Identification of a novel herpesvirus associated with cutaneous ulcers in a fisher (Martes pennanti).

On December 8, 2008, a male fisher (Martes pennanti) housed in a quarantine enclosure at the St-Félicien Zoo was found dead with multiple skin ulcers on the muzzle and plantar pads. At necropsy, no major findings were found, and a specific cause of death was not determined microscopically. However, at the borders of ulcerated sites, there were increased numbers of koilocytes, with perinuclear vacuolation and nuclear enlargement. A pan-herpesvirus nested polymerase chain reaction (PCR) assay was conducted, and an expected PCR product of 230 nucleotides was obtained within tissues collected from around the skin ulcers. Other tissues, including intestines and pool of lung, liver, and kidney, tested negative. The obtained PCR amplicon was sequenced and was highly related to the partial viral DNA polymerase (DPOL) gene of Mustelid herpesvirus 1. Virus isolation was negative, and no virion was detected by electron microscopy. The pathogenic potential of this novel herpesvirus and its role in the death of the fisher are unknown.

Non-hemagglutinating flaviviruses: molecular mechanisms for the emergence of new strains via adaptation to European ticks

Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers to fatal encephalitis but the factors that determine disease severity are currently undefined. TBEV is characteristically a hemagglutinating (HA) virus; the ability to agglutinate erythrocytes tentatively reflects virion receptor/fusion activity. However, for the past few years many atypical HA-deficient strains have been isolated from patients and also from the natural European host tick, Ixodes persulcatus. By analysing the sequences of HA-deficient strains we have identified 3 unique amino acid substitutions (D67G, E122G or D277A) in the envelope protein, each of which increases the net charge and hydrophobicity of the virion surface. Therefore, we genetically engineered virus mutants each containing one of these 3 substitutions; they all exhibited HA-deficiency. Unexpectedly, each genetically modified non-HA virus demonstrated increased TBEV reproduction in feeding Ixodes ricinus, not the recognised tick host for these strains. Moreover, virus transmission efficiency between infected and uninfected ticks co-feeding on mice was also intensified by each substitution. Retrospectively, the mutation D67G was identified in viruses isolated from patients with encephalitis. We propose that the emergence of atypical Siberian HA-deficient TBEV strains in Europe is linked to their molecular adaptation to local ticks. This process appears to be driven by the selection of single mutations that change the virion surface thus enhancing receptor/fusion function essential for TBEV entry into the unfamiliar tick species. As the consequence of this adaptive mutagenesis, some of these mutations also appear to enhance the ability of TBEV to cross the human blood-brain barrier, a likely explanation for fatal encephalitis. Future research will reveal if these emerging Siberian TBEV strains continue to disperse westwards across Europe by adaptation to the indigenous tick species and if they are associated with severe forms of TBE.

Proteornics analysis unravels the functional repertoire of coronavirus nonstructural protein 3

Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructurall protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

Cell entry by enveloped viruses: redox considerations for HIV and SARS-coronavirus

For enveloped viruses, genome entry into the target cell involves two major steps: virion binding to the cell-surface receptor and fusion of the virion and cell membranes. Virus-cell membrane fusion is mediated by the virus envelope complex, and its fusogenicity is the result of an active virus-cell interaction process that induces conformation changes within the envelope. For some viruses, such as influenza, exposure to an acidic milieu within the cell during the early steps of infection triggers the necessary structural changes. However, for other pathogens which are not exposed to such environmental stress, activation of fusogenicity can result from precise thiol/disulfide rearrangements mediated by either an endogenous redox autocatalytic isomerase or a cell-associated oxidoreductase. Study of the activation of HIV envelope fusogenicity has revealed new knowledge about how redox changes within a viral envelope trigger fusion. We discuss these findings and their implication for anti-HIV therapy. In addition, to compare and contrast the situation outlined for HIV with an enveloped virus that can fuse with the cell plasma membrane independent of the redox status of its envelope protein, we review parallel data obtained on SARS coronavirus entry.

The structure of echovirus type 12 bound to a two-domain fragment of its cellular attachment protein decay-accelerating factor (CD 55)

Echovirus type 12 (EV12), an enterovirus of the Picornaviridae family, uses the complement regulator, decay-accelerating factor (DAF, CD55) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV12 bound to a fragment of DAF, consisting of short consensus repeat domains 3 and 4, from cryo-negative stain electron microscopy data (EMD #1057). This shows that, as for an earlier reconstruction of the related echovirus type 7 bound to DAF, attachment is not within the viral canyon but occurs close to the two-fold symmetry axes. Despite this general similarity, our reconstruction reveals a receptor interaction that is quite different from that observed for EV7. Fitting of the crystallographic co-ordinates for DAF34 and EV11 into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB #1UPN). Our finding that the mode of virus-receptor interaction in EV12 is distinct from that seen for EV7 raises interesting questions regarding the evolution and biological significance of the DAF-binding phenotype in these viruses.

A structural analysis of M protein in coronavirus assembly and morphology

The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy, tomography and statistical analysis. We present evidence that suggests M can adopt two conformations and that membrane curvature is regulated by one M conformer. Elongated M protein is associated with rigidity, clusters of spikes and a relatively narrow range of membrane curvature. In contrast, compact M protein is associated with flexibility and low spike density. Analysis of several types of virus-like particles and virions revealed that S protein, N protein and genomic RNA each help to regulate virion size and variation, presumably through interactions with M. These findings provide insight into how M protein functions to promote virus assembly.

Arenavirus budding resulting from viral-protein-associated cell membrane curvature

Viral replication occurs within cells, with release (and onward infection) primarily achieved through two alternative mechanisms: lysis, in which virions emerge as the infected cell dies and bursts open; or budding, in which virions emerge gradually from a still living cell by appropriating a small part of the cell membrane. Virus budding is a poorly understood process that challenges current models of vesicle formation. Here, a plausible mechanism for arenavirus budding is presented, building on recent evidence that viral proteins embed in the inner lipid layer of the cell membrane. Experimental results confirm that viral protein is associated with increased membrane curvature, whereas a mathematical model is used to show that localized increases in curvature alone are sufficient to generate viral buds. The magnitude of the protein-induced curvature is calculated from the size of the amphipathic region hypothetically removed from the inner membrane as a result of translation, with a change in membrane stiffness estimated from observed differences in virion deformation as a result of protein depletion. Numerical results are based on experimental data and estimates for three arenaviruses, but the mechanisms described are more broadly applicable. The hypothesized mechanism is shown to be sufficient to generate spontaneous budding that matches well both qualitatively and quantitatively with experimental observations.

Purification of coronavirus virions for Cryo-EM and proteomic analysis

Purification of intact enveloped virus particles can be useful as a first step in understanding the structure and function of both viral and host proteins that are incorporated into the virion. Purified preparations of virions can be used to address these questions using techniques such as mass spectrometry proteomics. Recent studies on the proteome of coronavirus virions have shown that in addition to the structural proteins, accessory and non-structural virus proteins and a wide variety of host cell proteins associate with virus particles. To further study the presence of virion proteins, high quality sample preparation is crucial to ensure reproducible analysis by the wide variety of methods available for proteomic analysis.

Papilomavirus Humano (HPV) - Um estudo de revisao

Human Papillomaviruses (HPVs) are epitheliotropic viruses, that induce benign and malignant lesions on several body sites. It's a small circular DNA virus, non-enveloped and 75 types have been identified. Frequently HPV 6, 11 (benign lesions) and 16, 18 (malignant lesions) are occurred on mucosa. The infection takes place at the basal layer cells with microlesions, when the virus enters into the cells and looses the capsid. The benign HPV types is associated to cell's genome in epissomal way. In malignant lesions, it integrates into the cell's DNA. HPV viruses are sexually transmitted and responsable for malignant cell transformation. Thus this viruses have an extremely epidemiologic importance. This paper reports a HPV review study about: epidemiology, diagnostic methods and treatment to papillomavirus infection.

THE GLYCOPROTEINS OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS AND THEIR ROLE IN INFECTION AND IMMUNITY

The porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen of swine and is known to cause abortion and infertility in pregnant sows and respiratory distress in piglets. PRRSV contains a major glycoprotein (GP5) and three minor glycoproteins (GP2a, GP3, and GP4) on the virion envelope, all of which are required for infectious virus production. To study their interactions amongst each other and with a cellular receptor for PRRSV, CD163, I cloned each of the viral glycoproteins and CD163 in various expression vectors. My studies have shown that while the GP2a, GP3, and GP4 are co-translationally glycosylated, the GP5 is post-translationally glycosylated. By using co-immunoprecipitation (co-IP) assays, strong interaction was demonstrated between GP4 and GP5 proteins, although weak interactions among the other envelope glycoproteins were also detected. Further, GP4 was found to mediate interactions leading to formation of multiprotein glycoprotein complex. My results also show that GP2a and GP4 proteins are the only two GPs that specifically interact with the CD163 molecule and that glycosylation of these GPs is required for efficient interaction. Based on these studies, I have developed an interactome map of the viral GPs and CD163 and have proposed a model of the viral glycoprotein complex and its interaction with CD163. Studies reported here also show that glycan addition at residue 184 (N184) of GP2a, and residues N42, N50, and N131 of GP3 is essential for recovery of infectious virus. Although single site glycosylation mutants of GP4 had no effect on infectious virus production, introduction of double mutations was lethal. The loss of glycan moieties of GP2a, GP3, and GP4 proteins had no effect on host neutralizing antibody production. Overall, I conclude that the PRRSV glycoproteins are co-translationally and post-translationally glycosylated, the GP4 protein is central to mediating interglycoprotein interactions, and along with GP2a, serves as the viral attachment protein that is responsible for interactions with the viral receptor, CD163. Further, glycosylation of GP2a, GP3, and GP4 proteins is required for infectious virus production, efficient interaction with CD163, but does not play any role in neutralizing antibody response in infected animals.

Molecular basis oh herpes simplex virus entry into the cell and retargeting of the viral tropism for the design of oncolytic herpesviruses

Herpes simplex virus 1 (HSV-1) infects oral epitelial cells, then spreads to the nerve endings and estabilishes latency in sensory ganglia, from where it may, or may not reactivate. Diseases caused by virus reactivation include mild diseases such as muco-cutaneous lesions, and more severe, and even life-threatening encephalitis, or systemic infections affecting diverse organs. Herpes simplex virus represents the most comprehensive example of virus receptor interaction in Herpesviridae family, and the prototype virus encoding multipartite entry genes. In fact, it encodes 11-12 glycoproteins and a number of additional membrane proteins: five of these proteins play key roles in virus entry into subsceptible cells. Thus, glycoprotein B (gB) and glycoprotein C (gC) interact with heparan sulfate proteoglycan to enable initial attachment to cell surfaces. In the next step, in the entry cascade, gD binds a specific surface receptor such as nectin1 or HVEM. The interaction of glycoprotein D with the receptor alters the conformation of gD to enable the activation of gB, glycoprotein H, and glycoprotein L, a trio of glycoproteins that execute the fusion of the viral envelope with the plasma membrane. In this thesis, I described two distinct projects: I. The retargeting of viral tropism for the design of oncolytic Herpesviruses: • capable of infecting cells through the human epitelial growth factor receptor 2 (HER2), overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis; • detargeted from its natural receptors, HVEM and nectin1. To this end, we inserted a ligand to HER2 in gD. Because HER2 has no natural ligand, the selected ligand was a single chain antibody (scFv) derived from MAb4D5 (monoclonal antibody to HER2), herein designated scHER2. All recombinant viruses were targeted to HER2 receptor, but only two viruses (R-LM113 and R-LM249) were completely detargeted from HVEM and nectin1. To engineer R-LM113, we removed a large portion at the N-terminus of gD (from aa 6 to aa 38) and inserted scHER2 sequence plus 9-aa serine-glycine flexible linker at position 39. On the other hand, to engineer R-LM249, we replaced the Ig-folded core of gD (from aa 61 to aa 218) with scHER2 flanked by Ser-Gly linkers. In summary, these results provide evidence that: i. gD can tolerate an insert almost as big as gD itself; ii. the Ig-like domain of gD can be removed; iii. the large portion at the N-terminus of gD (from aa 6 to aa 38) can be removed without loss of key function; iv. R-LM113 and R-LM249 recombinants are ready to be assayed in animal models of mammary and ovary tumour. This finding and the avaibility of a large number of scFv greatly increase the collection of potential receptors to which HSV can be redirected. II. The production and purification of recombinant truncated form of the heterodimer gHgL. We cloned a stable insect cell line expressing a soluble form of gH in complex with gL under the control of a metalloprotein inducible promoter and purified the heterodimer by means of ONE-STrEP-tag system by IBA. With respect to biological function, the purified heterodimer is capable: • of reacting to antibodies that recognize conformation dependent epitopes and neutralize virion infectivity; • of binding a variety cells at cell surface. No doubt, the availability of biological active purified gHgL heterodimer, in sufficient quantities, will speed up the efforts to solve its crystal structure and makes it feasible to identify more clearly whether gHgL has a cellular partner, and what is the role of this interaction on virus entry.

Parapoxvirus: basi genomiche della patogenesi

Parapoxvirus (PPV) are member of a genus in the family poxviridae which currently encompasses four species: the prototype orf virus (OV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV) and parapoxvirus of New Zealand red deer (PVNZ). PPVs cause widespread, but localized diseases of small and large ruminants and they can also be transmitted to man. Knowledge of the molecular biology of PPV is still limited as compared to orthopoxviruses, especially vaccinia virus (VACV). The PPV genome displays a high G+C content and relatively small size for poxvirus. Coventional electron microscopy displays PPV virions with ovoid shape and slightly smaller in size than the brickshaped orthopoxviruses. The most striking feature, which readily enables identification of PPV, is a tubule-like structure that surrounds the particle in a spiral fashion. PPV genome organization and content is very similar to that of other poxviruses, the central region contain 88 genes which are present in all poxviruse, in contrast the terminal regions are variable and contain a set of genes unique to the genus PPV. Genes in the near-terminal regions of the genome are frequently not essential for growth in cultured cells encoding factors with important roles in virushost interactions including modulating host immune responses and determining host range. Recently it was suggested that the open reading frames (ORFs) 109 and 110 of the OV genome have a major role in determining species specificity during natural infection in sheep and goats. This hypothesis is based on the analysis of a few number of sequences of different sheep and goats viral isolates. PPV replicate into the cytoplasm of infected cells and produce three structurally different infectious particles: the intracellular mature virions (IMV), intracellular enveloped virions (IEV) and the extracellular enveloped virions (EEV). The vaccinia A33R and A34R hotologue proteins encoded by the ORFS 109 and 110 are expressed in the envelope of the IEV and EEV. The F1L immunodominant protein of orf virus is the major component of the surface tubule structure of the IMV and can post-translationaly insert into membranes via Cterminal, hydrofobic anchor sequence like its orthologue VACV H3L protein. Moreover the F1L protein binds to glycosaminoglycans on the cell surface and has an important role in IMV adsorption to mammalian cells. In this study we investigated the morphogenesis of the PPV through the construction of a mutant virus deleted of the F1L protein. A study of the deleted virus life cycle was conducted in different type of cells and its morphology was observed with electron microscopy. It was demonstared that F1L protein have important role in morphogenesis and infectivity. Moreover it is essential to determine the spiral fashion of the tubule like structure of the virion surface. Some pathogenetic aspects of the PPV infection were studied, in particular the protein implicated in the host range were analysed in detail. An experimental infection with OV and PCPV was conducted in goats and sheep. After infection, the severity of the lesions were comparable in both the animal species. The OV did not result in severe disease neither in sheep nor in goats, suggesting that host factors, rather than virus strain characteristics, may play an important role in the pathogenesis of the Parapoxvirus infections. The PCPV failed to produce any lesion in both sheep and goats, ruling out the possibility of any recombination between PCPV and OV during natural infection in these animal species. The phylogenetic analysis of the ORFs 109 and 110 from several goats and sheep viral isolates showed a clustering based on the antigenic content of the protein that was independent from species and geographic origin.

Untersuchungen zur posttranslationalen präS-Translokation des großen Hüllproteins des Hepatitis-B-Virus

Untersuchungen zur posttranslationalen präS-Translokation des großen Hüllproteins des Hepatitis-B-Virus. Das große (L) Hüllprotin des Hepatitis-B-Virus (HBV) besitzt die ungewöhnliche Eigenschaft, mittels partieller, posttranslationaler Translokation seiner präS-Domäne durch intrazelluläre Membranen zwei unterschiedliche Transmembrantopologieen auszubilden. Unter Berücksichtigung der Hypothese eines HBV-spezifischen Transmembrankanals, der sich möglicherweise während der Virusmorphogenese bilden und die präS-Translokation ermöglichen könnte, wurden Parameter untersucht, welche die L-Topologie beeinflussen. Dazu wurden Wildtyp-L-Proteine und L-Mutanten in Säugerzellen synthetisiert und deren Topologie mittels Proteaseschutzversuchen untersucht. Ich konnte zeigen, daß alle Faktoren, für die angenommen wurde, daß sie für die Ausbildung einer HBV-spezifischen Pore und die damit verbundene präS-Reorientierung wichtig seien, entbehrlich sind. Im einzelnen konnte nachgewiesen werden, daß die posttranslationale präS-Translokation weder die Helferfunktion der HBV S und M Proteine, noch die kovalente Dimerausbildung der Hüllproteine benötigt. Weiterhin ergaben die Untersuchungen, daß keine der amphipathischen Transmembrandomänen des L-Proteins an der präS-Reorientierung beteiligt ist. Vielmehr wurde die hydrophobe Transmembrandomäne 2 (TM2) als ausreichend und essentiell für diesen Prozeß identifiziert. Zellfraktionierungsstudien ergaben weiterhin, daß die präS-Reorientierung und damit die duale Topologie des L-Proteins innerhalb des Endoplasmatischen Retikulums (ER) herbeigeführt wird. Letztlich konnte eine Interaktion des L-Proteins mit zellulären Chaperonen (Hsc70, Hsp40, BiP) gezeigt werden, was eine Beteiligung dieser Proteine am Translokationsprozeß nahelegt.

Der Einfluss einer Cytomegalovirus-Infektion auf das Risiko eines Leukämie-Rezidivs nach Knochenmarktransplantation

Eine Erkrankung durch das humane Cytomegalovirus (hCMV) und ein Rezidiv des Ausgangstumors sind zwei gravierende Komplikationen im Rahmen der Therapie von Malignomen des hämatopoetischen Systems durch Knochenmarktransplantation (KMT). Eine mögliche pathogenetische Interaktion zwischen hCMV-Infektion und einem von einer Minimalen Residualen Leukämie (MRL) ausgehenden Rezidiv wurde bislang in klinischen Verlaufsstudien nach KMT noch nicht systematisch untersucht.In der vorliegenden Arbeit wurde der Einfluss von CMV auf ein Lymphom in einem Modellsystem untersucht. Dazu erweiterten wir das etablierte Modell der murinen CMV (mCMV) Infektion nach experimenteller syngener KMT mit dem Mausstamm BALB/c als Spender und Empfänger durch ein B-Zell-Lymphom als weiteren Parameter. Als Modell-Lymphom diente uns eine mit dem Reportergen lacZ transfizierte, ex tumore klonierte, in der Leber hochgradig tumorigene Variante (Klon E12E) des von BALB/c abgeleiteten B-Zell-Lymphoms A20. Die Arbeiten führten zur Erstbeschreibung einer anti-metastatischen Wirkung der mCMV-Infektion (Erlach at al., 2002; J. Virol. 76: 2857-2870).Im neu etablierten Tiermodell konnte erstmals gezeigt werden, dass eine CMV-Infektion die Ansiedelung eines murinen B-Zell-Lymphoms in der Leber deutlich verringert. Damit wurde die Entstehung einer letalen Tumorerkrankung, abhängig von der Ausgangslast an Tumorzellen signifikant verzögert oder sogar ganz verhindert. Die Suche nach Mechanismen, die diesen antitumoralen Effekt von mCMV verursachen, führte zu folgenden Ergebnissen: Im Unterschied zu onkolytischen Viren basiert die antitumorale Wirkung von mCMV nicht auf einer zytozidalen Infektion der Lymphomzellen. Darüber hinaus zeigt mCMV keine zytostatische oder Apoptose-induzierende Wirkung. Außerdem sind die rekonstituierenden Knochenmarkzellen als Effektoren für den anti-Tumoreffekt auszuschließen. Direkte zytotoxische, sowie systemische Effekte von TNF-alpha konnten als antitumorale Mechanismen ausgeschlossen werden. Die intravenöse Applikation von UV-inaktiviertem Virus zeigte ebenfalls eine Inhibition des Lymphomwachstums, so dass der antitumorale Effekt offenbar durch virale Strukturproteine (Virion-Proteine) ausgelöst wird.Auf der Basis dieser Daten vermuten wir eine Inhibition der Extravasation (transendotheliale Migration/Diapedese) der Tumorzellen aufgrund einer gestörten Kommunikation zwischen Tumorzelle und sinusoidalem Endothel der Leber. Das virale Hüll-Glykoprotein B kann aufgrund seiner signalinduzierenden Wirkung als ein guter Kandidat angesehen werden.

Structural and functional analysis of the hepatitis C virus non-structural protein 5A

Das Hepatitis C Virus (HCV) ist ein umhülltes RNA Virus aus der Familie der Flaviviridae. Sein Genom kodiert für ein ca. 3000 Aminosäuren langes Polyprotein, welches co- und posttranslational in seine funktionellen Einheiten gespalten wird. Eines dieser viralen Proteine ist NS5A. Es handelt sich hierbei um ein stark phosphoryliertes Protein, das eine amphipatische α-Helix im Amino-Terminus trägt, welche für die Membran-Assoziation von NS5A verantwortlich ist. Welche Rolle die Phosphorylierung für die Funktion des Proteins spielt, bzw. welche Funktion NS5A überhaupt ausübt, ist zur Zeit noch unklar. Beobachtungen lassen Vermutungen über eine Funktion von NS5A bei der Resistenz infizierter Zellen gegenüber Interferon-alpha zu. Weiterhin wird vermutet, das NS5A als Komponente des membranständigen HCV Replikasekomplexes an der RNA Replikation beteiligt ist. Das Ziel dieser Doktorarbeit war es, die Funktion von NS5A für die RNA Replikation zu untersuchen. Zu diesem Zweck wurde eine Serie von Phosphorylierungsstellen-Mutanten generiert, die auf Ihre Replikationsfähigkeit und den Phosphorylierungsstatus hin untersucht wurden. Wir fanden, dass bestimmte Serin-Substitutionen im Zentrum von NS5A zu einer gesteigerten RNA Replikation führten, bei gleichzeitig reduzierter NS5A Hyperphosphorylierung. Weiterhin studierten wir den Einfluß von Mutationen in der Amino-terminalen amphipatischen α-Helix von NS5A auf die RNA-Replikation, sowie Phosphorylierung und subzelluläre Lokalisation des Proteins. Wir fanden, dass geringfügige strukturelle Veränderungen der amphipatischen Helix zu einer veränderten subzellulären Lokalisation von NS5A führten, was mit einer reduzierten oder komplett inhibierten RNA Replikation einherging. Zudem interferierten die strukturellen Veränderungen mit der Hyperphosphorylierung des Proteins, was den Schluß nahe legt, dass die amphipatische Helix eine wichtige strukturelle Komponente des Proteins darstellt, die für die korrekte Faltung und Phosphorylierung des Proteins essentiell ist. Als weitere Aspekte wurden die Trans-Komplementationsfähigkeit der verschiedenen viralen Komponenten des HCV Replikasekomplexes untersucht, sowie zelluläre Interaktionspartner von NS5A identifiziert. Zusammenfassend zeigen die Ergebnisse dieser Doktorarbeit, dass NS5A eine wichtige Rolle bei der RNA-Replikation spielt. Diese Funktion wird wahrscheinlich über den Phosphorylierungszustand des Proteins reguliert.