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Tesis por compendio / [ES] Los viroides son los patógenos con replicación autónoma más simples y sólo se han encontrado de forma natural infectando plantas superiores. Desde que se descubrieron en los años setenta, se ha adquirido un conocimiento considerable sobre su naturaleza y mecanismos de replicación en las plantas huésped. Sin embargo, aún quedan por descubrir muchos aspectos de la biología de los viroides. Por lo tanto, un conocimiento más profundo de la naturaleza y el modo de acción de los viroides han sido los objetivos principales que engloban esta tesis. Para ello, es esencial contar con procedimientos sencillos y eficientes para la obtención de clones de ADNc infecciosos. Se desarrolló un nuevo método eficiente para construir clones de viroides infecciosos y se probó con un viroide de cada familia: El viroide latente de la berenjena (ELVd, Avsunviroidae) y el viroide del lúpulo (HSVd, Pospiviroidae). Esta aproximación se basó en enzimas de restricción de tipo IIS que cortan fuera del sitio de reconocimiento y supone un procedimiento universal para obtener clones infecciosos de un viroide independientemente de su secuencia, con una alta eficiencia. A pesar de que los viroides han sido considerados como ARN no codificantes desde su descubrimiento, nuestro análisis computacional predijo pequeños marcos de lectura abiertos en cada uno de los genomas de HSVd y ELVd. No se encontraron similitudes significativas con las proteínas de la base de datos de plantas superiores, pero algunos de estos péptidos predichos estaban altamente conservados entre todas las variantes de HSVd y ELVd. Curiosamente, la fusión de estas secuencias conservadas con una proteína fluorescente reveló una localización subcelular específica en el correspondiente orgánulo donde tiene lugar la replicación/acumulación para cada viroide: nucleolo y cloroplasto para HSVd y ELVd, respectivamente. Las mutaciones que truncan el dominio nucleolar de HSVd fueron perjudiciales para el viroide, mientras que el truncamiento de cualquiera de los dos ORF de ELVd que contiene una señal de localización al cloroplasto también disminuyó (pero en menor medida) la eficiencia biológica del viroide, tal vez debido a la redundancia funcional. Se encontraron formas circulares de los ARN de HSVd y ELVd en fracciones polisómicas, lo que revela su interacción física con la maquinaria de traducción de la célula vegetal. En conjunto, estas observaciones experimentales indican que no se puede descartar la capacidad de codificación de los viroides, aunque la prueba definitiva (la detección de los péptidos codificados por los circRNAs) es un reto tecnológico que deberá abordarse en futuras líneas de investigación. Finalmente, para estudiar qué cambios se producen en el huésped durante la infección con un viroide sintomático, se realizó un análisis integrador de las alteraciones genómicas de plantas de pepino infectadas con HSVd. Se integraron los transcriptomas, el sRNAnomas y el metilomas para determinar la respuesta temporal a la infección por el viroide. Nuestros resultados apoyan que el HSVd promueve el rediseño de las vías reguladoras del pepino afectando predominantemente a capas reguladoras específicas en diferentes fases de la infección. La respuesta inicial se caracterizó por una reconfiguración del transcriptoma del hospedador mediante el uso diferencial de exones, seguido de una predominante regulación a la baja de la actividad transcripcional modulada por los cambios epigenéticos del hospedador asociados a la infección y caracterizada por un aumento de la hipermetilación. Las alteraciones en el metabolismo de los ARN pequeños y microARNs del huésped fueron marginales y se produjeron principalmente en la fase tardía. En general, estos datos constituyen el primer mapa exhaustivo de las respuestas de la planta a la infección de un viroide. / [CA] Els viroids són els patògenes més simples amb replicació autònoma i només s'han identificat de forma natural infectant a plantes superiors. Des que es descobriren als anys setanta, s'ha adquirit un coneixement considerable sobre la seua natura i els mecanismes de replicació en plantes hoste. No obstant, encara queden per descobrir molts aspectes de la biologia dels viroids. Per tant, un coneixement més profund de la natura i el mode d'acció dels viroids han sigut els objectius principals que engloben aquesta tesi. Per a això, és essencial la disponibilitat de procediments senzills i eficients per a l'obtenció de clones infecciosos. Es va desenvolupar un nou mètode eficient per a construir clones infecciosos y es fa provar amb un viroid de cada família: el viroide latent de la albergínia (ELVd, Avsunviroidae) y el viroid del llúpol (HSVd, Pospiviroidae). Aquesta aproximació es basà en enzims de restricció de tipus IIS que tallen fora del lloc de reconeixement i suposa un procediment universal per obtenir clones infecciosos de un viroid independentment de la seua seqüencia amb una elevada eficiència. Tot i que els viroids s'han considerat com ARNs no codificants des del seu descobriment, el nostre anàlisi computacional va predir xicotets ORF als genomes de HSVd y ELVd. No es trobaren similituds significatives amb proteïnes depositades a les bases de dades, però alguns d'aquest pèptids estaven altament conservats a les variants de HSVd y ELVd. Curiosament, la fusió d'aquestes seqüencies conservades amb una proteïna fluorescent revelà una localització subcel·lular especifica al orgànul on te lloc la replicació/acumulació de cada viroid: nuclèol i cloroplast per a HSVd i ELVd, respectivament. Les mutacions que trunquen el domini nucleolar de HSVd foren perjudicials per al viroid, mentre que el truncament de qualsevol de les dos ORF de ELVd que contenen una senyal de localització al cloroplast també va disminuir (però en menor mesura) l'eficiència biològica del viroid, el que pot ser degut a una redundància funcional. Es detectaren formes d'ARN circular de HSVd i ELVd a les fraccions polisòmiques, el que revela la seua interacció física amb la maquinaria de traducció cel·lular. En conjunt, aquestes observacions experimentals indiquen que no es pot descartar la capacitat codificants dels viroids, encara que la evidencia definitiva (la detecció del pèptids codificats per ARN circulars) es un repte tecnològic que s'haurà d'adreçar en línies d'investigació futures. Finalment, per tal d'estudiar que canvis es produeixen a l'hoste durant la infecció amb un viroid simptomàtic, es va realitzar un anàlisi integrador de les alteracions genòmiques de les plantes de cogombre infectades amb HSVd. S'integraren els transcriptomes, sARNomes i metilomes per determinar la resposta temporal a la infecció per viroid. Els resultats obtinguts suporten que HSVd promou un redisseny de les vies reguladores de cogombre afectant predominantment a nivells reguladors específics a les diferents etapes de la infecció. La resposta inicial es caracteritzà per una reconfiguració del transcriptoma de l'hoste mitjançant l'ús diferencial d'exons, seguit d'una repressió transcripticional modulada per canvis epigenètics de l'hoste caracteritzats per una major hipermetilació. Les alteracions al metabolisme de ARN xicotets i microARNs de l'hoste van ser marginals i es produïren principalment al final de la infecció. En general, aquestes dades constitueixen el primer mapa exhaustiu de les respostes de la planta a la infecció per un viroid. / [EN] Viroids are the simplest pathogens with autonomous replication and have only been found naturally infecting higher plants. Since viroids were discovered in the seventies, we have gained considerable knowledge about their nature and replication mechanisms in host plants. However, many aspects of viroid biology are yet to be discovered. Therefore, a deeper understanding of the nature and mode of action of viroids have been the encompassing main goals of this thesis. For this purpose, simple and efficient procedures for obtaining infectious cDNA clones are essential. A new efficient method for constructing infectious viroid clones was developed and tested with one viroid of each family: eggplant latent viroid (ELVd, Avsunviroidae) and hop stunt viroid (HSVd, Pospiviroidae). This procedure was based on type IIS restrictions enzymes that cut outside of the recognition site and supposes a universal procedure for obtaining infectious clones of a viroid independently of its sequence, with a high efficiency. Despite viroids have been considered as plant-pathogenic non-coding RNAs since their discovery, our computational analysis predicted small open reading frames in each of the HSVd and ELVd genomes. No significant similarities with proteins in the database of higher plants were found, but some of these predicted peptides were highly conserved among all HSVd and ELVd variants. Interestingly, the fusion of these conserved sequences to a fluorescent protein revealed a specific subcellular localization in the corresponding organelle where replication/accumulation takes place for each viroid: nucleolus and chloroplast for HSVd and ELVd, respectively. Mutations that truncate the nucleolar domain of HSVd were detrimental for the viroid while truncating any of the two ELVd ORF that contains a chloroplast transit signal also diminished (but to a lesser extent) viroid biological efficiency, maybe because of functional redundancy. Circular forms of both, HSVd and ELVd RNAs were found in polysome fractions, revealing their physical interaction with the translational machinery of the plant cell. Altogether, these experimental observations indicate that the coding capacity of viroids cannot be ruled out, although the definitive evidence (detection of the circRNA-encoded peptides) is a technological challenge to be addressed in future research lines. Finally, to study the host changes that are produced during a symptomatic viroid infection, an integrative analysis of the timing and intensity of the genome-wide alterations in cucumber plants infected with HSVd was performed. Differential host transcriptome, sRNAnome and methylome were integrated to determine the temporal response to viroid-infection. Our results support that HSVd promotes the redesign of the cucumber regulatory-pathways predominantly affecting specific regulatory layers at different infection-phases. The initial response was characterized by a reconfiguration of the host-transcriptome by differential exon usage, followed by a predominant down-regulation of the transcriptional activity modulated by the host epigenetic changes associated to infection and characterized by increased hypermethylation. The alterations in host sRNA and microRNA metabolism were marginal and mainly occurred at the late stage. Overall, these data constitute the first comprehensive map of the plant responses to a viroid infection. / La Conselleria d’Educació, Investigació, Cultura i Esports (Generalitat Valenciana) y el Fondo Social Europeo (FSECV 2014-2020) han cofinanciado la contratación del doctorando como personal investigador de carácter predoctoral (ACIF/2017/114) y unas estancias predoctorales fuera de la Comunitat Valenciana (BEFPI/2020). La realización de esta tesis doctoral también se ha realizado en el marco de dos proyectos de investigación del Ministerio de Ciencia, Innovación y Universidades, con cofinanciación de fondos FEDER [BIO2017-88321-R y AGL2016-79825-R] . / Márquez Molins, J. (2022). Novel Insights of Viroid Biology and Host Responses to Their Infection [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/183479 / TESIS / Premios Extraordinarios de tesis doctorales / Compendio

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

The satellite RNA of Lucerne Transient Streak Virus (LTSV) is a 322-nucleotide, single-stranded circular RNA that has a rod-like structure very similar to that of viroids. As it does not encode any translation products and cannot replicate independently of a helper virus, the satellite RNA is proposed to rely on viral-encoded proteins for the replication and/or cell-to-cell movement that facilitate its systemic infection in a host. To investigate the requirements for replication of the LTSV satellite RNA, transgenic plant systems were generated to express the viral RNA-dependent RNA polymerase and predicted viral transport protein independently as well as in combination. Results of infectivity assays of these transgenic lines demonstrated for the first time that the viral-encoded RNA-dependent RNA polymerase is necessary and sufficient for the replication of LTSV satellite RNA, and that no additional viral proteins are required for its cell-to-cell or systemic transport.

The satellite RNA of Lucerne Transient Streak Virus (LTSV) is a 322-nucleotide, single-stranded circular RNA that has a rod-like structure very similar to that of viroids. As it does not encode any translation products and cannot replicate independently of a helper virus, the satellite RNA is proposed to rely on viral-encoded proteins for the replication and/or cell-to-cell movement that facilitate its systemic infection in a host. To investigate the requirements for replication of the LTSV satellite RNA, transgenic plant systems were generated to express the viral RNA-dependent RNA polymerase and predicted viral transport protein independently as well as in combination. Results of infectivity assays of these transgenic lines demonstrated for the first time that the viral-encoded RNA-dependent RNA polymerase is necessary and sufficient for the replication of LTSV satellite RNA, and that no additional viral proteins are required for its cell-to-cell or systemic transport.

Thesis (M.S.)--Oregon State University, 2011. / Printout. Includes bibliographical references (leaves 55-59). Also available on the World Wide Web.

Universiẗat, Diss., 2003--Düsseldorf.

The aim of this work was to identify potential mRNA targets of PTGS triggered by viroid-derived small RNAs (vsRNAs) in PSTVd-infected tomato plants (S. lycopersicum L.). We selected 47 possible gene targets using data provided by Prof. Dr. Steger (Heinrich Heine Universität, Düsseldorf, Germany) - the list of 1633 possible target mRNAs from tomato based on vsRNA:mRNA duplex prediction. The vsRNA sequences were obtained by Illumina sequencing of small RNA libraries from healthy and PSTVd-infected tomato plants. By qRT-PCR analysis we identified 6 genes with significantly altered levels of mRNA in PSTVd-infected tomato plants: CUL1 (protein ubiquitination), ERF4 (transcription factor of abiotic stress signalling pathway), H/ACA1 (rRNA pseudouridylation), NPH3 (transcription factor of fototropic signalling pathway), Sl-MYB (transcription factor regulating leaf development) and TCP3 (transcription factor regulating leaf development). The binary vector pLV07 with inserted expression cassette containing coding sequence of Sl-MYB was prepared for experiments in ?leaf factory? system in N. benthamiana plants. Expression analyses in ?leaf factory? system after 1,5 DPI using qRT-PCR and RNA blots revealed strong inhibition of expression of Sl-MYB in leaf sectors infiltrated with severe PSTVd AS1 strain, while mild PSTVd QFA strain showed minimal change in expression comparing to control sectors. Moreover, the overexpression of Sl-MYB in leaf sectors resulted in development of necroses after 2,5-3 DPI, in presence of silencing suppressor p19 after 2 DPI. The development of necroses was largely inhibited in PSTVd AS1-infiltrated leaf sectors in comparison with PSTVd QFA- and control-infiltrated sectors.