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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22832号 / 医博第4671号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 安達 泰治, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Tobacco mosaic virus (TMV) is a simple and frequently used model virus which has been studied already more over than 130 years. Due to the intensive study of this virus the details of its infectious cycle, genomic information and also the structure of the created viral particle as well as the mechanism of its creation are known today. The process of encapsidation (viral particle formation) is sufficiently described in the in vitro conditions. In the in vitro conditions the origin of assembly (OAS) was also described. The OAS was identified in the coding sequence of the gene for the movement protein (MP). The importance of replication centers (replication factories) has also been supposed. The aim of the diploma thesis was to study the specificity of the interaction of RNA and coat protein in the process of the particle assembly taking place directly inside the plants. The experiments were performed to verify the necessity of presence of OAS sequence in process of initiation of viral encapsidation. The effect of the cell compartmentation on this process has also been studied. Based on several viral systems (the Tobacco mosaic virus, the Potato virus X, the Bean yellow dwarf virus and Cowpea mosaic virus) gene constructs were created. These constructs enables to study this idea at the molecular...

A unique mechanism of protein oligomerization is domain swapping. It is a feature found in some proteins wherein a dimer or a higher oligomer is formed by the exchange of identical structural segments between protomers. Domain swapping is thought to have played a key role in the evolution of stable oligomeric proteins and in oligomerization of amyloid proteins. This thesis deals with studies to understand the significance of hinges involved in domain swapping for protein oligomerization and function. The stationary phase survival protein SurE from Salmonella typhimurium (StSurE) and Sesbania mosaic virus (SeMV) coat protein have been used as models for studies on domain swapping. This thesis has been divided into eight chapters. Chapter 1 provides a brief introduction to domain swapping, while Chapters 2 to 6 describes the studies carried out on StSurE protein, Chapter 7 deals with studies on SeMV coat protein. The final Chapter 8 provides brief descriptions of various experimental techniques employed during these investigations. Chapter 1 deals with a brief introduction to domain swapping in proteins. Examples where different domains are exchanged are cited. Then it describes physiological relevance of domain swapping in proteins and probable factors which promote swapping. Finally it also discusses the uncertainties that are inevitable in protein structure prediction and design. Chapter 2 describes the structure of Salmonella typhimurium SurE (StSurE; Pappachan et al., 2008) determined at a higher resolution. The chapter also deals with the sequence and structure based comparison of StSurE with other known SurE homolog structures. A comparative analysis of the relative conservation of N- and C-terminal halves of SurE protomer and variations observed in the quaternary structures of SurE homologs are presented. Then a brief introduction is provided on function of StSurE. The conserved active site of StSurE that might be important for its phosphatase activity is described. A plausible mechanism for the phosphatase activity as proposed by Pappachan et al. (2008) is presented. Crystal structures of StSurE bound with AMP, pNPP and pNP that was determined with the view of better understanding the mechanism of enzyme function is presented. These structures provide structural evidence for the mechanism proposed by Pappachan et al. (2008). Finally a substrate entry channel inferred from these structures is discussed. SurE from Salmonella typhimurium (StSurE) was selected for studies on domain swapping as there is at least one homologous structure (Pyrobaculum aerophilum - PaSurE) in which swapping of the C-terminal helices appears to have been avoided without leading to the loss of oligomeric structure or function. It was of interest to examine if an unswapped dimer of StSurE resembling PaSurE dimer could be constructed by mutagenesis. To achieve this objective, a crucial hydrogen bond in the hinge involved in C-terminal helix swapping was abolished by mutagenesis. These mutants were constructed with the intention of increasing the flexibility of the hinge which might bring the C-terminal helices closer to the respective protomer as in PaSurE. Chapter 3 presents a comparative analysis of the hinges involved in C-terminal helix swapping in PaSurE and StSurE. Based on the comparison of structure and sequence, crucial residues important for C-terminal helix swapping in StSurE were identified as D230 and H234. The chapter describes the construction of mutants obtained by substituting D230 and H234 by alanine and their biophysical characterization. Finally it describes structural studies carried out on these mutants. The mutation H234A and D230A/H234A resulted in highly distorted dimers, although helix swapping was not avoided. Comparative analysis of the X-ray crystal structures of native StSurE and mutants H234A and D230A/H234A reveal large structural changes in the mutants relative to the native structure. However the crystal structures do not provide information on the changes in dynamics of the protein resulting from these mutations. To gain better insights into the dynamics involved in the native and mutants H234A and D230A/H234A, MD simulations were carried on using GROMACS 4.0.7. Chapter 4 deals with a brief description of the theory of molecular dynamics, followed by results of simulation studies carried out on monomeric and dimeric forms of StSurE and dimeric forms of its mutants H234A and D230A/H234A. The conformational changes and dynamics of different swapped segments are discussed. Crystal structures of H234A and D230A/H234A mutants reveal that they form highly distorted dimers with altered dimeric interfaces. Chapter 5 focuses on comparison of dimeric interfaces of the native StSurE and hinge mutants H234A and D230A/H234A. Based on the analysis, three sets of interactions were selected to investigate the importance of the interface formed by swapped segments in StSurE mutants H234A and D230A/H234A. One of the selected sites corresponds to a novel interaction involving tetramerization loop in the hinge mutants H234A and D230A/H234A resulting in a salt bridge between E112 – R179’ and E112’ – H180 (prime denotes residue from the other chain of the dimeric protein). This salt bridge seems to stabilize the distorted dimer. It is shown by structural studies that the loss of this salt bridge due to targeted mutation restores symmetry and dimeric organization of the mutants. Loss of a crucial hydrogen bond in the hinge region involved in C-terminal helix swapping in SurE not only leads to large structural changes but also alters the conformation of a loop near the active site. It is of interest to understand functional consequences of these structural changes. StSurE is a phosphatase, and its activity could be conveniently monitored using the synthetic substrate para nitrophenyl phosphate (pNPP) at pH 7 and 25 ºC. Chapter 6 deals with the functional studies carried out with various StSurE mutants. The studies suggest that there is a drastic loss in phosphatase activity in hinge mutants D230A, H234A and D230A/H234A, while in the salt bridge mutants the function seems to have been restored. Few of these mutants also exhibit positive cooperativity, which could probably be due to altered dynamics of domains. Sesbania mosaic virus (SeMV) is a plant virus, belonging to genus sobemovirus. SeMV is a T=3 icosahedral virus (532 symmetry) made up of 180 coat protein (CP) subunits enclosing a positive-sense RNA genome. The asymmetric unit of the icosahedral capsid is composed of chemically identical A, B and C subunits occupying quasi-equivalent environments. Residues 48 – 59 of the N-terminal arms of the C subunits interact at the nearby icosahedral three-fold axes through a network of hydrogen bonds to form a structure called the “β-annulus”. Residues 60 – 73 form the “βA-arm” that connects the N-terminal β-annulus to the rest of the protomer. Various studies on SeMV-CP suggest that different lengths of the N-terminal segments affect the assembly of virus. It might be possible to exploit this flexibility of the N-terminus in SeMV-CP to introduce swapping of this segment between two 2-fold related C subunits as is found in Rice yellow mottle virus (RYMV), another sobemovirus, with which SeMV shares significant sequence similarity. Chapter 7 focuses on attempts made to examine the mutational effects planned to introduce domain swapping. The strategy used for introducing swapping in SeMV-CP was based on the sequence of the βA-arm or the hinge involved in swapping of β-annulus in RYMV. TEM images of the mutant virus like particles obtained suggest that they are heterogeneous. These mutants could not be crystallized, probably due to the heterogeneity. However, the assembly of the expressed proteins to virus like particles was profoundly influenced by the mutations. Chapter 8 discusses various crystallographic, biophysical and biochemical techniques used during these investigations. Finally the thesis concludes with Conclusions and Future perspectives of the various studies reported in the thesis. In summary, I have addressed the importance of amino acid residues and interactions of hinges involved in domain swapping for the quaternary structure and function of proteins.

[ES] Los virus de plantas son los agentes causales de un gran número de enfermedades en plantas que ocasionan grandes pérdidas económicas. El virus de las manchas necróticas del melón (MNSV), es un pequeño virus de RNA monocatenario de polaridad positiva, perteneciente al género Gammacarmovirus, cuyo genoma codifica cinco proteínas. La proteína de cubierta (CP), está formada por tres dominios distintos. El descubrimiento de un péptido de transito dual en la región amino-terminal de la CP fue el punto de partida de esta tesis. Al inicio de una infección por MNSV, la CP nuevamente sintetizada es transportada al interior de cloroplastos y mitocondrias mientras que, una parte mucho menor se mantiene en el citoplasma aumentando a medida que avanza la infección. La inhibición de este transporte dual conlleva un aumento de la actividad supresora del silenciamiento del RNA de la CP. Sin embargo, la infección sistémica se ve particularmente afectada. Por tanto, la acumulación de la CP en el citoplasma puede provocar un aumento de la replicación viral pero a su vez una sobreexpresión de la p29, puede provocar una explosión oxidativa y una necrosis que restringe el movimiento viral. De este modo, el transporte de la CP a los orgánulos podría evitar una replicación viral excesiva mediante la modulación de la actividad supresora para gestionar el equilibrio entre la defensa de la planta y la contradefensa viral favoreciendo una interacción compatible entre ambos. Desafortunadamente, Arabidopsis thaliana no es huésped para el MNSV. Por tanto, para entender mejor el transporte de la CP a estos orgánulos, se identificaron los receptores y los poros de los translocones de las membranas externas de las mitocondrias y los cloroplastos en Nicotiana benthamiana. Esta caracterización funcional se realizó principalmente mediante VIGS y RT-qPCR, que mostró una redundancia funcional mayor que la observada entre los homólogos de Arabidopsis. Además, esta herramienta también se utilizó para evaluar la relevancia de cada componente bajo la infección por MNSV, y junto con los estudios de interacción CP-receptor realizados mediante BiFC y Y2H, nos permitió identificar NbToc159A para cloroplastos y NbOm64 para mitocondrias, como principales receptores. A su vez, el silenciamiento de NbToc34, NbToc75 o NbTom40 resultó en una resistencia generalizada no solo a MNSV sino también al virus del arrugamiento del nabo (TCV) y al virus del moteado del clavel (CarMV), lo que respalda la idea actualmente aceptada y que involucra el estado fisiológico del cloroplasto y la mitocondria en la señalización temprana de la respuesta defensiva. Finalmente, se realizó una búsqueda de factores del huésped que interaccionasen con la CP mediante con TurboID, una ligasa de biotina, que permite la detección de interacciones tanto directas e indirectas como transitorias y estables. Así, se obtuvo un gran número de proteínas candidatas utilizando la CP de MNSV y su mutante de localización citoplásmica, ∆NtCP. Tres de ellas, NbSIK1, NbSMU2 y NbMAP3K mostraron un efecto perjudicial constante y repetitivo sobre la acumulación del RNA viral. Después de la validación de las interacciones mediante otro método, y el análisis de la localización subcelular de la CP bajo el silenciamiento del interactor correspondiente, se establecieron dos hipótesis principales. En primer lugar, dado que la función principal de NbSMU2 está relacionada con el procesamiento y regulación del RNA mensajero, esta proteína podría ser secuestrada por la CP provocando la expresión de genes provirales. Por otro lado, NbSIK1 y NbMAP3K, actúan como reguladores positivo y negativo de la respuesta PTI a la infección, respectivamente. Además, ambas proteínas interaccionan entre sí y forman parte de la cascada de MAP quinasas, por lo que en nuestra segunda hipótesis, la CP interaccionaría con este complejo, promoviendo una regulación negativa de la PTI que facilitaría el desarrollo de la infección. / [CA] Els virus de plantes són els principals causants de la major part de malalties en plantes i les consegüents pèrdues econòmiques. El virus de les taques necròtiques del meló (MNSV) és un virus menut d'RNA monocatenari de polaritat positiva, pertanyent al gènere Gammacarmovirus, el genoma del qual codifica cinc proteïnes. La proteïna de coberta (CP) està formada per tres dominis diferents. El descobriment d'un pèptid de trànsit dual a la part aminoterminal de la CP va ser el punt de partida d'aquesta tesi. A l'inici d'una infecció per MNSV, la CP novament sintetitzada és transportada a l'interior dels cloroplasts i mitocondris mentre que, una part molt menor es manté al citoplasma augmentant a mesura que avança la infecció. La inhibició d'aquest transport dual comporta un augment de l'activitat supressora del silenciament de l'RNA de la CP. No obstant això, la infecció sistèmica es va frenar. Per tant, l'acumulació de la CP al citoplasma pot provocar un augment de la replicació viral però alhora una sobreexpressió de la p29, una replicasa auxiliar que ocasiona alteracions morfològiques als mitocondris, una explosió oxidativa i necrosi que restringeix el moviment viral. D'aquesta manera, el transport de la CP als orgànuls podria evitar una replicació viral excessiva mitjançant la modulació de l'activitat supressora per gestionar l'equilibri entre defensa de la planta i contradefensa viral que condueixen a una interacció compatible entre tots dos. Per entendre millor el mecanisme molecular que regeix el transport de la CP a aquests orgànuls, es van identificar els receptors i els porus dels translocon de les membranes externes dels mitocondris i els cloroplasts a Nicotiana benthamiana. Aquesta caracterització funcional es va realitzar principalment mitjançant VIGS i RT-qPCR, que va mostrar una redundància funcional més gran que l'observada entre els homòlegs d'Arabidopsis. A més, aquesta eina també es va utilitzar per avaluar la rellevància de cada component sota la infecció per MNSV, i juntament amb els estudis d'interacció CP-receptor realitzats mitjançant BiFC i Y2H, ens va permetre identificar a NbToc159A per a cloroplasts i NbOm64 per a mitocondris, com els principals receptors implicats en el transport de la CP a aquests orgànuls. Alhora, el silenciament de NbToc34, NbToc75 o NbTom40 va resultar en una resistència generalitzada a MNSV, TCV i CarMV, la qual cosa recolza la idea que circula actualment i que involucra l'estat fisiològic del cloroplast i el mitocondri en la senyalització primerenca de la resposta defensiva. Finalment, es va fer una cerca de factors de l'hoste que interaccionessin amb la CP mitjançant la innovadora tècnica de marcatge de proximitat amb TurboID, una lligasa de biotina, que permet la detecció d'interaccions tant directes i indirectes com transitòries i estables. Així, es va obtenir un gran nombre de proteïnes candidates utilitzant la CP de MNSV i el seu mutant de localització citoplàsmica, ∆NtCP. Tres de elles, NbSIK1, NbSMU2 i NbMAP3K van mostrar un efecte perjudicial constant i repetitiu sobre l'acumulació de l'RNA viral. Després de la validació de les interaccions mitjançant un altre mètode, i l'examen de la localització subcel·lular de la CP sota el silenciament de cada interactor, es van establir dues hipòtesis principals. En primer lloc, atès que la funció principal de NbSMU2 està relacionada amb el processament i la regulació de l'RNA missatger, aquesta proteïna podria ser segrestada per la CP provocant l'expressió de gens provirals. D'altra banda, NbSIK1 i NbMAP3K actuen com a reguladors positiu i negatiu de la resposta PTI a la infecció, respectivament. A més, les dos proteïnes interaccionen entre si i formen part de la cascada de MAP quinases, per la qual cosa en la nostra segona hipòtesi, la CP interaccionaria amb aquest complex, promovent una regulació negativa de la PTI que facilitaria el desenvolupament de la infecció. / [EN] Plant viruses are the causal agents of many plant diseases and the subsequent economic losses, estimated to be US$60 billion worldwide each year. The melon necrotic spot virus (MNSV) is a small, single-stranded, positive-sense RNA virus that belongs to the genus Gammacarmovirus and encodes five proteins. The coat protein (CP) is composed of three distinct domains. The discovery of a dual transit peptide in the amino-terminal part of the CP was the starting point of this thesis. Early in MNSV infection, the new synthesized CP is imported into chloroplasts and mitochondria, while the cytoplasmic pool increases as the infection progresses. Inhibiting this dual transport leads to an increase in the RNA silencing suppressor activity of the CP. However, far from resulting in an enhanced infection development, systemic spread was impaired. Therefore, the accumulation of cytoplasmic CP may cause an increase in viral replication and overexpression of p29, an auxiliary replicase that causes morphological alterations, ROS, and necrosis that may restrict viral movement. Thus, a new role for CP targeting would be to avoid excessive viral replication by modulating the suppressor activity to manage the balance between plant defense and viral counter-defense, leading to a compatible interaction. Unfortunately, Arabidopsis thaliana is not a host for MNSV. Thus, to better understand the molecular mechanism behind the CP dual targeting, the receptors and pores of the Nicotiana benthamiana mitochondrial and chloroplast outer membrane translocons were genome identified, and some functional characterization was carried out. We assigned the following names NbToc75-III, NbToc34, NbToc90, NbToc120, NbToc159A, NbToc159B, NbTic22-III for chloroplast translocon components, and NbTom40, NbTom20-1, NbTom20-2, NbOm64 for mitochondrion translocon components. The functional characterization was mainly carried out by virus-induced gene silencing (VIGS) and RT-qPCR, revealing a functional redundancy higher than that reported for Arabidopsis homologs. Additionally, VIGS was also used to evaluate the relevance of each translocon component in MNSV infection, and together with CP-receptor interaction studies performed by BiFC and Y2H, allowed us to identify NbToc159A for chloroplasts and NbOm64 for mitochondria as the main receptors involved in the CP organelle import. Moreover, silencing of NbToc34, NbToc75, or NbTom40 resulted in a generalized resistance not only to MNSV but also to turnip crinkle virus (TCV), and carnation mottle virus (CarMV), supporting the current idea that involves the chloroplast and mitochondrion physiological state in early defense response signaling. Finally, a search for host factors interacting with the CP was performed by the innovative TurboID proximity labeling tool, which allows the detection of both direct/indirect and transient/stable interactions. Thus, a large number of candidate proteins were obtained that interacted either with the MNSV CP or with ∆NtCP, a cytoplasm-localized mutant. Three of them, NbSIK1, NbSMU2, and NbMAP3K, showed a consistent and repetitive detrimental effect on MNSV RNA accumulation. After the validation of the interactions using another method and the analysis of the subcellular localization of the MNSV CP under each interactor silencing, two main hypotheses were proposed. Firstly, since the main function of NbSMU2 is related to messenger RNA regulation by splicing, this protein could be sequestered by the CP, causing the expression of proviral genes. On the other hand, NbSIK1 and NbMAP3K act as positive and negative regulators of the PTI response to infection, respectively. Moreover, both proteins interact with each other and are part of the MAP kinase cascade, so in our second hypothesis, CP would interact with this complex, promoting a negative regulation of PTI that would facilitate viral infection. / La autora ha disfrutado de un contrato predoctoral de formación de personal investigador (FPI) (PRE-2018-84130) otorgado por el Ministerio de Ciencia e Innovación asociado al proyecto BIO2017-88321-R. Este trabajo de tesis doctoral ha sido realizado con el apoyo económico de los proyectos de investigación del Ministerio de Ciencia e Innovación, BIO2017-88321-R y PID2020-115571RB- I00. / Sáiz Bonilla, M. (2023). Deciphering the intracellular dual targeting of the melon necrotic spot virus coat protein, its interaction with host factors and their roles in plant defense [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/195836