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Potyvirusten HCpro-proteiinin yhteydet kasvisolun mikrotubulusverkostoon ja viruksen replikaatiovesikkeleihin
Get PDFViruses, as intracellular parasites, occupy and rearrange the host cell. Potyviruses (genus Potyvirus), the largest group of plant RNA viruses, infect plants of several families, including the nightshade family (Solanaceae). Potyviruses modify the internal organization of host cell membranes and membranous organelles for the purposes of viral multiplication and movement. At the same time, they have to deceive the receptors of the host cell that guard against external and internal alterations. Virus factories are formed in the perinuclear regions by deploying and concentrating endoplasmic reticulum, Golgi and chloroplasts. The peripheries of the cell, containing cortical microtubules (MTs), are involved in secretion and endocytosis-mediated defence signalling.
Helper component proteinase (HCpro) is a multifunctional protein of potyviruses that participates in subversion of host defence, including RNA silencing. HCpro of Potato virus A (PVA) interacts with HCpro-interacting protein 2 (HIP2) of potato (Solanum tuberosum) and also with viral proteins that accumulate in or near viral replication vesicles. Host eukaryotic translation initiation factors eIF4E or eIF(iso)4E are required for replication, accumulation or systemic movement of potyviruses and are recruited to viral replication vesicles during infection. In this study, the interaction of HCpro with HIP2 and a new interaction of HCpro with eIF4E proteins were studied and their localizations in infected cells were described. Structural predictions of HCpro and HIP2 were applied for more detailed understanding of these interactions.
HIP2 from potato and tobacco (Nicotiana tabacum) were found to be functionally related to a homologous MT-associated protein of Arabidopsis thaliana, SPIRAL2. Virus-induced silencing of HIP2 in Nicotiana benthamiana caused twisted growth, a phenotype typical of defects in cortical MT organization. A similar spiral phenotype of an Arabidopsis spiral2 line was complemented by transgenic expression of potato HIP2. Self-interactions regulated localization and spatial distribution of HIP2 on the cortical MT array. Predicted structural domains, localizations and self-interactions of the plant-specific HIP2 and SPIRAL2 suggested similarities to the eukaryotic multi-TOG-domain proteins CLASP and MAP215 that regulate MT dynamics, and its interactions with the plasma membrane and cell organelles, and participate in endosomal signalling.
Interactions of HCpro and the host proteins were studied in PVA-infected plant cells using bimolecular fluorescence complementation (BiFC). Signals of HIP2 and HCpro interaction occurred in the cell cortex, along MTs and at MT intersections, but also in granules near the chloroplasts. The signals of HCpro interaction with eIF(iso)4E were concentrated in foci that associated with chloroplasts and viral replication vesicles. Replication vesicles of PVA were detected near chloroplasts and occasionally with HCpro and HIP2 at cortical MTs.
Detailed yeast two-hybrid system (YTHS) analysis of HCpro interactions showed that those with HIP2 were regulated by a highly variable region (HVR) in a hinge region of a central domain of HCpro. Sequence comparisons and YTHS assays revealed a conserved eIF4E binding motif (4EBD) at the beginning of a carboxyl-terminal domain of HCpro. Structural models of HCpro made in silico suggested that the HVR formed an exposed loop or coil structure, while 4EBD was within an alpha-helical structure.
Accumulation of PVA was significantly reduced in the HIP2-silenced leaves of N. benthamiana. Similarly, mutations in HVR of HCpro reduced interaction with HIP2 and accumulation of PVA in potato and Nicotiana plants. The silencing-suppression capacity of HVR-mutated HCpro was increased (in 3 types of mutants) or strongly reduced (in one type). The single HVR mutant type that compromised the ability of HCpro to suppress silencing or interact with HIP2 in YTHS greatly reduced accumulation of PVA, but did not prevent replication and movement of the mutated virus. These results confirmed that interaction of HCpro with HIP2 enhances virus accumulation.
Three of the HVR-mutated viruses were hypervirulent, causing necrotic symptoms novel to PVA in systemically infected leaves of Nicotiana. A microarray analysis showed activation of the hypersensitive response and signalling by jasmonic acid and ethylene. Structural modelling of HCpro indicated that mutations in HVR altered the conformation of its whole hinge region while the fold of 4EBD remained unaffected. Together with aberrant or disrupted HIP2 interactions, the conformational changes may have triggered host defence.
Several discoveries were made during the course of this thesis. It was found, for instance, that the HCpro interactions with HIP2 are determined by a structurally important but variable region, while those with eIF4E proteins are determined by a highly conserved motif, 4EBD, that is amongst the first of its kind identified in viruses. It was also found that HIP2 has similarities to conserved eukaryotic MT proteins involved in MT dynamics and signalling. MTs are a well-exploited area of research in animal virology, but this is the first report of a role of an MT interaction in virus accumulation in plants and also the first report of an interaction of a potyvirus protein with an MT-associated protein, and its localization with MTs. Considering that HCpro located both at MTs and near the viral replication vesicles, and that it interacted with eIF4E proteins targeted to those vesicles, HCpro may be involved in allocation of intracellular resources or regulation of host signalling for sustained infection. Future developments have potential for the improvement of virus resistance in plants.Potyvirukset ovat suuri virusryhmä, joka aiheuttaa sato- ja laatutappioita kaikissa viljelykasveissa, mm. perunalla (Solanaceae-heimo). Virustauteja ei voida torjua kemiallisesti, joten virusvapaa lisäysmateriaali ja perinnöllinen viruskestävyys ovat kasvinsuojelun kulmakiviä. Virukset ovat solunsisäisiä loisia, jotka käyttävät ja muokkaavat solun rakenteita ja resursseja lisääntymiseensä ja samalla tyrehdyttävät solun viruspuolustuksen. Täten virusten ja kasvien tuottamien proteiinien väliset suorat vuorovaikutukset ovat avainasemassa kasvien alttiuden ymmärtämiseksi ja kestävyysominaisuuksien löytämiseksi.
Potyvirusten perimän monistus eli replikaatio tapahtuu solulimakalvostosta irtoavissa vesikkeleissä, joita kuljetetaan viherhiukkasten läheisyyteen. Potyvirusten monitoimintainen HCpro-proteiini toimii viruksen monistumisessa ja häiritsee kasvin puolustusta, etenkin RNA-hiljennystä, joka on virusten torjunnan perusmekanismi. Perunan A-viruksen (PVA) HCpro-proteiini kykenee tarrautumaan perunan HIP2-mikrotubulusproteiiniin. Kasvisoluissa mikrotubulukset muodostavat solukalvon alaisen verkoston, joka osallistuu soluelinten ankkuroitumiseen ja solun puolustuksen herättäviin viestiketjuihin. Joitakin kasviproteiineja tiedetään kaapattavan replikaatiovesikkeleihin ja tarvittavan viruksen lisääntymiseen. Tällaisia ovat translaation aloitustekijä eIF4E proteiinit, joiden viruksille sopimattomat muodot tekevät kasvista virusta kestäviä.
Tässä työssä tutkittiin PVA:n HCpro-proteiinin ja kasviproteiinien vuorovaikutusta elävissä, viroottisissa kasvisoluissa. Niiden solunsisäistä sijaintia havainnoitiin fluoresoivien merkkiproteiinien avulla. HCpro-proteiini oli PVA:n tartuttamissa soluissa kosketuksissa sekä mikrotubulusproteiini HIP2:n että translaatiotekijä eIF4E:n kanssa. Vuorovaikuttaessaan eIF4E:n kanssa HCpro sijaitsi viruksen replikaatiovesikkelien läheisyydessä. HIP2:n yhteydessä HCpro sijaitsi mikrotubuluksilla ja mikrotubulusverkostossa havaittiin replikaatiovesikkeleitä.
Vuorovaikutuksille tärkeitä proteiinirakenteita tarkasteltaessa löydettiin HCpro:ssa seitsemän aminohapon pituinen alue, joka säätelee vuorovaikutusta eIF4E:n kanssa ja jonka aminohapot ovat konservoituneita potyvirusten välillä. HIP2-vuorovaikutusta sääteli muunteleva alue. Kun kasvin HIP2-proteiinin määrää vähennettiin estämällä ao. geenin ilmentymistä tai muutettiin viruksen HCpro-proteiinia HIP2:een heikommin tarttuvaksi, HIP2:n voitiin todeta olevan välttämätön PVA:n tehokkaalle monistumiselle. HIP2-vuorovaikutuksen suhteen heikennetty PVA herätti kasvin puolustuksen, johtaen kuolio-oireisiin. HIP2-proteiinin rakenteessa havaittiin yhteneväisyyttä mikrotubuluksia sääteleviin ja solunsisäisen viestinnän proteiineihin. Rakennemallinnuksen perusteella HCpro-muunnokset muuttivat HCpro:n laskostumista. Osa muunnetuista HCpro-proteiineista esti RNA-hiljennystä pidempään kuin muuntamaton HCpro. Nämä HCpro-muodot lisäsivät lehdissä tuotetun vierasproteiinin saantoa, mitä voidaan soveltaa esimerkiksi lääkeaineiden tuotantoon kasveissa.
Saatua uutta tietoa HCpro:n vuorovaikutuksista HIP2:n ja eIF4E:n kanssa voidaan käyttää näiden proteiinien virukselle sopimattomien geenimuotojen etsimiseksi ja sellaisten hyödyntämiseksi viruskestävien kasvien jalostuksessa.ei saavutettav
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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