obésité et diabète: une interaction cliniquement importante [Smoking, obesity and diabetes: a clinically important interaction].

Smoking, obesity and diabetes are among the leading cause of premature death worldwide. Smokers have globally a lower body weight compared with non smokers but they tend to accumulate more fat in the abdomen. Most smokers gain weight when they quit smoking, however this does not seem to diminish the health benefits associated with smoking cessation. Smoking increases the risk of developing type 2 diabetes. Among people with diabetes, smoking significantly increases the risks of complications and mortality. Interventions with pharmacologic help should be offered to all smokers, with or without diabetes, in order to increase smoking cessation rates and limit weight gain.

NS4B Self-Interaction through Conserved C-Terminal Elements Is Required for the Establishment of Functional Hepatitis C Virus Replication Complexes.

Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.