Insights to Protein Pathogenicity from the Lens of Protein Evolution

As protein sequences evolve, differences in selective constraints may lead to outcomes ranging from sequence conservation to structural and functional divergence. Evolutionary protein family analysis can illuminate which protein regions are likely to diverge or remain conserved in sequence, structure, and function. Moreover, nonsynonymous mutations in pathogens may result in the emergence of protein regions that affect the behavior of pathogenic proteins within a host and host response. I aimed to gain insight on pathogenic proteins from cancer and viruses using an evolutionary perspective. First, I examined p53, a conformationally flexible, multifunctional protein mutated in ~50% of human cancers. Multifunctional proteins may experience rapid sequence divergence given trade-offs between functions, while proteins with important functions may be more constrained. How, then, does a protein like p53 evolve? I assessed the evolutionary dynamics of structural and regulatory properties in the p53 family, revealing paralog-specific patterns of functional divergence. I also studied flaviviruses, like Dengue and Zika virus, whose conformational flexibility contributes to antibody-dependent enhancement (ADE). ADE has long complicated vaccine development for these viruses, making antiviral drug development an attractive alternative. I identified fitness-critical sites conserved in sequence and structure in the proteome of flaviviruses with the potential to act as broadly neutralizing antiviral drug target sites. I later developed Epitopedia, a computational method for epitope-based prediction of molecular mimicry. Molecular mimicry occurs when regions of antigenic proteins resemble protein regions from the host or other pathogens, leading to antibody cross-reactivity at these sites which can result in autoimmunity or have a protective effect. I applied Epitopedia to the antigenic Spike protein from SARS-CoV-2, the causative agent of COVID-19. Molecular mimicry may explain the varied symptoms and outcomes seen in COVID-19 patients. I found instances of molecular mimicry in Spike associated with COVID-19-related blood-clotting disorders and cardiac disease, with implications on disease treatment and vaccine design

The Prediction of miRNAs in SARS-CoV-2 Genomes: hsa-miR Databases Identify 7 Key miRs Linked to Host Responses and Virus Pathogenicity-Related KEGG Pathways Significant for Comorbidities.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a member of the betacoronavirus family, which causes COVID-19 disease. SARS-CoV-2 pathogenicity in humans leads to increased mortality rates due to alterations of significant pathways, including some resulting in exacerbated inflammatory responses linked to the "cytokine storm" and extensive lung pathology, as well as being linked to a number of comorbidities. Our current study compared five SARS-CoV-2 sequences from different geographical regions to those from SARS, MERS and two cold viruses, OC43 and 229E, to identify the presence of miR-like sequences. We identified seven key miRs, which highlight considerable differences between the SARS-CoV-2 sequences, compared with the other viruses. The level of conservation between the five SARS-CoV-2 sequences was identical but poor compared with the other sequences, with SARS showing the highest degree of conservation. This decrease in similarity could result in reduced levels of transcriptional control, as well as a change in the physiological effect of the virus and associated host-pathogen responses. MERS and the milder symptom viruses showed greater differences and even significant sequence gaps. This divergence away from the SARS-CoV-2 sequences broadly mirrors the phylogenetic relationships obtained from the whole-genome alignments. Therefore, patterns of mutation, occurring during sequence divergence from the longer established human viruses to the more recent ones, may have led to the emergence of sequence motifs that can be related directly to the pathogenicity of SARS-CoV-2. Importantly, we identified 7 key-microRNAs (miRs 8066, 5197, 3611, 3934-3p, 1307-3p, 3691-3p, 1468-5p) with significant links to KEGG pathways linked to viral pathogenicity and host responses. According to Bioproject data (PRJNA615032), SARS-CoV-2 mediated transcriptomic alterations were similar to the target pathways of the selected 7 miRs identified in our study. This mechanism could have considerable significance in determining the symptom spectrum of future potential pandemics. KEGG pathway analysis revealed a number of critical pathways linked to the seven identified miRs that may provide insight into the interplay between the virus and comorbidities. Based on our reported findings, miRNAs may constitute potential and effective therapeutic approaches in COVID-19 and its pathological consequences

A novel interaction between the 5′ untranslated region of the virus genome and Musashi homolog 2 is essential for Chikungunya virus genome replication

Chikungunya virus (CHIKV) is a single-stranded, positive-sense alphavirus of the Togaviridae family and is transmitted among humans via Aedes spp. mosquitos. Typical symptoms of CHIKV infection include debilitating arthralgia which can persist for months or years. The recent re-emergence of CHIKV raises serious global health concerns due to high rates of morbidity and the lack of licensed antiviral drugs or clinically approved vaccines. Current knowledge about the molecular mechanisms controlling CHIKV replication and virus-host interactions is limited. Previous studies from our group have mapped six stem-loops within the 5′ untranslated region (5′ UTR) and the first ~200nt of ORF-1. Phenotypic analysis demonstrated that they are RNA replication elements (RREs) required for virus genome replication through structuredependent mechanisms, which involve vertebrate and invertebrate-specific factors. However, the aspect of molecular virology of how the RREs function or through what interactions are yet to be investigated. In this study, reverse genetics and biochemical approaches were used to identify and confirm a specific interaction between cellular RNA binding protein Musashi homolog 2 and this structured region of the CHIKV genome. Using electromobility shift assay, I confirmed the direct interaction between MSI2 and the 5′ UTR of the CHIKV genome, with the binding site being the single-stranded region upstream of the AUG start codon. Using infectious virus and sub-genomic replicon systems, combined with RNA silencing and drug inhibition assays, it was demonstrated for the first time that MSI2 is required for CHIKV genome replication. A CHIKV trans-complementation system and strand-specific qRT-PCR were used to show that MSI2 is required for the initiation of negative-strand synthesis, possibly by functioning as a molecular switch for translation and replication as MSI2 also interacts directly or indirectly with viral non-structural proteins nsP1 and nsP3 – both are essential components of the viral replication complex. These findings provide novel insights into how CHIKV exploits cellular components for its replication and identify potential targets for antiviral therapy

Insights into the Molecular Mechanisms of the N6-Methyladenosine (m6A) Methylation Machinery in the Regulation of the Infection Cycle of RNA Plant Viruses

[ES] La N6-metiladenosina (m6A) es una modificación generalizada en los ARN celulares de diferentes organismos que puede afectar muchos procesos y vías celulares. En las plantas, ocurre mediante un complejo de metilación que contiene varias proteínas: MTA, MTB, FIP37, VIR y HAKAI. Esta modificación es eliminada por desmetilasas de la familia AlkB, mientras que los miembros de la familia ETC son las proteínas mejor descritas que reconocen y procesan los ARN m6A-modificados. Estudios de epitransciptómica viral han revelado un papel igualmente importante de m6A durante la infección por virus; sin embargo, no existe una función pro- o antiviral de m6A generalizada. El laboratorio donde se ha llevado a cabo este trabajo ha sido pionero en el estudio del efecto de m6A en la interacción planta-virus, utilizando como virus modelo el AMV. El AMV pertenece a la familia Bromoviridae, y su genoma está formado por tres (+)ssARN. Los ARN1/2 codifican las subunidades de replicasa (P1 y P2), mientras que el ARN3 codifica la proteína de movimiento (MP) y sirve como molde para la síntesis del sgARN4, que codifica la proteína de cubierta (CP). Al comienzo de esta tesis, nuestro laboratorio ya había informado sobre: la presencia de supuestos motivos m6A en el 3'UTR/RNA3, una región crítica para la replicación de AMV, la primera m6A-desmetilasa de Arabidopsis (ALKBH9B), la relevancia funcional de ALKBH9B para mantener niveles adecuados de m6A/A para la correcta replicación de AMV, la capacidad de la CP de AMV para interactuar con ALKBH9B, posiblemente para usurpar la actividad de ALKBH9B, y la capacidad de las proteínas de Arabidopsis ECT2/3/5 para interactuar con el ARNv de AMV que contienen m6A. Dada la relevancia funcional de m6A en la biología de AMV, en esta tesis se decidió profundizar en el conocimiento de las implicaciones del mecanismo de regulación de m6A en el ciclo infeccioso viral de AMV. Para ello, se decidió: profundizar en la comprensión funcional de la m6A-desmetilasa ALKBH9B, evaluar la función in vivo de los supuestos dos sitios m6A presentes en el 3'UTR/ARN3, y explorar una posible implicación de algunas m6A metiltransferasas en la infección causada por AMV. El mapeo de los subdominios funcionales de atALKBH9B determinó la presencia de IDRs en la región N-terminal, dentro del dominio interno similar a AlkB y en la región C-terminal. Alrededor del 78% del RBD identificado en ALKBH9B está contenido en el IDR C-terminal. Debido a que las IDRs se localizan con frecuencia en proteínas que se someten a LLPS, un proceso que probablemente contribuye a la formación y estabilidad de los gránulos de ARN, es posible que las IDR y la RBD de ALKBH9B puedan actuar de manera cooperativa para promover la formación de gránulos de ARN. El análisis de los putativos motivos DRACH localizados en el bucle de hpB y en el tallo inferior de hpE del 3'UTR/ARN3 de AMV demostró que son sitios críticos involucrados en la replicación in vivo de AMV. La identidad de los residuos 2012A, 2013A y 2014A en el bucle hpB parece ser un requisito estructural clave para la replicación y/o acumulación de AMV. Con respecto a hpE, nuestros resultados determinaron que el supuesto residuo de m6A (1902A), así como el apareamiento de bases del tallo inferior de hpE, también son requisitos esenciales para la síntesis in vivo de ARNs de cadena positiva en AMV. Hasta donde sabemos, esta es la primera evidencia en AMV que muestra que el bucle de hpB y el tallo inferior de hpE están involucrados en la replicación/acumulación viral y la síntesis de ARNs de cadena positiva, respectivamente. Finalmente, en cuanto al estudio de la influencia de las m6A-metiltransferasas en el ciclo de infección viral de AMV, no se determinó un efecto proviral y/o antiviral en el complejo m6A-ARNm metiltransferasa conformado por atMTA:atMTB, ni en el putativo complejo m6A- ARNr metiltransferasa conformado por atMETTL5-like:atTRMT112-like sobre la biología de AMV.[CA] La N6-metiladenosina (m6A) és una modificació generalitzada en els ARN cellulars de diferents organismes que pot afectar molts processos i vies cellulars. En les plantes, ocorre mitjançant un complex de metilació que conté diverses proteïnes: MTA, MTB, FIP37, VIR i HAKAI. Aquesta modificació és eliminada per desmetilasas de la família AlkB, mentre que els membres de la família ETC són les proteïnes més ben descrites que reconeixen i processen els ARN m6A-modificats. Estudis de epitransciptómica viral han revelat un paper igualment important de m6A durant la infecció per virus; no obstant això, no existeix una funció pro- o antiviral de m6A generalitzada. El laboratori on s'ha dut a terme aquest treball ha sigut pioner en l'estudi de l'efecte de m6A en la interacció planta-virus, utilitzant com a virus model el AMV. El AMV pertany a la família Bromoviridae, i el seu genoma està format per tres (+) ssARN. Els ARN1/2 codifiquen les subunitats de replicasa (P1 i P2), mentre que l'ARN3 codifica la MP i serveix com a motle per a la síntesi del sgARN4, que codifica la CP. Al començament d'aquesta tesi, el nostre laboratori ja havia informat sobre: la presència de suposats motius m6A en el 3'UTR/RNA3, una regió crítica per a la replicació de AMV, la primera m6A-desmetilasa de Arabidopsis (ALKBH9B), la rellevància funcional d'ALKBH9B per a mantindre nivells adequats de m6A/A per a la correcta replicació de AMV, la capacitat de la CP de AMV per a interactuar amb ALKBH9B, possiblement per a usurpar l'activitat d'ALKBH9B, i la capacitat de les proteïnes de Arabidopsis ECT2/3/5 per a interactuar amb el ARNv de AMV que contenen m6A. Donada la rellevància funcional de m6A en la biologia de AMV, en aquesta tesi es va decidir aprofundir en el coneixement de les implicacions del mecanisme de regulació de m6A en el cicle infecciós viral de AMV. Per a això, es va decidir: aprofundir en la comprensió funcional de la m6A-desmetilasa ALKBH9B, avaluar la funció in vivo dels supòsits dos llocs m6A presents en el 3'UTR/ARN3, i explorar una possible implicació d'algunes m6A metiltransferasas en la infecció causada per AMV. El mapatge dels subdominis funcionals de atALKBH9B va determinar la presència de IDRs a la regió N-terminal, dins del domini intern similar a AlkB i a la regió C-terminal. Al voltant del 78% del RBD identificat en ALKBH9B està contingut en el IDR C-terminal. Pel fet que les IDRs es localitzen amb freqüència en proteïnes que se sotmeten a LLPS, un procés que probablement contribueix a la formació i estabilitat dels grànuls d'ARN, és possible que les IDR i la RBD d'ALKBH9B puguen actuar de manera cooperativa per a promoure la formació de grànuls d'ARN. L'anàlisi dels putatius motius DRACH localitzats en el bucle de hpB i en la tija inferior de hpE del 3'UTR/ARN3 de AMV va demostrar que són llocs crítics involucrats en la replicació in vivo de AMV. La identitat dels residus 2012A, 2013A i 2014A en el bucle hpB sembla ser un requisit estructural clau per a la replicació i/o acumulació de AMV. Respecte a hpE, els nostres resultats van determinar que el suposat residu de m6A (1902A), així com l'aparellament de bases de la tija inferior de hpE, també són requisits essencials per a la síntesi in vivo de ARNs de cadena positiva en AMV. Fins on sabem, aquesta és la primera evidència en AMV que mostra que el bucle de hpB i la tija inferior de hpE estan involucrats en la replicació/acumulació viral i la síntesi de ARNs de cadena positiva, respectivament. Finalment, quant a l'estudi de la influència de les m6A-metiltransferasas en el cicle d'infecció viral de AMV, no es va determinar un efecte proviral i/o antiviral en el complex m6A-ARNm metiltransferasa conformat per atMTA:atMTB, ni en el putatiu complex m6A-ARNr metiltransferasa conformat per atMETTL5-like:atTRMT112-like sobre la biologia de AMV.[EN] N6-methyladenosine (m6A) is a widespread modification on cellular RNAs of different organisms that can impact many cellular processes and pathways. In plants, m6A-methylation is mainly installed by a methylation complex containing several proteins: MTA, MTB, FIP37, VIR, and HAKAI. This modification is removed by demethylases of the AlkB family, and members of the ECT family are the best described proteins that recognize and process m6A-modified RNAs. Studies of viral epitransciptomics have revealed an equally important role of m6A during virus infection; however, there is no global pro- or antiviral role of m6A that can be generalized. The laboratory where this work was carried out has been a pioneer in the study of the effect of m6A on plant-viruses, using AMV as a model-virus. AMV belongs to the Bromoviridae family and, as the rest of the members of this family, its genome consists of three (+)ssRNAs. RNA1 and RNA2 encode the replicase subunits (P1 and P2), whereas RNA 3 encodes the MP and serves as a template for the synthesis of sgRNA 4, which encodes CP. At the beginning of this thesis, our laboratory had already reported on: the presence of putative m6A-motifs in the 3'UTR RNA3, a critical region for AMV replication, the first Arabidopsis m6A-demethylase (ALKBH9B), the functional relevance of ALKBH9B to maintain adequate m6A/A levels for correct AMV replication, the ability of AMV-CP to interact with ALKBH9B, possibly to usurp ALKBH9B activity, and the capability of Arabidopsis ECT2/3/5 to interact with m6A-containing AMV vRNAs. Given the functional relevance of m6A on the biology of AMV, in this thesis it was decided to deepen the knowledge of the implications of the m6A regulation mechanism on the viral infectious cycle of AMV. For this, it was decided: deepen the functional understanding of the m6A-demethylase ALKBH9B, evaluate the in vivo function of the putative two m6A-sites present in the 3'UTR-RNA 3, and explore a possible involvement of some m6A-methyltransferases in infection caused by AMV. We mapped functional subdomains in the atALKBH9B m6A-demethylase required for its binding to the vRNA and to the CP of AMV. Remarkably, it was observed the presence of IDRs in the N-terminal region, within the internal domain like AlkB and in the C-terminal region. About 78% of the RBD identified in ALKBH9B is contained in the C-terminal IDR. In this context, it has been proposed that the capability to specifically target different RNAs in RBPs containing IDRs is due to conformational flexibility as well as the establishment of extended conserved electrostatic interfaces with RNAs. Additionally, due that IDRs are frequently localized in proteins that undergo LLPS, a process that likely contributes to the formation and stability of RNA granules, it's possible that the IDRs and the RBD of ALKBH9B could act cooperatively to promote RNA granule formation. The analysis of the putative DRACH-motifs located in the hpB loop and the lower-stem of hpE in the 3'UTR RNA 3 present hot sites involved in AMV replication in vivo. The identity of residues 2012A, 2013A and 2014A in the hpB loop appears to be a key structural requirement for AMV replication and/or accumulation. Regarding hpE, our results determined that the putative m6A-residue 1902A, as well as the base pairing of the lower-stem of hpE, are also essential requirements for the in vivo plus-strand synthesis in AMV. To our knowledge, this is the first evidence in AMV to show that the hpB loop and the lower-stem of hpE are involved in viral replication/accumulation and plus-strand synthesis, respectively. Finally, regarding the study of the influence of m6A-methyltransferases on the viral infection cycle of AMV, a non-proviral and/or antiviral effect was determined in the m6A-mRNA methyltransferase complex made up of atMTA:atMTB, nor of the putative m6A-rRNA methyltransferase complex made up of atMETTL5-like:atTRMT112-like on the biology of AMV.Alvarado Marchena, LF. (2022). Insights into the Molecular Mechanisms of the N6-Methyladenosine (m6A) Methylation Machinery in the Regulation of the Infection Cycle of RNA Plant Viruses [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185122TESI

Alphavirus-host interactions : how to unravel the proviral activity of G3BP

The genus Alphavirus belongs to the virus family Togaviridiae, a group of arthropod-borne enveloped RNA viruses with single-stranded, positive-sense genome. Members of this genus can be found worldwide and are geographically distinguished into Old World and New World alphaviruses. Old World alphaviruses include the re-emerging human pathogen chikungunya virus (CHIKV) and the model virus Semliki Forest virus (SFV). Upon cellular infection, the released viral genome is directly translated to produce the non-structural polyprotein, which is subsequently processed into four non-structural proteins. The non-structural protein 3 (nsP3) of Old World alphaviruses contains two FGDF motifs, which facilitate binding to the NTF2-like domain of the host protein G3BP and its recruitment to viral replication complexes. G3BP1 and G3BP2 (hereafter jointly referred to as G3BP) are homologous proteins, best characterized for their ability to assemble stress granules in response to a variety of cellular stressors, such as viral infection. In paper I we investigated the structural and functional significance of the two FGDF motifs for the Old World alphaviruses SFV and CHIKV. A 3-dimensional structure of the NTF2-like domain of G3BP1 in complex with a SFV nsP3-derived peptide showed that the two FGDF motifs crosslink dimers of G3BP1 into a nsP3:G3BP1 oligomer. Mutational analysis of the FGDF motifs furthermore revealed that both motifs are required for efficient growth of SFV and suggest a critical role for the formation of nsP3:G3BP oligomeric structures for SFV. CHIKV is non-viable if the nsP3:G3BP1 interaction is abrogated through mutation of both motifs. The presence of a single functional FGDF motif is sufficient to rescue CHIKV replication, albeit to a lesser extent than in the presence of both motifs. Together, the results of this paper highlight similarities, but also discrepancies between SFV and CHIKV for the two G3BP-binding motifs. In paper II we studied potential proviral roles of G3BP for SFV and CHIKV. To this end, we used a panel of human osteosarcoma (U2OS) cell lines lacking endogenous G3BP proteins and stably expressing G3BP1 mutants and truncation variants. SFV replication is attenuated in the absence of G3BP and efficiently rescued by the presence of only the NTF2-like domain of G3BP1, which is accompanied by clustering of replication complexes. On the contrary, CHIKV strictly depends on the presence of the NTF2-like and the RGG domains of G3BP1. By immunoprecipitation we show that the RGG domain of G3BP1 facilitates binding of nsP3:G3BP1 complexes to 40S ribosomal, which correlates with enhanced localized translational activity in close proximity to viral replication complexes. The results suggest that G3BP exerts several proviral activities by mediating clustering of viral replication complexes and the recruitment of the translation initiation machinery. The results of paper III demonstrate that Old Wold alphaviruses differ remarkably in their dependence on G3BP. We describe a role for the P4 residue of the cleavage site between nsP1 and nsP2 (1/2 site) of the alphavirus non-structural polyprotein in conferring the extent of G3BP sensitivity. An Arg residue at the P4 position of the 1/2 site, as for CHIKV, is associated with fast a cleavage rate at this site and a high sensitivity towards G3BP deletion. An His residue at this position, as for SFV4, confers a slower cleavage rate at this site, accompanied with partial resistance towards G3BP deletion. Arg-to-His substitution of the P4 residue of CHIKV allows partially rescues replication even in the absence of G3BP. However, our data suggest that G3BP proteins do not influence the processing of the non-structural polyprotein. Instead we propose a critical role for G3BP proteins during the initiation of viral RNA replication. In summary, the work presented in this thesis improves our understanding of alphavirus-host interactions and highlights previously unanticipated proviral roles of G3BP for Old World alphavirus infections. The results mark G3BP as a potential target for the development of antivirals and provide a platform for future investigations

THE NON-STRUCTURAL ROLES OF DENGUE VIRUS STRUCTURAL CAPSID PROTEIN

Ph.DDOCTOR OF PHILOSOPH

Characterisation of nanobodies directed against emerging viruses

The emergence of new viral pathogens, such as SARS-CoV-2, or re-emergence of known pathogens, like CHIKV, point out the need for further understanding of the biology behind viruses, as well as the urgent need for the development of therapeutic and diagnostic tools. Nanobodies, small antigen-binding fragments derived from camelid heavy-chain antibodies, have gained attention for their use in viral research due to their wide range of applications: from the study of protein-protein interactions, uncovering of new viral targets, to the generation of new diagnostic tools or therapeutics. In paper I, we isolated a nanobody, Ty1, targeting the receptor binding domain (RBD) of SARS-CoV-2. We showed the ability of Ty1 to neutralise SARS-CoV-2 pseudotyped lentivirus potently (IC50 of 0.77 μg mL-1). The highly neutralising ability of Ty1 was likely due to its ability to bind the RBD in the ‘up’ and ‘down’ conformations, causing direct blocking to the cellular receptor and steric hindrance, respectively. Moreover, staining of SARS-CoV-2-infected cells with Ty1 confirmed its high specificity. In paper II we made use of a novel and rapid strategy to create nanobody multimers. We first functionalised the nanobodies using sortase A ligation to attach click chemistry functional groups. Then, the functionalised nanobodies were used to create C-to-C terminal bi- and tetravalent nanobody constructs by Cu-free strain-promoted azide-alkyne click chemistry (SPAAC). The bivalent and tetrameric nanobody constructs showed an increased potency with respect to the monomeric Ty1 of 150-fold and 4000-fold, respectively. This was true both for SARS-CoV-2 spike pseudotyped lentivirus and infectious SARS-CoV-2. In paper III, we generated nanobodies targeting the spike complex of CHIKV. We used a combinatory immunisation strategy with a cDNA prime followed by a protein boost. The CHIKV spike complex is formed by homotrimers of heterodimers of the E1 and E2 proteins. While E2 binds to the cellular receptor, E1 is responsible for the fusion of viral and cellular membranes, both essential steps of viral entry. We made use of a bivariate mining approach coupled to NGS and calculation of enrichment (fold difference in frequency between basal and enriched libraries) for the quick selection of nanobodies targeting either protein. We identified 12 nanobodies that detected cells infected with the 3 CHIKV lineages (ECSA, Asian and WA). Surprisingly, neutralisation of the ECSA and Asian genotypes was below 50% for all tested nanobodies, while 2 nanobodies, Dy010 and Dy059 could neutralise the WA lineage above 50% with PRNT50 values of 563 and 722 nM, respectively. Fusion to an Fc fragment produced an increase in potency of 130- and 63-fold for Dy010 and Dy059. Moreover, 4 of the nanobodies, Dy009, Dy025, Dy027 and Dy201 cross-reacted with other alphaviruses including ONNV, RRV and SFV, while one nanobody, Dy007, showed great specificity for CHIKV. These nanobodies expand the toolbox for research of this important human pathogen and could form a basis for the development of therapeutic or diagnostic tools