Nucleocapsid formation and RNA synthesis of Marburg virus is dependent on two coiled coil motifs in the nucleoprotein

The nucleoprotein (NP) of Marburg virus (MARV) is responsible for the encapsidation of viral genomic RNA and the formation of the helical nucleocapsid precursors that accumulate in intracellular inclusions in infected cells. To form the large helical MARV nucleocapsid, NP needs to interact with itself and the viral proteins VP30, VP35 and L, which are also part of the MARV nucleocapsid. In the present study, a conserved coiled coil motif in the central part of MARV NP was shown to be an important element for the interactions of NP with itself and VP35, the viral polymerase cofactor. Additionally, the coiled coil motif was essential for the formation of NP-induced intracellular inclusions and for the function of NP in the process of transcription and replication of viral RNA in a minigenome system. Transfer of the coiled coil motif to a reporter protein was sufficient to mediate interaction of the constructed fusion protein with the N-terminus of NP. The coiled coil motif is bipartite, constituted by two coiled coils which are separated by a flexible linker

The Use of a Mobile Laboratory Unit in Support of Patient Management and Epidemiological Surveillance during the 2005 Marburg Outbreak in Angola

A mobile laboratory unit (MLU) was deployed to Uige, Angola as part of the World Health Organization response to an outbreak of viral hemorrhagic fever caused by Marburg virus (MARV). Utilizing mainly quantitative real-time PCR assays, this laboratory provided specific MARV diagnostics in the field. The MLU operated for 88 consecutive days allowing MARV-specific diagnostic response in <4 hours from sample receiving. Most cases were found among females in the child-bearing age and in children less than five years of age including a high number of paediatric cases implicating breastfeeding as potential transmission route. Oral swabs were identified as a useful alternative specimen source to the standard whole blood/serum specimens for patients refusing blood draw. There was a high concordance in test results between the MLU and the reference laboratory in Luanda operated by the US Centers for Disease Control and Prevention. The MLU was an important outbreak response asset providing valuable support in patient management and epidemiological surveillance. Field laboratory capacity should be expanded and made an essential part of any future outbreak investigation

Human Practice. Digital Ecologies. Our Future. : 14. Internationale Tagung Wirtschaftsinformatik (WI 2019) : Tagungsband

Erschienen bei: universi - Universitätsverlag Siegen. - ISBN: 978-3-96182-063-4Aus dem Inhalt: Track 1: Produktion & Cyber-Physische Systeme Requirements and a Meta Model for Exchanging Additive Manufacturing Capacities Service Systems, Smart Service Systems and Cyber- Physical Systems—What’s the difference? Towards a Unified Terminology Developing an Industrial IoT Platform – Trade-off between Horizontal and Vertical Approaches Machine Learning und Complex Event Processing: Effiziente Echtzeitauswertung am Beispiel Smart Factory Sensor retrofit for a coffee machine as condition monitoring and predictive maintenance use case Stakeholder-Analyse zum Einsatz IIoT-basierter Frischeinformationen in der Lebensmittelindustrie Towards a Framework for Predictive Maintenance Strategies in Mechanical Engineering - A Method-Oriented Literature Analysis Development of a matching platform for the requirement-oriented selection of cyber physical systems for SMEs Track 2: Logistic Analytics An Empirical Study of Customers’ Behavioral Intention to Use Ridepooling Services – An Extension of the Technology Acceptance Model Modeling Delay Propagation and Transmission in Railway Networks What is the impact of company specific adjustments on the acceptance and diffusion of logistic standards? Robust Route Planning in Intermodal Urban Traffic Track 3: Unternehmensmodellierung & Informationssystemgestaltung (Enterprise Modelling & Information Systems Design) Work System Modeling Method with Different Levels of Specificity and Rigor for Different Stakeholder Purposes Resolving Inconsistencies in Declarative Process Models based on Culpability Measurement Strategic Analysis in the Realm of Enterprise Modeling – On the Example of Blockchain-Based Initiatives for the Electricity Sector Zwischenbetriebliche Integration in der Möbelbranche: Konfigurationen und Einflussfaktoren Novices’ Quality Perceptions and the Acceptance of Process Modeling Grammars Entwicklung einer Definition für Social Business Objects (SBO) zur Modellierung von Unternehmensinformationen Designing a Reference Model for Digital Product Configurators Terminology for Evolving Design Artifacts Business Role-Object Specification: A Language for Behavior-aware Structural Modeling of Business Objects Generating Smart Glasses-based Information Systems with BPMN4SGA: A BPMN Extension for Smart Glasses Applications Using Blockchain in Peer-to-Peer Carsharing to Build Trust in the Sharing Economy Testing in Big Data: An Architecture Pattern for a Development Environment for Innovative, Integrated and Robust Applications Track 4: Lern- und Wissensmanagement (e-Learning and Knowledge Management) eGovernment Competences revisited – A Literature Review on necessary Competences in a Digitalized Public Sector Say Hello to Your New Automated Tutor – A Structured Literature Review on Pedagogical Conversational Agents Teaching the Digital Transformation of Business Processes: Design of a Simulation Game for Information Systems Education Conceptualizing Immersion for Individual Learning in Virtual Reality Designing a Flipped Classroom Course – a Process Model The Influence of Risk-Taking on Knowledge Exchange and Combination Gamified Feedback durch Avatare im Mobile Learning Alexa, Can You Help Me Solve That Problem? - Understanding the Value of Smart Personal Assistants as Tutors for Complex Problem Tasks Track 5: Data Science & Business Analytics Matching with Bundle Preferences: Tradeoff between Fairness and Truthfulness Applied image recognition: guidelines for using deep learning models in practice Yield Prognosis for the Agrarian Management of Vineyards using Deep Learning for Object Counting Reading Between the Lines of Qualitative Data – How to Detect Hidden Structure Based on Codes Online Auctions with Dual-Threshold Algorithms: An Experimental Study and Practical Evaluation Design Features of Non-Financial Reward Programs for Online Reviews: Evaluation based on Google Maps Data Topic Embeddings – A New Approach to Classify Very Short Documents Based on Predefined Topics Leveraging Unstructured Image Data for Product Quality Improvement Decision Support for Real Estate Investors: Improving Real Estate Valuation with 3D City Models and Points of Interest Knowledge Discovery from CVs: A Topic Modeling Procedure Online Product Descriptions – Boost for your Sales? Entscheidungsunterstützung durch historienbasierte Dienstreihenfolgeplanung mit Pattern A Semi-Automated Approach for Generating Online Review Templates Machine Learning goes Measure Management: Leveraging Anomaly Detection and Parts Search to Improve Product-Cost Optimization Bedeutung von Predictive Analytics für den theoretischen Erkenntnisgewinn in der IS-Forschung Track 6: Digitale Transformation und Dienstleistungen Heuristic Theorizing in Software Development: Deriving Design Principles for Smart Glasses-based Systems Mirroring E-service for Brick and Mortar Retail: An Assessment and Survey Taxonomy of Digital Platforms: A Platform Architecture Perspective Value of Star Players in the Digital Age Local Shopping Platforms – Harnessing Locational Advantages for the Digital Transformation of Local Retail Outlets: A Content Analysis A Socio-Technical Approach to Manage Analytics-as-a-Service – Results of an Action Design Research Project Characterizing Approaches to Digital Transformation: Development of a Taxonomy of Digital Units Expectations vs. Reality – Benefits of Smart Services in the Field of Tension between Industry and Science Innovation Networks and Digital Innovation: How Organizations Use Innovation Networks in a Digitized Environment Characterising Social Reading Platforms— A Taxonomy-Based Approach to Structure the Field Less Complex than Expected – What Really Drives IT Consulting Value Modularity Canvas – A Framework for Visualizing Potentials of Service Modularity Towards a Conceptualization of Capabilities for Innovating Business Models in the Industrial Internet of Things A Taxonomy of Barriers to Digital Transformation Ambidexterity in Service Innovation Research: A Systematic Literature Review Design and success factors of an online solution for cross-pillar pension information Track 7: IT-Management und -Strategie A Frugal Support Structure for New Software Implementations in SMEs How to Structure a Company-wide Adoption of Big Data Analytics The Changing Roles of Innovation Actors and Organizational Antecedents in the Digital Age Bewertung des Kundennutzens von Chatbots für den Einsatz im Servicedesk Understanding the Benefits of Agile Software Development in Regulated Environments Are Employees Following the Rules? On the Effectiveness of IT Consumerization Policies Agile and Attached: The Impact of Agile Practices on Agile Team Members’ Affective Organisational Commitment The Complexity Trap – Limits of IT Flexibility for Supporting Organizational Agility in Decentralized Organizations Platform Openness: A Systematic Literature Review and Avenues for Future Research Competence, Fashion and the Case of Blockchain The Digital Platform Otto.de: A Case Study of Growth, Complexity, and Generativity Track 8: eHealth & alternde Gesellschaft Security and Privacy of Personal Health Records in Cloud Computing Environments – An Experimental Exploration of the Impact of Storage Solutions and Data Breaches Patientenintegration durch Pfadsysteme Digitalisierung in der Stressprävention – eine qualitative Interviewstudie zu Nutzenpotenzialen User Dynamics in Mental Health Forums – A Sentiment Analysis Perspective Intent and the Use of Wearables in the Workplace – A Model Development Understanding Patient Pathways in the Context of Integrated Health Care Services - Implications from a Scoping Review Understanding the Habitual Use of Wearable Activity Trackers On the Fit in Fitness Apps: Studying the Interaction of Motivational Affordances and Users’ Goal Orientations in Affecting the Benefits Gained Gamification in Health Behavior Change Support Systems - A Synthesis of Unintended Side Effects Investigating the Influence of Information Incongruity on Trust-Relations within Trilateral Healthcare Settings Track 9: Krisen- und Kontinuitätsmanagement Potentiale von IKT beim Ausfall kritischer Infrastrukturen: Erwartungen, Informationsgewinnung und Mediennutzung der Zivilbevölkerung in Deutschland Fake News Perception in Germany: A Representative Study of People’s Attitudes and Approaches to Counteract Disinformation Analyzing the Potential of Graphical Building Information for Fire Emergency Responses: Findings from a Controlled Experiment Track 10: Human-Computer Interaction Towards a Taxonomy of Platforms for Conversational Agent Design Measuring Service Encounter Satisfaction with Customer Service Chatbots using Sentiment Analysis Self-Tracking and Gamification: Analyzing the Interplay of Motivations, Usage and Motivation Fulfillment Erfolgsfaktoren von Augmented-Reality-Applikationen: Analyse von Nutzerrezensionen mit dem Review-Mining-Verfahren Designing Dynamic Decision Support for Electronic Requirements Negotiations Who is Stressed by Using ICTs? A Qualitative Comparison Analysis with the Big Five Personality Traits to Understand Technostress Walking the Middle Path: How Medium Trade-Off Exposure Leads to Higher Consumer Satisfaction in Recommender Agents Theory-Based Affordances of Utilitarian, Hedonic and Dual-Purposed Technologies: A Literature Review Eliciting Customer Preferences for Shopping Companion Apps: A Service Quality Approach The Role of Early User Participation in Discovering Software – A Case Study from the Context of Smart Glasses The Fluidity of the Self-Concept as a Framework to Explain the Motivation to Play Video Games Heart over Heels? An Empirical Analysis of the Relationship between Emotions and Review Helpfulness for Experience and Credence Goods Track 11: Information Security and Information Privacy Unfolding Concerns about Augmented Reality Technologies: A Qualitative Analysis of User Perceptions To (Psychologically) Own Data is to Protect Data: How Psychological Ownership Determines Protective Behavior in a Work and Private Context Understanding Data Protection Regulations from a Data Management Perspective: A Capability-Based Approach to EU-GDPR On the Difficulties of Incentivizing Online Privacy through Transparency: A Qualitative Survey of the German Health Insurance Market What is Your Selfie Worth? A Field Study on Individuals’ Valuation of Personal Data Justification of Mass Surveillance: A Quantitative Study An Exploratory Study of Risk Perception for Data Disclosure to a Network of Firms Track 12: Umweltinformatik und nachhaltiges Wirtschaften Kommunikationsfäden im Nadelöhr – Fachliche Prozessmodellierung der Nachhaltigkeitskommunikation am Kapitalmarkt Potentiale und Herausforderungen der Materialflusskostenrechnung Computing Incentives for User-Based Relocation in Carsharing Sustainability’s Coming Home: Preliminary Design Principles for the Sustainable Smart District Substitution of hazardous chemical substances using Deep Learning and t-SNE A Hierarchy of DSMLs in Support of Product Life-Cycle Assessment A Survey of Smart Energy Services for Private Households Door-to-Door Mobility Integrators as Keystone Organizations of Smart Ecosystems: Resources and Value Co-Creation – A Literature Review Ein Entscheidungsunterstützungssystem zur ökonomischen Bewertung von Mieterstrom auf Basis der Clusteranalyse Discovering Blockchain for Sustainable Product-Service Systems to enhance the Circular Economy Digitale Rückverfolgbarkeit von Lebensmitteln: Eine verbraucherinformatische Studie Umweltbewusstsein durch audiovisuelles Content Marketing? Eine experimentelle Untersuchung zur Konsumentenbewertung nachhaltiger Smartphones Towards Predictive Energy Management in Information Systems: A Research Proposal A Web Browser-Based Application for Processing and Analyzing Material Flow Models using the MFCA Methodology Track 13: Digital Work - Social, mobile, smart On Conversational Agents in Information Systems Research: Analyzing the Past to Guide Future Work The Potential of Augmented Reality for Improving Occupational First Aid Prevent a Vicious Circle! The Role of Organizational IT-Capability in Attracting IT-affine Applicants Good, Bad, or Both? Conceptualization and Measurement of Ambivalent User Attitudes Towards AI A Case Study on Cross-Hierarchical Communication in Digital Work Environments ‘Show Me Your People Skills’ - Employing CEO Branding for Corporate Reputation Management in Social Media A Multiorganisational Study of the Drivers and Barriers of Enterprise Collaboration Systems-Enabled Change The More the Merrier? The Effect of Size of Core Team Subgroups on Success of Open Source Projects The Impact of Anthropomorphic and Functional Chatbot Design Features in Enterprise Collaboration Systems on User Acceptance Digital Feedback for Digital Work? Affordances and Constraints of a Feedback App at InsurCorp The Effect of Marker-less Augmented Reality on Task and Learning Performance Antecedents for Cyberloafing – A Literature Review Internal Crowd Work as a Source of Empowerment - An Empirical Analysis of the Perception of Employees in a Crowdtesting Project Track 14: Geschäftsmodelle und digitales Unternehmertum Dividing the ICO Jungle: Extracting and Evaluating Design Archetypes Capturing Value from Data: Exploring Factors Influencing Revenue Model Design for Data-Driven Services Understanding the Role of Data for Innovating Business Models: A System Dynamics Perspective Business Model Innovation and Stakeholder: Exploring Mechanisms and Outcomes of Value Creation and Destruction Business Models for Internet of Things Platforms: Empirical Development of a Taxonomy and Archetypes Revitalizing established Industrial Companies: State of the Art and Success Principles of Digital Corporate Incubators When 1+1 is Greater than 2: Concurrence of Additional Digital and Established Business Models within Companies Special Track 1: Student Track Investigating Personalized Price Discrimination of Textile-, Electronics- and General Stores in German Online Retail From Facets to a Universal Definition – An Analysis of IoT Usage in Retail Is the Technostress Creators Inventory Still an Up-To-Date Measurement Instrument? Results of a Large-Scale Interview Study Application of Media Synchronicity Theory to Creative Tasks in Virtual Teams Using the Example of Design Thinking TrustyTweet: An Indicator-based Browser-Plugin to Assist Users in Dealing with Fake News on Twitter Application of Process Mining Techniques to Support Maintenance-Related Objectives How Voice Can Change Customer Satisfaction: A Comparative Analysis between E-Commerce and Voice Commerce Business Process Compliance and Blockchain: How Does the Ethereum Blockchain Address Challenges of Business Process Compliance? Improving Business Model Configuration through a Question-based Approach The Influence of Situational Factors and Gamification on Intrinsic Motivation and Learning Evaluation von ITSM-Tools für Integration und Management von Cloud-Diensten am Beispiel von ServiceNow How Software Promotes the Integration of Sustainability in Business Process Management Criteria Catalog for Industrial IoT Platforms from the Perspective of the Machine Tool Industry Special Track 3: Demos & Prototyping Privacy-friendly User Location Tracking with Smart Devices: The BeaT Prototype Application-oriented robotics in nursing homes Augmented Reality for Set-up Processe Mixed Reality for supporting Remote-Meetings Gamification zur Motivationssteigerung von Werkern bei der Betriebsdatenerfassung Automatically Extracting and Analyzing Customer Needs from Twitter: A “Needmining” Prototype GaNEsHA: Opportunities for Sustainable Transportation in Smart Cities TUCANA: A platform for using local processing power of edge devices for building data-driven services Demonstrator zur Beschreibung und Visualisierung einer kritischen Infrastruktur Entwicklung einer alltagsnahen persuasiven App zur Bewegungsmotivation für ältere Nutzerinnen und Nutzer A browser-based modeling tool for studying the learning of conceptual modeling based on a multi-modal data collection approach Exergames & Dementia: An interactive System for People with Dementia and their Care-Network Workshops Workshop Ethics and Morality in Business Informatics (Workshop Ethik und Moral in der Wirtschaftsinformatik – EMoWI’19) Model-Based Compliance in Information Systems - Foundations, Case Description and Data Set of the MobIS-Challenge for Students and Doctoral Candidates Report of the Workshop on Concepts and Methods of Identifying Digital Potentials in Information Management Control of Systemic Risks in Global Networks - A Grand Challenge to Information Systems Research Die Mitarbeiter von morgen - Kompetenzen künftiger Mitarbeiter im Bereich Business Analytics Digitaler Konsum: Herausforderungen und Chancen der Verbraucherinformati

Die Rolle der Homooligomerisierung des Polymerasekofaktors VP35 im Vermehrungszyklus des Marburg-Virus

Das Marburg-Virus verursacht eine fieberhafte hämorrhagische Erkrankung bei menschlichen und nichtmenschlichen Primaten, die mit hohen Letalitätsraten einhergeht. Taxonomisch bildet das Marburg-Virus (MARV) zusammen mit dem Ebola-Virus (EBOV) die Familie der Filoviridae. Die Virionen sind aus einem Nukleokapsidkomplex, der über eine Proteinmatrix mit der Virushülle verbunden ist, aufgebaut. Der Nukleokapsidkomplex besteht aus der viralen RNA sowie den vier Nukleokapsidproteinen NP, VP35, VP30 und L, die untereinander durch verschiedene Protein-Protein-Interaktionen verbunden sind. Der Komplex aus VP35 und dem L-Protein fungiert als virale RNA-abhängige RNA Polymerase, wobei L den katalytischen Teil des Enzyms darstellt. In der vorliegenden Arbeit wurde die Homooligomerisierung des Polymerasekofaktors VP35 des MARV hinsichtlich ihrer Funktion für die Synthese der viralen RNA und die Morphogenese neuer Nukleokapsidkomplexe sowie die Interaktion des VP35 mit dem NP untersucht. Die Homooligomerisierung des MARV VP35 wird über eine Coiled-Coil-Struktur im N-Terminus des Proteins vermittelt, die im Bereich der Aminosäuren 70 bis 120 lokalisiert ist. Zwei Leucinreste an den Positionen 90 und 104 waren ausschlaggebend für die Stabilität der Coiled-Coil-Struktur und somit für die Homooligomerisierung des VP35. Ferner konnte gezeigt werden, dass das Coiled-Coil-Motiv des VP35 ausreichend war, um die Protein-Protein-Interaktion zu vermitteln. Die Homooligomerisierung des VP35 bildete die Voraussetzung für die Interaktion mit dem L-Protein und die Ausbildung eines funktionellen Polymerasekomplexes. Im Gegensatz dazu schien die Homooligomerisierung des VP35 für die Bindung an das NP, der strukturgebenden Komponente des Nukleokapsidkomplexes, nicht notwendig zu sein. Über die Interaktion des NP mit dem N-Terminus des VP35 könnte das NP durch das VP35 in Lösung gehalten und während der Replikation der viralen RNA als Substrat für deren Verpackung zur Verfügung gestellt werden. Eine weitere NP-Bindungsstelle auf dem C-Terminus des VP35 scheint eine wichtige Rolle beim Zusammenbau neuer Nukleokapsidkomplexe zu spielen. Die Integrität der Coiled-Coil-Strukturen des VP35 und des NP hatte einen wesentlichen Einfluss auf die Ausbildung und die Struktur der intrazytoplasmatischen Einschlusskörper, die die Reifezentren neuer Nukleokapsidkomplexe darstellen. Zusammenfassend lässt sich festhalten, dass die Homooligomerisierung des MARV VP35 essentiell für dessen Interaktion mit dem L und für die virale Transkription und Replikation ist. Für die direkte Interaktion von VP35 mit NP scheint die Homooligomerisierung des VP35 keine Rolle zu spielen. Es zeichnet sich allerdings ab, dass für die Entstehung von funktionellen Nukleokapsiden die Homooligomerisierung des VP35 notwendig ist

Die Rolle der Homooligomerisierung des Polymerasekofaktors VP35 im Vermehrungszyklus des Marburg-Virus

Das Marburg-Virus verursacht eine fieberhafte hämorrhagische Erkrankung bei menschlichen und nichtmenschlichen Primaten, die mit hohen Letalitätsraten einhergeht. Taxonomisch bildet das Marburg-Virus (MARV) zusammen mit dem Ebola-Virus (EBOV) die Familie der Filoviridae. Die Virionen sind aus einem Nukleokapsidkomplex, der über eine Proteinmatrix mit der Virushülle verbunden ist, aufgebaut. Der Nukleokapsidkomplex besteht aus der viralen RNA sowie den vier Nukleokapsidproteinen NP, VP35, VP30 und L, die untereinander durch verschiedene Protein-Protein-Interaktionen verbunden sind. Der Komplex aus VP35 und dem L-Protein fungiert als virale RNA-abhängige RNA Polymerase, wobei L den katalytischen Teil des Enzyms darstellt. In der vorliegenden Arbeit wurde die Homooligomerisierung des Polymerasekofaktors VP35 des MARV hinsichtlich ihrer Funktion für die Synthese der viralen RNA und die Morphogenese neuer Nukleokapsidkomplexe sowie die Interaktion des VP35 mit dem NP untersucht. Die Homooligomerisierung des MARV VP35 wird über eine Coiled-Coil-Struktur im N-Terminus des Proteins vermittelt, die im Bereich der Aminosäuren 70 bis 120 lokalisiert ist. Zwei Leucinreste an den Positionen 90 und 104 waren ausschlaggebend für die Stabilität der Coiled-Coil-Struktur und somit für die Homooligomerisierung des VP35. Ferner konnte gezeigt werden, dass das Coiled-Coil-Motiv des VP35 ausreichend war, um die Protein-Protein-Interaktion zu vermitteln. Die Homooligomerisierung des VP35 bildete die Voraussetzung für die Interaktion mit dem L-Protein und die Ausbildung eines funktionellen Polymerasekomplexes. Im Gegensatz dazu schien die Homooligomerisierung des VP35 für die Bindung an das NP, der strukturgebenden Komponente des Nukleokapsidkomplexes, nicht notwendig zu sein. Über die Interaktion des NP mit dem N-Terminus des VP35 könnte das NP durch das VP35 in Lösung gehalten und während der Replikation der viralen RNA als Substrat für deren Verpackung zur Verfügung gestellt werden. Eine weitere NP-Bindungsstelle auf dem C-Terminus des VP35 scheint eine wichtige Rolle beim Zusammenbau neuer Nukleokapsidkomplexe zu spielen. Die Integrität der Coiled-Coil-Strukturen des VP35 und des NP hatte einen wesentlichen Einfluss auf die Ausbildung und die Struktur der intrazytoplasmatischen Einschlusskörper, die die Reifezentren neuer Nukleokapsidkomplexe darstellen. Zusammenfassend lässt sich festhalten, dass die Homooligomerisierung des MARV VP35 essentiell für dessen Interaktion mit dem L und für die virale Transkription und Replikation ist. Für die direkte Interaktion von VP35 mit NP scheint die Homooligomerisierung des VP35 keine Rolle zu spielen. Es zeichnet sich allerdings ab, dass für die Entstehung von funktionellen Nukleokapsiden die Homooligomerisierung des VP35 notwendig ist

Homo-Oligomerization of Marburgvirus VP35 Is Essential for Its Function in Replication and Transcription

The nucleocapsid protein VP35 of Marburgvirus, a filovirus, acts as the cofactor of the viral polymerase and plays an essential role in transcription and replication of the viral RNA. VP35 forms complexes with the genome encapsidating protein NP and with the RNA-dependent RNA polymerase L. In addition, a trimeric complex had been detected in which VP35 bridges L and the nucleoprotein NP. It has been presumed that the trimeric complex represents the active polymerase bound to the nucleocapsid. Here we present evidence that a predicted coiled-coil domain between amino acids 70 and 120 of VP35 is essential and sufficient to mediate homo-oligomerization of the protein. Substitution of leucine residues 90 and 104 abolished (i) the probability to form coiled coils, (ii) homo-oligomerization, and (iii) the function of VP35 in viral RNA synthesis. Further, it was found that homo-oligomerization-negative mutants of VP35 could not bind to L. Thus, it is presumed that homo-oligomerization-negative mutants of VP35 are unable to recruit the polymerase to the NP/RNA template. In contrast, inability to homo-oligomerize did not abolish the recruitment of VP35 into inclusion bodies, which contain nucleocapsid-like structures formed by NP. Finally, transcriptionally inactive mutants of VP35 containing the functional homo-oligomerization domain displayed a dominant-negative phenotype. Inhibition of VP35 oligomerization might therefore represent a suitable target for antiviral intervention

VP24 of Marburg Virus Influences Formation of Infectious Particles

The highly pathogenic enveloped Marburg virus (MARV) is composed of seven structural proteins and the nonsegmented negative-sense viral RNA genome. Four proteins (NP, VP35, VP30, and L) make up the helical nucleocapsid, which is surrounded by a matrix that is composed of the viral proteins VP40 and VP24. VP40 is functionally homologous to the matrix proteins of other nonsegmented negative-strand RNA viruses. As yet, the function of VP24 remains elusive. In the present study we found that VP24 colocalized with inclusions in MARV-infected cells that contain preformed nucleocapsids and with nucleocapsids outside the inclusions. Coexpression studies revealed that VP24 is recruited into the inclusions by the presence of NP. Furthermore, VP24 displayed membrane-binding properties and was recruited into filamentous virus-like particles (VLPs) that are induced by VP40. The incorporation of VP24 altered neither the morphology of VLPs nor the budding efficiency of VLPs. When VP24 was silenced in MARV-infected cells by small interfering RNA technology, the release of viral particles was significantly reduced while viral transcription and replication were unimpaired. Our data support the idea that VP24 is essential for a process that takes place after replication and transcription and before budding of virus progeny. It is presumed that VP24 is necessary for the formation of transport-competent nucleocapsids and/or the interaction between the nucleocapsids and the budding sites at the plasma membrane

Inhibition of Filovirus Replication by the Zinc Finger Antiviral Protein

The zinc finger antiviral protein (ZAP) was recently shown to inhibit Moloney murine leukemia virus and Sindbis virus replication. We tested whether ZAP also acts against Ebola virus (EBOV) and Marburg virus (MARV). Antiviral effects were observed after infection of cells expressing the N-terminal part of ZAP fused to the product of the zeocin resistance gene (NZAP-Zeo) as well as after infection of cells inducibly expressing full-length ZAP. EBOV was inhibited by up to 4 log units, whereas MARV was inhibited between 1 to 2 log units. The activity of ZAP was dependent on the integrity of the second and fourth zinc finger motif, as tested with cell lines expressing NZAP-Zeo mutants. Heterologous expression of EBOV- and MARV-specific sequences fused to a reporter gene suggest that ZAP specifically targets L gene sequences. The activity of NZAP-Zeo in this assay was also dependent on the integrity of the second and fourth zinc finger motif. Time-course experiments with infectious EBOV showed that ZAP reduces the level of L mRNA before the level of genomic or antigenomic RNA is affected. Transient expression of ZAP decreased the activity of an EBOV replicon system by up to 95%. This inhibitory effect could be partially compensated for by overexpression of L protein. In conclusion, the data demonstrate that ZAP exhibits antiviral activity against filoviruses, presumably by decreasing the level of viral mRNA

: Autocrine TGF-β and melanoma cell tumorigenicity

We previously identified constitutive Smad signaling in human melanoma cells despite resistance to transforming growth factor-beta (TGF-beta) control of cell proliferation. This led us to investigate the effect of inhibitory Smad7 overexpression on melanoma cell behavior. Using the highly metastatic cell line, 1205-Lu, we thus generated melanoma cell clones constitutively expressing Smad7, and their mock-transfected counterparts. Stable expression of Smad7 resulted in an inhibition of constitutive Smad2/3 phosphorylation, and in a reduced TGF-beta response of Smad3/Smad4-driven gene transactivation, as measured using transfected Smad3/4-specific reporter gene constructs. Smad7 overexpression, however, did not alter their proliferative capacity and resistance to TGF-beta-driven growth inhibition. On the other hand, expression of Smad7 efficiently reduced the capacity of human melanoma cells to invade Matrigel in Boyden migration chambers, while not affecting their motility and adhesion to collagen and laminin. Gelatin zymography identified reduced MMP-2 and MMP-9 secretion by Smad7-expressing melanoma cells as compared with their control counterparts. Smad7-expressing melanoma cells exhibited a dramatically reduced capacity to form colonies under anchorage-independent culture conditions, and, when injected subcutaneously into nude mice, were largely delayed in their ability to form tumors. These results suggest that TGF-beta production by melanoma cells not only affects the tumor environment but also directly contributes to tumor cell aggressiveness through autocrine activation of Smad signaling