Involvement of the host RNA N 6-adenosine methylation (m6A) pathway in the infection cycle of Alfalfa mosaic virus Mireya Martínez Pérez September 2020 Directors: Prof. Vicente Pallás Benet Dr. Frederic Aparicio Herrero Dr. Jesús Ángel Sánchez Navarro Don Vicente Pallás Benet, Doctor en Ciencias Biológicas, Profesor de Investigación del Consejo Superior de Investigaciones Científicas del Instituto de Biología Molecular y Celular de Plantas (Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas) de Valencia. Don Frederic Aparicio Herrero, Doctor en Biología, Profesor Contratado Doctor (Universidad Politécnica de Valencia) de Valencia. Don Jesús Ángel Sánchez Navarro, Doctor en Biología, Científico Titular del Consejo Superior de Investigaciones Científicas del Instituto de Biología Molecular y Celular de Plantas (Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Científicas) de Valencia. CERTIFICAN: Que Doña Mireya Martínez Pérez, Licenciada en Biotecnología por la Universidad Politécnica de Valencia, ha realizado bajo su dirección el trabajo que con título “Involvement of the host RNA N6- adenosine methylation (m6A) pathway in the infection cycle of Alfalfa mosaic virus” presenta para optar al grado de Doctor en Biotecnología por la Universidad Politécnica de Valencia. Y para que así conste a los efectos oportunos, firman el presente certificado en Valencia a ____ de _____________ de 2020. Vicente Pallás Benet Frederic Aparicio Herrero Jesús Ángel Sánchez Navarro Summary .............................................................................................................................. 12 Resumen ............................................................................................................................... 13 Resum ................................................................................................................................... 16 Abbreviations ...................................................................................................................... 21 General introduction .......................................................................................................... 25 1. Virus concept .................................................................................................................. 27 2. Alfalfa mosaic virus (AMV) .......................................................................................... 28 2.1. Origin, geographical distribution and economic impact ................................................ 28 2.2. Viral transmission and symptomatology ........................................................................... 29 2.3. Genome organization and life cycle .................................................................................. 30 2.3.1. Replication, translation and encapsidation .............................................................................. 32 2.3.2. Cell-to-cell and systemic movement ......................................................................................... 34 2.4. CP interaction proteins ........................................................................................................ 36 3. RNA granules ................................................................................................................. 37 3.1. Interplay between cytoplasmic RNA granules and viral infections .............................. 39 4. RNA m6A methylation .................................................................................................. 41 4.1. m6A machinery...................................................................................................................... 42 4.1.1. m6A writers ..................................................................................................................................... 43 4.1.2. m6A erasers ..................................................................................................................................... 44 4.1.3. m6A readers ..................................................................................................................................... 45 4.2. Roles of m6A ......................................................................................................................... 48 4.2.1. Molecular functions ..................................................................................................................... 48 4.2.2. Biological functions ..................................................................................................................... 50 5. m6A modification in animal viral infections ............................................................... 52 5.1. Viral regulation mediated by m6A-modified vRNA ........................................................ 53 5.1.1. Positive m6A-mediated viral regulation .................................................................................... 53 5.1.2. Negative m6A-mediated viral regulation .................................................................................. 56 5.1.3. Mixed m6A-mediated viral regulation ....................................................................................... 56 5.2. Indirect m6A-mediated viral regulation ............................................................................. 61 6. m6A methylation in plant viral infections: everything is about to be done ............ 63 Motivation and objectives .................................................................................................. 67 Chapter I .............................................................................................................................. 71 Arabidopsis m6A demethylase activity modulates viral infection of a plant virus and the m6A abundance in its genomic RNAs .......................................................................................... 73 Chapter II ........................................................................................................................... 103 The Arabidopsis m6A readers ECT2, ECT3 and ECT5 restrict infection of Alfalfa mosaic virus .................................................................................................................................................. 105 General discussion ............................................................................................................ 143 Conclusions ....................................................................................................................... 153 References .......................................................................................................................... 157 Summary/Resumen/Resum Summary Alfalfa mosaic virus (AMV) affects the global production of alfalfa and other important economical crops, e.g. potato (Solanum tuberosum), tomato (Solanum lycopersicum) or soybean (Glycine max). This virus is the only member of Alfamovirus genus, belonging to the Bromoviridae family, and its genome consists of three positive-sense single-stranded RNA molecules. AMV RNAs are capped (m7GG) at the 5’UTR and present a homologous sequence at the 3’UTR that adopts two mutually exclusive conformations, which were proposed to act as a molecular switch from translation to replication. The two subunits of the viral replicase, P1 and P2, are encoded from the RNA 1 and RNA 2, respectively. RNA 3 encodes the movement protein (MP) and the capsid protein (CP), although the latest is translated from a subgenomic RNA (sgRNA 4). Many studies show that viral CPs are multifunctional proteins implied in diverse steps of the infection cycle and, additionally, AMV and ilarvirus CPs acquire a special relevance, since the presence of CP or sgRNA 4 molecules are required to onset the infection. Therefore, the identification of host CP-interactors is a prevalent strategy to shed light on the molecular mechanisms governing viral infections. Post-transcriptional chemical modifications can affect intramolecular interactions or the RNA recognition by different RNA-binding proteins and, thus, entail a new level of gene expression modulation. N6-methylation of adenosine (m6A) is the most abundant internal modification of mRNA across eukaryotes. Proteins known as m6A writers and erasers define the methylation state of an mRNA, whereas m6A readers recognize this modified nucleotide and drive the fate of methylated mRNAs. In mammals, erasers belong to the AlkB (2- oxoglutarate- and Fe(II)-dependent oxygenase) protein family, which in Escherichia coli protects nucleic acids against methylation damage, whereas m6A readers comprise RNA-binding proteins with a so-called YT521-B homology (YTH) domain. At the beginning of this Thesis, some components of the methylation complex had been characterized in plants, whereas 13 homologs of AlkB (atALKBH1-10B) and 13 proteins of the YTH family (EVOLUTIONARILY CONSERVED C-TERMINAL REGION proteins 1-11, ECT1-11, At4g11970 and Cleavage and Polyadenylation Specificity Factor 30 (CPSF30)) had been identified in the Arabidopsis genome. However, unlike mammals and yeast, no functional roles had been described for any of these proteins. Besides, since the early 70s, several reports have brought to light the presence of m6A residues in viral RNAs from mammalian viruses with RNA and DNA genomes, and the critical roles that this modification and the m6A host machinery play regulating viral infections. However, no m6A-containing plant virus had been reported so