The viral coat protein is regulated by HSP70 and HSP40 in Potato virus A infection

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Title: The viral coat protein is regulated by HSP70 and HSP40 in Potato virus A infection
Author: Hafrén, Anders
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences, Department of Biosciences
University of Helsinki, Faculty of Agriculture and Forestry, Department of Food and Environmental Sciences,
Publisher: Helsingin yliopisto
Date: 2010-11-26
Thesis level: Doctoral dissertation (article-based)
Abstract: Plus-stranded (plus) RNA viruses multiply within a cellular environment as tightly integrated units and rely on the genetic information carried within their genomes for multiplication and, hence, persistence. The minimal genomes of plus RNA viruses are unable to encode the molecular machineries that are required for virus multiplication. This sets requisites for the virus, which must form compatible interactions with host components during multiplication to successfully utilize primary metabolites as building blocks or metabolic energy, and to divert the protein synthesis machinery for production of viral proteins. In fact, the emerging picture of a virus-infected cell displays tight integration with the virus, from simple host and virus protein interactions through to major changes in the physiological state of the host cell. This study set out to develop a method for the identification of host components, mainly host proteins, that interact with proteins of Potato virus A (PVA; Potyvirus) during infection. This goal was approached by developing affinity-tag based methods for the purification of viral proteins complexed with associated host proteins from infected plants. Using this method, host membrane-associated viral ribonucleoprotein (RNP) complexes were obtained, and several host and viral proteins could be identified as components of these complexes. One of the host proteins identified using this strategy was a member of the heat shock protein 70 (HSP70) family, and this protein was chosen for further analysis. To enable the analysis of viral gene expression, a second method was developed based on Agrobacterium-mediated virus genome delivery into plant cells, and detection of virally expressed Renilla luciferase (RLUC) as a quantitative measure of viral gene expression. Using this method, it was observed that down-regulation of HSP70 caused a PVA coat protein (CP)-mediated defect associated with replication. Further experimentation suggested that CP can inhibit viral gene expression and that a distinct translational activity coupled to replication, referred to as replication-associated translation (RAT), exists. Unlike translation of replication-deficient viral RNA, RAT was dependent on HSP70 and its co-chaperone CPIP. HSP70 and CPIP together regulated CP turnover by promoting its modification by ubiquitin. Based on these results, an HSP70 and CPIP-driven mechanism that functions to regulate CP during viral RNA replication and/or translation is proposed, possibly to prevent premature particle assembly caused by CP association with viral RNA.Virus är obligata parasiter och kräver en cellulär miljö för att kopiera sig själv. Denna miljö kan erbjudas av bakterier eller högre organismer som växter och djur. Alla celler eller organismer fungerar inte som värd för alla virus, specifika virus klarar sig endast i specifika värdorganismer. Dessa kallas för kompatibla virus och värd kombinationer. Då en kompatibel samverkan mellan virus och värd existerar, uppstår en infektion, och under infektionens lopp samverkar virusets komponenter, dvs. virus genomet i sig och proteiner som kodas av det, med komponenter som erbjuds av värd cellen, inklusive proteiner och membran. Samtidigt utgör den cellulära miljön en resurs varifrån virus erhåller materia och energi för att kopiera sig själv och utvecklas. Denna studie har koncentrerat sig på att identifiera värd proteiner som deltar och reglerar en kompatibel infektion mellan modellväxten Nicotiana benthamiana och Potatis virus A. Ett av växtproteinerna som kunde identifieras, ser ut att delta i infektionen genom att destinera virusets höljes protein för nedbrytning, och denna process krävs för effektiv kopiering av viruset. Därmed föreslås att modellviruset i denna studie har inkorporerat växtkomponenter som fungerar i nedbrytning av växtens egna proteiner, för att degradera sitt höljes protein i ett tidigt skede av infektionen, och därigenom uppnå temporär kontroll över ackumulering av detta protein. Virusproteinet ifråga kan verka negativt på vissa kritiska processer i virusets infektionsförlopp, trots att det är absolut nödvändigt för viruset att spridas både i och emellan växter.
Subject: biologia
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