New hepatic cell lines for research, drug testing and vaccine development: Molecular tools for high-throughput screening of HCV replicating cells

Hepatitis C virus (HCV) is a main contributor to chronic liver disease, causing a major social and economic burden worldwide. There is a demand for the development of a vaccine to prevent transmission and eliminate HCV infection. This development has been limited by the lack of research and improved tools, including in vitro hepatic cell model permissive to HCV wild-type infection. This work aimed at developing and validating the molecular tools for a high-throughput screening of highly competent hepatic cell lines in supporting the HCV life-cycle, and implementing and optimizing protocols to produce and handle HCV. This molecular tool consisted of a full-length replicon based on the J6/C tagged with a GFP reporter, enabling to identify HCV permissive cells. Since the tagged replicon consists of a RNA molecule, we started by optimizing method for delivering into Huh-7.5 cells, using Lipofectamine MessengerMAX, and by implementing an in vitro transcription (IVT) protocol. Additionally, we establish reporter plasmids to produce control transcripts, suiting the purpose of IVT validation and to function as a transfection internal control. As a mechanism for translation initiation in these reporter constructions, two possibilities were evaluated: a cap-dependent mechanism, using ARCA, and a capindependent mechanism, based on four types of IRES. After establishing the protocols to produce and deliver the tagged replicon into cells, the functionality of this RNA construction was confirmed by the reporter gene expression, when transfected in Huh-7.5 cell line. After the validation of the reporter capacity of the tagged replicon, to continuo in the follow-up of this thesis, new hepatic cell lines established by immortalization from primary human hepatocytes will be assessed their ability to support the HCV life cycle. This work contributed to create a methodology for screening improved hepatic cell lines to better serve research, drug testing and vaccine development against HCV

Internal Ribosome Entry Site (IRES)-Mediated Translation and Its Potential for Novel mRNA-Based Therapy Development

This article belongs to the Special Issue mRNA Metabolism in Health and Disease 2.0.Many conditions can benefit from RNA-based therapies, namely, those targeting internal ribosome entry sites (IRESs) and their regulatory proteins, the IRES trans-acting factors (ITAFs). IRES-mediated translation is an alternative mechanism of translation initiation, known for maintaining protein synthesis when canonical translation is impaired. During a stress response, it contributes to cell reprogramming and adaptation to the new environment. The relationship between IRESs and ITAFs with tumorigenesis and resistance to therapy has been studied in recent years, proposing new therapeutic targets and treatments. In addition, IRES-dependent translation initiation dysregulation is also related to neurological and cardiovascular diseases, muscular atrophies, or other syndromes. The participation of these structures in the development of such pathologies has been studied, yet to a far lesser extent than in cancer. Strategies involving the disruption of IRES-ITAF interactions or the modification of ITAF expression levels may be used with great impact in the development of new therapeutics. In this review, we aim to comprehend the current data on groups of human pathologies associated with IRES and/or ITAF dysregulation and their application in the designing of new therapeutic approaches using them as targets or tools. Thus, we wish to summarise the evidence in the field hoping to open new promising lines of investigation toward personalised treatments.This work was partially supported by UID/MULTI/04046/2019 Research Unit grant (to BioISI)info:eu-repo/semantics/publishedVersio

Studying and targeting the functions of p53 mRNA in carcinogenesis

Tese de mestrado, Biologia Humana e Ambiente, Universidade de Lisboa, Faculdade de Ciências, 2019A maioria da tradução do ácido ribonucleico mensageiro (mRNA, do inglês ribonucleic acid) é assegurada pela tradução canónica dependente da cap (guanina metilada adicionada à extremidade 5' do pré-mRNA), processo que constitui o maior gasto de energia e recursos na célula. Assim, em resposta a várias condições adversas, como hipóxia, infecção viral, privação de nutrientes e fatores de crescimento, stresse do retículo endoplasmático, entre outras, o mecanismo de tradução dependente da cap é suprimido. No entanto, nestas situações adversas, é de extrema importância a expressão de certas proteínas que regulam a adaptação e a reparação de danos na célula. Sob estas condições limitantes, alguns mRNAs garantem a tradução destas proteínas essenciais através de mecanismos alternativos de iniciação da tradução não canónica. O supressor tumoral p53, codificado pelo gene TP53, é uma proteína reguladora fulcral em condições celulares muito desfavoráveis. Através de processos de tradução canónica, iniciação interna da tradução e de splicing alternativo (excisão de diferentes exões do transcrito), o gene TP53 promove a expressão de pelo menos quinze isoformas da proteína p53. As isoformas da p53 têm alvos únicos de transcrição, geralmente vinculados a funções não redundantes, e demonstram capacidade de resposta a diferentes sinais de stresse. As diferentes características das isoformas da p53 permitem a sua participação em várias vias de regulação celular, nomeadamente a reparação do ácido desoxirribonucleico (DNA, do inglês deoxyribonucleic acid), regulação do ciclo celular, apoptose, metabolismo e envelhecimento. A expressão da p53 é regulada por vários mecanismos de degradação que incluem a poliubiquitinação mediada por Hdm2 (do inglês murine double minute 2 human homolog) e consequente degradação proteossomal. Apesar da rigorosa regulação da p53 é frequente a associação desta proteína com fenómenos carcinogénicos que levam à formação de tumores particularmente agressivos e resistentes a tratamentos. O gene TP53 constitui um alvo estratégico para as células transformadas, uma vez que a expressão aberrante das isoformas da p53 lhes permite superar as adversidades impostas pelo microambiente desfavorável característico no desenvolvimento tumoral. A investigação da nossa equipa está grandemente direcionada para o estudo da isoforma Δ160p53, especificamente o estudo do IRES (do inglês, internal ribosome entry site) da Δ160p53 recentemente identificado, uma vez que as células que sobre-expressam esta isoforma registam elevada proliferação celular e capacidade de invasão particularmente agressiva, em comparação com as características induzidas por outras isoformas. De acordo com a linha de investigação da nossa equipa, a abordagem que propomos para o estudo do mecanismo de iniciação da tradução mediado por IRES Δ160p53 prende-se na identificação de pequenos compostos farmacológicos que inibem o IRES Δ160p53. Com esse objectivo, começámos por estabelecer as condições ideais, a linha celular e o sistema mais indicado para a triagem desses compostos. Simultaneamente, clonámos plasmídeos resistentes à neomicina contendo um sistema bicistrónico com dois genes repórteres RLuc e FLuc (do inglês, Renilla Luciferase e Firefly Luciferase, respetivamente), usando sequências contendo Δ160p53 IRES já disponíveis no laboratório, a fim de obter células eucarióticas estáveis para os ensaios de seleção de compostos farmacológicos. No período em que decorreram estes procedimentos não foi possível testar os plasmídeos clonados e avaliar o seu desempenho em ensaios de seleção dos compostos farmacológicos, no entanto apresentamos a melhor estratégia de clonagem entre as duas abordagens aplicadas. Paralelamente, o nosso laboratório investe na identificação de outros mRNAs de proteínas que frequentemente se encontram alteradas no cancro, para além do mRNA da p53, e que são traduzidos através de IRES e especificamente regulados pelo Hdm2, uma vez que este pode atuar como ITAF (do inglês, IRES transacting factor), regulando positivamente estas proteínas. Deste modo, modificámos a sequência de Hdm2 disponível no laboratório através de mutagénese direcionada ao codão 395. O codão 395 possui uma Serina que é fosforilada após danos infligidos no DNA, o que por sua vez, desencadeia a atividade do Hdm2 como ITAF. Assim, alterámos a serina para uma alanina, que não pode ser fosforilada e portanto, não será capaz de se ligar aos mRNAs. A segunda mutagénese modificou a Serina para Ácido Aspártico, dado que este mimetiza a Serina fosforilada, induzindo permanentemente a atividade do Hdm2 como ITAF. Estas sequências foram enviadas para o laboratório do Departamento de Genética Humana do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), em Lisboa, Portugal, para realizar co-imunoprecipitação (co-IP) dos mRNAs ligados a Hdm2. Inicialmente, os investigadores focaram-se na regulação da p53 ao nível proteico, no entanto com o surgimento de evidências relativas às funções reguladoras dos mRNAs, para além do seu conhecido papel na tradução, uma nova atenção direcionou-se para o mRNA da p53. De facto, foi demonstrado que o mRNA da p53 tem uma função protetora sobre a proteína p53, bloqueando a sua degradação sob stresse celular. Adicionalmente, descobertas recentes correlacionaram a modificação pós-transcripcional m6A (modificação do mRNA de mamíferos em bases de adenina por adição de um grupo metilo na posição 6 do nitrogénio) com o início de tradução independente da cap. Dado o recente surgimento de indícios que apontam para existência de modificações m6A localizadas nas proximidades de IRES, e a sua potencial relação com o início da tradução mediada por IRES, propusemos a determinação desta modificação na adenina do codão 213, um codão comummente mutado na isoforma Δ160p53. Para este fim, optimizámos a ligação de sondas de DNA híbrido (sondas de DNA modificadas com ribonucleótidos) ao RNA total extraído de HeLa (linha celular derivada de cancro de colo do útero humano) e A549 (linha celular derivada de carcinoma de pulmão humano). Foram desenhados dois conjuntos de sondas para cada uma de três sequências: para a sequência de interesse Δ160p53 contendo o codão 213, para o controlo positivo MALAT1 e para o controlo negativo composto pela sequência Δ160p53 contendo o codão de iniciação AUG160. Os produtos obtidos foram amplificados por PCR e separados por separação em gel de agarose, em que a observação de bandas indica a ausência de metilação da adenina nas sequências analisadas. Apesar de não ter sido possível determinar se a adenina do codão 213 se encontra metilada, demonstrámos que as sondas projectadas se ligam devidamente às sequencias seleccionadas e que ocorreu ligação entre as sondas por via da T3 ligase utilizada. Com o cumprimento dos objetivos definidos para a presente dissertação, procuramos discernir quais são as melhores abordagens para a triagem dos compostos farmacológicos direcionados à inibição do IRES Δ160p53, para além de produzir ferramentas essenciais para os ensaios de Hdm2-coIP e para a triagem dos fármacos. Por outro lado, com a adoção de um novo método para a determinação da modificação m6A em mRNA da isoforma Δ160p53, ambicionamos estabelecer as bases para os estudos da função desta modificação na tradução não-canónica do mRNA. Por fim, com a presente dissertação, esperamos contribuir para a valiosa investigação sobre os mecanismos de tradução mediada pelo IRES e fornecer novos dados que auxiliem o desenvolvimento de novas estratégias terapêuticas.The majority of mRNA translation is ensured by the canonical cap-dependent translation, which represents the cellular process with the biggest expense of energy and resources. Thus, in response to various adverse conditions such as hypoxia, viral infection, nutrients and growth factors starvation, endoplasmic-reticulum stress (ER-stress), among others, the canonical cap-dependent translation mechanism is supressed. However, in these adverse situations the expression of certain proteins that mediate adaptation and damage repair is of extreme importance. Under these limiting conditions some key mRNAs ensure the translation of these pivotal proteins through alternative noncanonical translation initiation mechanisms. One well-known example of a regulatory protein that is expressed even in very unfavourable conditions is the tumour suppressor p53. The TP53 gene, containing three promoters and together with alternative internal initiation and alternative splicing, expresses at least fifteen reported isoforms. The p53 isoforms have been shown to have unique transcription targets, often linked to non-redundant functions, and demonstrate responsiveness to different specific stress signals. These findings help to better understand how p53 integrates such a variety of pathways including DNA repair, cell cycle regulation, apoptosis, metabolism and even aging. Having such a wide range of functions that deeply impact the cellular survival, p53 expression is regulated through a variety of mechanisms that include the Hdm2 (murine double minute 2 human homolog) mediated poly-ubiquitination and 26S-mediated proteasomal degradation. However, despite its tight regulation, p53 proteins are frequently reported to grant cells with carcinogenic-like features, causing particularly aggressive and treatment-resistant tumours. Indeed, TP53 gene is the most commonly mutated gene in cancer, representing a strategic target for transformed cells, as the aberrant expression of the p53 protein isoforms offers the means to overcome the extremely challenging microenvironments, characteristic in cancer onset and tumour development. Our team's research has been greatly focused on the Δ160p53 isoform, specifically the study of the recently identified Δ160p53 IRES, as this isoform promotes, to a greater extent than other p53 isoforms, nefarious carcinogenic-like functions, namely invasion and cellular proliferation. One approach we propose to better understand the Δ160p53 IRES-mediated translation initiation mechanism is the identification of small drug compounds that inhibit Δ160p53 IRES. For this purpose, we started by establishing the optimal conditions, cell line and the system for this drug screening. Simultaneously, we cloned neomycin resistant plasmid constructs containing a bicistronic system with two reporter genes (Renilla Luciferase and Firefly Luciferase), using already available Δ160p53 IRES containing sequences, in order to select stable eukaryotic cells for the drug screening assays. Even though at the time of this thesis' experiments we did not have the chance to test the cloned plasmids and evaluate their performance in the drug screening assays, we present the best cloning strategy out of the two approaches used. In parallel, our laboratory has invested in the identification of other cancer-related IREStranslated mRNAs specifically regulated by Hdm2, as it is known that Hdm2 can act as an ITAF, positively regulating commonly deregulated proteins in cancer. With this in mind, we modified the Hdm2 sequence using site directed mutagenesis to create two missense mutations in the 395th codon. The 395th codon has a Serine that is phosphorylated upon DNA damage, triggering the Hdm2's activity as an ITAF. Thus, we altered the Serine to an Alanine, which cannot be phosphorylated, therefore it will not be able to bind to mRNAs. The second mutagenesis aimed the modification of the Serine to Aspartic acid. The aspartic acid mimics the phosphorylated Serine, thereby permanently inducing the Hdm2's activity as an ITAF. These sequences were sent to the partner laboratory of the Department of Human Genetics in Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), in Lisbon, Portugal, to perform co-immunoprecipitation (co-IP) assays of Hdm2-bound mRNAs. For a long time, researchers focused on the p53 regulation at the protein level however, attentions started to turn to the p53 mRNA as more evidences of mRNA's regulatory functions, beyond the well studied role in translation, appeared. Indeed, it was demonstrated that p53 mRNA has a protective function over p53 protein by blocking its degradation under cellular stress, resembling a regulatory "noncoding"-like function. Furthermore, recent findings have correlated the mammalian mRNA post-transcriptional modification on adenine bases by a methyl group in the nitrogen-6 position (m6A) with cap-independent translation initiation. We were intrigued by reports that pointed to the existence of m6A modifications located in the vicinities of IRESs, suggesting a potential correlation of this alteration with IRES-mediated translation initiation. Therefore, we proposed to determine if the adenine of the 213th codon, a commonly mutated codon of the Δ160p53 isoform, bears the m6A modification. To identify the methylation on Δ160p53 mRNA at one-nucleotide resolution we optimized hybrid-DNA probe binding and T3 ligase experiments, using total RNA extracted from HeLa (Human cervical cancer-derived cell line) and A549 (Human lung carcinomaderived cell line). Two sets of probes were custom made for the Δ160p53 segment of interest containing the 213th codon, and also for both MALAT1 positive control and Δ160p53 sequence containing AUG160, used as negative control. The obtained products were amplified through PCR, and separated by agarose gel separation. Even though it was not possible to determine if the adenine of the 213th codon is methylated, we were able to demonstrate the designed probes are binding to the complementary sequences, and also that the T3 ligase is successfully joining the left and right probes. With the fulfilment of the objectives defined for the present thesis, we seek to discern which are the best approaches for the drug screening, targeted to the inhibition of the Δ160p53 IRES, as well as offer essential tools for the Hdm2-coIP assays and drug screening. Additionally, by undertaking a new method in our laboratory to determine the existence of the still poorly understood m6A mRNA modification in Δ160p53, we ambition to lay the foundations for further studies in regard to this modification's function in non-canonical mRNA translation. Ultimately, with the present thesis we hope to assist the valuable investigation regarding the mechanisms of IRES-mediated translation, and to provide new insights that support the development of new therapeutic strategies

Unanticipated Antigens: Translation Initiation at CUG with Leucine

Major histocompatibility class I molecules display tens of thousands of peptides on the cell surface for immune surveillance by T cells. The peptide repertoire represents virtually all cellular translation products, and can thus reveal a foreign presence inside the cell. These peptides are derived from not only conventional but also cryptic translational reading frames, including some without conventional AUG codons. To define the mechanism that generates these cryptic peptides, we used T cells as probes to analyze the peptides generated in transfected cells. We found that when CUG acts as an alternate initiation codon, it can be decoded as leucine rather than the expected methionine residue. The leucine start does not depend on an internal ribosome entry site–like mRNA structure, and its efficiency is enhanced by the Kozak nucleotide context. Furthermore, ribosomes scan 5′ to 3′ specifically for the CUG initiation codon in a eukaryotic translation initiation factor 2–independent manner. Because eukaryotic translation initiation factor 2 is frequently targeted to inhibit protein synthesis, this novel translation mechanism allows stressed cells to display antigenic peptides. This initiation mechanism could also be used at non-AUG initiation codons often found in viral transcripts as well as in a growing list of cellular genes

Improving virus production through quasispecies genomic selection and molecular breedings

Virus production still is a challenging issue in antigen manufacture, particularly with slow-growing viruses. Deep-sequencing of genomic regions indicative of efficient replication may be used to identify high-fitness minority individuals suppressed by the ensemble of mutants in a virus quasispecies. Molecular breeding of quasispecies containing colonizer individuals, under regimes allowing more than one replicative cycle, is a strategy to select the fittest competitors among the colonizers. A slow-growing cell culture-adapted hepatitis A virus strain was employed as a model for this strategy. Using genomic selection in two regions predictive of efficient translation, the internal ribosome entry site and the VP1-coding region, high-fitness minority colonizer individuals were identified in a population adapted to conditions of artificially-induced cellular transcription shut-off. Molecular breeding of this population with a second one, also adapted to transcription shut-off and showing an overall colonizer phenotype, allowed the selection of a fast-growing population of great biotechnological potential

Structure of the 5′ Untranslated Region of Enteroviral Genomic RNA

Enteroviral RNA genomes share a long, highly structured 5= untranslated region (5= UTR) containing a type I internal ribosome entry site (IRES). The 5= UTR is composed of stably folded RNA domains connected by unstructured RNA regions. Proper folding and functioning of the 5= UTR underlies the efficiency of viral replication and also determines viral virulence. We have characterized the structure of 5= UTR genomic RNA from coxsackievirus B3 using selective 2=-hydroxyl acylation analyzed by primer extension (SHAPE) and base-specific chemical probes in solution. Our results revealed novel structural features, including realignment of major domains, newly identified long-range interactions, and an intrinsically disordered connecting region. Together, these newly identified features contribute to a model for enteroviral 5= UTRs with type I IRES elements that links structure to function during the hierarchical processes directed by genomic RNA during viral infection. IMPORTANCE: Enterovirus infections are responsible for human diseases, including myocarditis, pancreatitis, acute flaccid paralysis, and poliomyelitis. The virulence of these viruses depends on efficient recognition of the RNA genome by a large family of host proteins and protein synthesis factors, which in turn relies on the threedimensional folding of the first 750 nucleotides of the molecule. Structural information about this region of the genome, called the 5= untranslated region (5= UTR), is needed to assist in the process of vaccine and antiviral development. This work presents a model for the structure of the enteroviral 5= UTR. The model includes an RNA element called an intrinsically disordered RNA region (IDRR). Intrinsically disordered proteins (IDPs) are well known, but correlates in RNA have not been proposed. The proposed IDRR is a 20-nucleotide region, long known for its functional importance, where structural flexibility helps explain recognition by factors controlling multiple functional states

Real-Time Temporal Dynamics of Bicistronic Expression Mediated by Internal Ribosome Entry Site and 2A Cleaving Sequence

Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.National Research Foundation of Korea (NRF) of the Ministry of Science and ICT [NRF-2017R1C1B2008775, NRF-2017R1A4A1015534, NRF-2018M3C7A1022310]; KBRI basic research program through Korea Brain Research Institute - Ministry of Science and ICT [17-BR-04]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Bioinformatic analyses of mammalian 5'-UTR sequence properties of mRNAs predicts alternative translation initiation sites

<p>Abstract</p> <p>Background</p> <p>Utilization of alternative initiation sites for protein translation directed by non-AUG codons in mammalian mRNAs is observed with increasing frequency. Alternative initiation sites are utilized for the synthesis of important regulatory proteins that control distinct biological functions. It is, therefore, of high significance to define the parameters that allow accurate bioinformatic prediction of alternative translation initiation sites (aTIS). This study has investigated 5'-UTR regions of mRNAs to define consensus sequence properties and structural features that allow identification of alternative initiation sites for protein translation.</p> <p>Results</p> <p>Bioinformatic evaluation of 5'-UTR sequences of mammalian mRNAs was conducted for classification and identification of alternative translation initiation sites for a group of mRNA sequences that have been experimentally demonstrated to utilize alternative non-AUG initiation sites for protein translation. These are represented by the codons CUG, GUG, UUG, AUA, and ACG for aTIS. The first phase of this bioinformatic analysis implements a classification tree that evaluated 5'-UTRs for unique consensus sequence features near the initiation codon, characteristics of 5'-UTR nucleotide sequences, and secondary structural features in a decision tree that categorizes mRNAs into those with potential aTIS, and those without. The second phase addresses identification of the aTIS codon and its location. Critical parameters of 5'-UTRs were assessed by an Artificial Neural Network (ANN) for identification of the aTIS codon and its location. ANNs have previously been used for the purpose of AUG start site prediction and are applicable in complex. ANN analyses demonstrated that multiple properties were required for predicting aTIS codons; these properties included unique consensus nucleotide sequences at positions -7 and -6 combined with positions -3 and +4, 5'-UTR length, ORF length, predicted secondary structures, free energy features, upstream AUGs, and G/C ratio. Importantly, combined results of the classification tree and the ANN analyses provided highly accurate bioinformatic predictions of alternative translation initiation sites.</p> <p>Conclusion</p> <p>This study has defined the unique properties of 5'-UTR sequences of mRNAs for successful bioinformatic prediction of alternative initiation sites utilized in protein translation. The ability to define aTIS through the described bioinformatic analyses can be of high importance for genomic analyses to provide full predictions of translated mammalian and human gene products required for cellular functions in health and disease.</p