Descoberta de novos vírus vegetais e estudo da diversidade viral intrahospedeiro a partir de dados gerados por sequenciamento em larga escala

Dissertação (mestrado)—Universidade de Brasília, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2018.As tecnologias de sequenciamento em larga escala permitem a caracterização genômica das comunidades virais presentes em tecidos vegetais e animais e em amostras ambientais com alta sensibilidade e acurácia. Devido ao sequenciamento simultâneo de várias sequências genômicas, essa técnica também permite o estudo da alta diversidade genética intra-hospedeiro apresentada pelos vírus de RNA. Nesse trabalho, estudamos e estabelecemos um pipeline para a análise de viroma em planta utilizando o modelo de pepino, reportamos a descoberta de dois novos vírus em videiras, Grapevine enamovirus1 (GEV-1) e Grapevine virga-like virus (GVLV). Após ensaios de amplificação rápida das extremidades do cDNA (rapid amplification of cDNA ends – RACE) da extremidade 5' do genoma do GEV-1, foi descrito a sequência genômica quase completa desse vírus (6227 bp), possibilitando a sua classificação como um membro do gênero Enamovirus (família Luteoviridae) com base na sua organização genômica, estudos filogenéticos e critérios estabelecidos pelo Comitê Internacional de Taxonomia de Vírus (International Committee on Taxonomy of Viruses – ICTV). Entretanto, o genoma do GVLV permanece parciamente sequenciado em duas partes: um contig de 3348 bp que contém os domínios metiltransferase (Met) e helicase (Hel); e um contig de 1272 bp que corresponde à RNA polimerase dependente de RNA (RdRp) parcial. Com base em estudos filogenéticos não foi possível classificar esse vírus, que mostra baixa identidade com ambas as famílias Virgaviridae e Bromoviridae. Adicionalmente, esse trabalho apresenta um estudo da diversidade genética intra-hospedeiro dos vírus associados ao enrolamento da folha da videira (Grapevine leafroll-associated virus – GLRaV), com foco na poliproteína dos GLRaV-2 e -3 (gêneros Closterovirus e Ampelovirus, respectivamente), assim como a detecção in silico de uma molécula defectiva de RNA do GLRaV-4 (Ampelovirus), a partir de dados gerados por HTS. As populações intra-hospedeiro encontradas em dois isolados de GLRaV-2 mostraram apenas 11 polimorfismos de único nucleotídeo (single nucleotide polymorphisms – SNPs) em comum (~14% dos SNPs em cada isolado). A diversidade intra-hospedeiro encontrada em dois isolados de GLRaV-3 foi baixa se comparada com os isolados de GLRaV-2.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).High-throughput sequencing technologies allow for the genomic characterization of viral communities present in plant and animal tissues and environmental samples with high accuracy and sensibility. The simultaneous sequencing of various genomic sequences by this technique also makes it useful for the study of the high intrahost genetic diversity presented by RNA viruses. In this work, we studied and established the conditions of analysis of plant virome using the cucumber model, the discovery of two novel grapevine viruses, Grapevine enamovirus-1 (GEV-1) and Grapevine virga-like virus (GVLV). After rapid amplification of cDNA ends (RACE) assays of the 5' end of GEV-1 genome, we obtained the near full genomic sequence of this virus (6227 bp), enabling its classification as a member of the genus Enamovirus (family Luteoviridae) based on its genomic properties, phylogenetic studies and criteria stablished by the International Committee on Taxonomy of Viruses (ICTV). However, the genome of GVLV remains only partially sequenced, separated in two parts: a 3348 bp contig containing the methyltranferase (Met) and helicase (Hel) domains; and a 1272 bp contig which corresponds to the partial RNA dependent RNA polimerase (RdRp). Based on phylogenetic studies, were not able to classify this novel virus, which shows low identity with viruses in the families Virgaviridae and Bromoviridae. Additionally, this works presents a study on the intrahost genetic diversity of Grapevine leafroll-associated viruses (GLRaVs), focusing on the polyprotein of GLRaV-2 and -3 (genera Closterovirus and Ampelovirus, respectively), as well as an in silico detection of a defective RNA molecule of GLRaV-4 (Ampelovirus). The intrahost population of two isolates of GLRaV-2 showed only 11 single nucleotide polymorphisms (SNPs) in common (~14 of the SNPs found on each isolate). The intrahost genetic diversity found on two isolates of GLRaV3 was low compared to GLRaV-2

Exploring the biochemical roles of TbeIF4E-3 AND TbeIF4E-5 in T. brucei cell cycle

African Sleeping Sickness afflicts millions of people in sub-Saharan areas of the continent. The disease causing agent, the unicellular eukaryotic protozoan Trypanosoma brucei, is intensely studied, yet no safe cure has been developed and has become a public health issue. Because gene expression control in T. brucei exhibits unique patterns, it is attractive for drug targeting. Our lab aims to explore the cellular and molecular role(s) of TbEIF4E-3 (4E-3p) and TbeIF4E-5 (4E-5p), structurally divergent homologs of eukaryotic translation initiation factor 4E (eIF4E), a canonical mRNA 5’ cap binding protein involved in post-translational regulation of gene expression. The 4E-3 and 4E-5 proteins are essential in cell cycle in trypanosomes. We hypothesize 4E-3p and 4E-5p to be key regulons, proteins that regulate the translation of distinct sets of multiple, functionally related mRNAs required for normal cell cycle. Evidence from immunofluorescence microscopy (IMF) by other labs and our own revealed cytoplasmic localization of 4E-3p in mRNP granules. Tandem co-immunoprecipitation (co-IP) and mass spectrometry (MS) revealed 4E-3p association with canonical protein partners found in other eukaryotes known to be involved in translation initiation and control such as eIF4G and eIF4A, consistent with earlier studies. Significantly, this present work further identified unique uncharacterized polypeptides, implying both conserved as well as novel functions. Additionally, RNAseq analysis from co-IPs revealed that 4E-3p also associates with various mRNAs, some of which are involved in the cell cycle, providing evidence towards potential translational regulation of cell cycle. By contrast, 4E-5p appeared more associated with the mitochondria from IMF studies, consistent with findings by others. Combined co-IP and MS analysis showed association with translational regulators, whereas RNAseq revealed association with mRNAs regulating cell division and replication control distinct from those associated with 4E-3p. These findings suggest that both 4E-3p and 4E-5p may act separately to regulate cell cycle, replication, and gene expression in T. brucei in a manner consistent with a proposed Parallel RNA Regulon Model. Among potential drug targets being analyzed are translational regulating proteins, of which 4E-3p and 4E-5p are attractive candidates due to their discovered role in normal cell cycle

A transcriptomics approach to understanding polymorphic and transcript level differences linked to isoquinoline alkaloid production in triploid varieties of narcissus pseudonarcissus

The Amaryllidaceae have characteristic isoquinoline alkaloids including galanthamine that is approved for treatment of Alzheimer’s disease. The daffodil (Narcissus pseudonarcissus) is an industrial source of this alkaloid. This project undertook analysis of the daffodil transcriptome as an approach to understanding this alkaloid biosynthetic pathway. Material from the basal plate of var. Carlton was analysed using the Roche 454 GS FLX Titanium and Illumina HiSeq platforms to assemble reference transcripts (45324 transcripts from 454, 165065 from Illumina). Annotation was via a bespoke BLAST pipeline utilizing UniProt, TAIR, Rfam and RefSeq. Further functional annotation and enrichment studies were carried out using the DAVID platform encompassing KEGG, GO and EC annotations. Illumina HiSeq sequencing of a second variety, Andrew’s Choice, was used alongside the reference transcripts to identify SNPs and transcript level differences. A bioinformatics method to determine ploidy indicated both varieties were triploid, in agreement with microscopy results. The level of selected transcripts was also assessed using qPCR. Several transcripts putatively involved in alkaloid biosynthesis were identified. Comp75950_c0_s1 showed homology to a C4H gene from peppers and could be involve in protocatechuic acid biosynthesis in daffodils. Two transcripts, Daff106212 and Contig1404, were predicted to catalyse the synthesis of norbelladine from protocatechuic acid and tyramine, and its subsequence conversion to 4’-O-methylnorbelladine. Finally, transcripts HDA57HA0AK3FX and Daff88927 were suggested for the final step in galanthamine biosynthesis, an intermolecular phenol coupling. This is the first transcriptomic comparison of two daffodil varieties and is an important resource for further investigation into genes involved in Amaryllidaceae alkaloid biosynthesis