Sweet potato is one of the 15 most important food crops worldwide. At least 30 different virus species, belonging to different taxonomic groups affect sweet potato. Little is known about the viruses present in sweet potato crops in Central America, which is the primary origin of sweet potato. The objective of this study was to design and implement primers for use in polymerase chain reaction (PCR) and Reverse transcription-PCR (RT-PCR) to identify and survey the diversity of plant viruses infecting sweet potato in Honduras and Guatemala. Primers were designed and used to amplify, clone, and sequence a taxonomically informative fragment of the coat protein (CP) gene for whitefly-transmitted geminiviruses (herein, sweepoviruses) and potyviruses, and of the heat shock protein 70 (HSP70) for the Crinivirus, Sweet potato chlorotic stunt virus (SPCSV). The partial genome sequences were used for identification based on phylogenetic relationships with reference sequences available in the GenBank database. All three of the plant virus groups identified in this study were found to occur either in single or in multiple infections. Results of the sequence analyses indicated that the genomic regions amplified in this study were capable of discriminating among potyvirus species, and strains of SPCSV. With respect to potyvirus, all isolates were identified as Sweet potato feathery mottle virus (SPFMV) species, except for two, which grouped phylogenetically with Sweet potato virus G (SPVG) and Sweet potato virus C (SPVC). All sweepoviruses detected in sweet potato plants belonged to a single phylogenetically, well-supported group that contains all other previously described geminiviruses (sweepoviruses) associated with sweet potato or closely related host species. These results demonstrate that the primers designed for amplification of plant virus species commonly recognized to infect sweet potato, effectively detected the viruses singly and in mixtures from symptomatic plants, and that the resultant fragment, when subjected to cloning and DNA sequenced, was phylogenetically informative at the species and/or strain levels, depending on the virus group.

The chicken eggshell possesses physical and chemical barriers to protect the embryo from pathogens. The avian eggshell cuticle is the outmost layer of the eggshell whose protein constituents remain largely unknown. Since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination, we hypothesize that cuticle protein components play an important role in microbial resistance. In our study, at least 47 proteins were identified by LC/MS/MS in the non-calcified cuticle layer. Similar to Kunitz-like protease inhibitor (also annotated as ovocalyxin-25, OCX-25) and ovocalyxin-32 (OCX-32) were two of most abundant proteins of the cuticle proteins. Some proteins that have antimicrobial activity were also detected in the proteomic results, such as lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor. This study represents the first comprehensive report of the cuticle proteome. Since the sequence similarity of the kunitz motif in OCX-25 is similar to that of BPTI, it is predicted that it will have the same trypsin inhibitory and antimicrobial activity against Gram-positive and/or Gram-negative bacteria. In order to test the antimicrobial property and trypsin inhibitor activity of OCX-25, cuticle proteins were extracted by 1N HCl. Antimicrobial activity was monitored using the Bioscreen C instrument; and antimicrobial activity was identified primarily against Staphylococcus aureus. Trypsin inhibitor activity was studied by using a specific trypsin assay, and the assay indicated that the cuticle proteins could inhibit the reaction of trypsin and substrate. Therefore, the current research has provided some insight into the antimicrobial and enzymatic aspects of the cuticle proteins, and its function for egg protection. Eggshell membranes are another important component of the chicken eggshell.Due to its insoluble and stable properties, there are still many questions regarding formation and constituents of the eggshell membranes. The purpose of our study was to identify eggshell membrane proteins, particularly these responsible for its structural features, by examining the transcriptome of the white isthmus during its formation. Bioinformatics tools were applied to analyze the differentially expressed genes as well as their encoded proteins. Some interesting proteins were encoded by the over-expressed genes in the white isthmus during the formation of eggshell membranes, such as Collagen X, and similar to spore coat protein SP75. These proteins may have potential applications. Our study provides a detailed description of the chicken white isthmus transcriptome during formation of the eggshell membranes; it could lead to develop the strategies to improve food safety of the table egg.

The chicken eggshell possesses physical and chemical barriers to protect the embryo from pathogens. The avian eggshell cuticle is the outmost layer of the eggshell whose protein constituents remain largely unknown. Since eggs with incomplete or absent cuticle are more susceptible to bacterial contamination, we hypothesize that cuticle protein components play an important role in microbial resistance. In our study, at least 47 proteins were identified by LC/MS/MS in the non-calcified cuticle layer. Similar to Kunitz-like protease inhibitor (also annotated as ovocalyxin-25, OCX-25) and ovocalyxin-32 (OCX-32) were two of most abundant proteins of the cuticle proteins. Some proteins that have antimicrobial activity were also detected in the proteomic results, such as lysozyme C, ovotransferrin, ovocalyxin-32, cystatin, ovoinhibitor. This study represents the first comprehensive report of the cuticle proteome. Since the sequence similarity of the kunitz motif in OCX-25 is similar to that of BPTI, it is predicted that it will have the same trypsin inhibitory and antimicrobial activity against Gram-positive and/or Gram-negative bacteria. In order to test the antimicrobial property and trypsin inhibitor activity of OCX-25, cuticle proteins were extracted by 1N HCl. Antimicrobial activity was monitored using the Bioscreen C instrument; and antimicrobial activity was identified primarily against Staphylococcus aureus. Trypsin inhibitor activity was studied by using a specific trypsin assay, and the assay indicated that the cuticle proteins could inhibit the reaction of trypsin and substrate. Therefore, the current research has provided some insight into the antimicrobial and enzymatic aspects of the cuticle proteins, and its function for egg protection. Eggshell membranes are another important component of the chicken eggshell.Due to its insoluble and stable properties, there are still many questions regarding formation and constituents of the eggshell membranes. The purpose of our study was to identify eggshell membrane proteins, particularly these responsible for its structural features, by examining the transcriptome of the white isthmus during its formation. Bioinformatics tools were applied to analyze the differentially expressed genes as well as their encoded proteins. Some interesting proteins were encoded by the over-expressed genes in the white isthmus during the formation of eggshell membranes, such as Collagen X, and similar to spore coat protein SP75. These proteins may have potential applications. Our study provides a detailed description of the chicken white isthmus transcriptome during formation of the eggshell membranes; it could lead to develop the strategies to improve food safety of the table egg.

The importance of interactions between transmembrane domains of integral membrane proteins has been well-established in a range of essential cellular functions. Most integral membrane proteins also possess regions that lie on the exterior of the membrane that may influence the ability of these transmembrane domains to interact. We sought to test this hypothesis by quantifying the energetics of transmembrane helix self-association in the absence and presence of an amphipathic helix that can bind to the membrane surface. The model chosen for this study was the major coat protein (MCP) of M13 bacteriophage, which has an N-terminal amphipathic helix linked to its single transmembrane segment via a flexible linker. Dimerization of both full-length MCP and a peptide containing only the transmembrane domain (MCPTM) was studied by solution NMR in SDS micelles. We found that there was an increase in the apparent dimerization affinity in the absence of the N-terminal helix. However, this increase in apparent affinity could be attributed to differences in detergent-binding properties of the two polypeptides in monomeric versus dimeric states when the empty micelle was considered to be a participant in the dimer dissociation. Preliminary results from the integral membrane protein, p7 of the hepatitis C virus are also presented in this thesis. It has been demonstrated that p7 enhances viral infectivity and accumulation, and that this function may require oligomerization in the membrane. While we encountered limitations due to challenges in the generation of sufficient quantities of pure p7 samples, we were able to perform circular dichroism spectroscopy under conditions that may favor different oligomeric states. These studies suggest that there is a change in the degree of helicity upon oligomerization, and suggest that SDS could be a suitable system to characterize the interactions of the p7 oligomer in the future.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-21T13:55:26Z No. of bitstreams: 1 Cicero Nicolini.pdf: 1169299 bytes, checksum: e932282294084c1947939ed928971bcb (MD5) / Made available in DSpace on 2017-02-21T13:55:26Z (GMT). No. of bitstreams: 1 Cicero Nicolini.pdf: 1169299 bytes, checksum: e932282294084c1947939ed928971bcb (MD5) Previous issue date: 2011-09-01 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Brazil is the world’s largest passion fruit producer. The sanitary problems, mainly diseases caused by viruses are among the limiting factors for increasing productivity and longevity of the plantations. In Brazil, passion fruit woodiness, caused by Cowpea aphid-borne mosaic virus (CABMV) is the major viral disease of this crop. CABMV is also causing agent of the potyvirus mosaic, a viral disease that produces serious damages in cowpea. This crop in Brazil is important mainly in the Northeastern region and represents a relevant source of protein and starch for the population. Some years ago, in the state of Pernambuco it was found a potyvirus naturally infecting “lava-pratos”, leguminous plant native to Atlantic Forest commonly found on the margins of passionflower and cowpea crops. In the city of Paudalho it was found a group of “lava-pratos” plants presenting very intense symptoms with yellow spot on the leaves. From those plants, preparations examined in a transmission electron microscope showed the presence of elongated flexuous particles and cylindrical inclusions typical of potyvirus as well as isometric particles in high concentration and vesicles on the periphery of the chloroplasts which suggested the occurrence of a tymovirus. Serological analyses and coat protein sequencing to identify the potyvirus as well as studies on host adaptation in passionflower and cowpea, using isolates from these crops were undertaken. The virus with isometric particles was chemically purified to produce polyclonal antiserum and subjected to serological studies, host range and genome sequencing for characterization. It was concluded that the potyvirus found in “lava-pratos” is CABMV and phylogenetically observed the host adaptation of this virus to the families Fabaceae and Passifloraceae. For some strains geographical distance was not the strongest factor for genetic grouping and the Brazilian isolates form a large group distinct to other countries. According to the criteria of taxonomy, using amino acid sequence of coat protein and conserved region "tymobox", it was determined the presence of a tymovirus in “lava-pratos”, for which was proposed to be a new species named Cassia yellow mosaic-associated virus (CAYMaV). / O Brasil é o maior produtor mundial de maracujá. Os problemas fitossanitários especialmente as doenças ocasionadas por vírus estão entre os fatores limitantes ao aumento de produtividade e longevidade dos plantios. A principal virose do maracujazeiro no Brasil é o endurecimento dos frutos causado por Cowpea aphid-borne mosaic virus (CABMV). O CABMV também é o agente do mosaico de potyvírus em feijão-caupi, virose que provoca sérios danos nessa cultura. No Brasil, o feijão-caupi é produzido principalmente na Região Nordeste e representa fonte importante de proteína e amido para a população. Há alguns anos atrás, um potyvírus foi encontrado em Pernambuco infectando naturalmente o lava-pratos, uma leguminosa nativa da Mata Atlântica comumente encontrada nas bordas de cultivos de maracujá e feijão-caupi. No município de Paudalho foi observado um grupo de plantas de lava-pratos apresentando sintomas muito intensos com manchas amarelas nas folhas. Dessas plantas, ao ser analisadas preparações em microscópio eletrônico de transmissão, além da presença de partículas alongadas flexuosas e de inclusões cilíndricas típicas de potyvírus foram detectadas partículas isométricas em alta concentração e vesículas nas bordas dos cloroplastos, o que sugeria ser de um tymovírus. Foram realizadas análises sorológicas e seqüenciamento da capa protéica para identificação do potyvírus, assim como estudar a adaptação hospedeira a maracujazeiro e feijão-caupi, utilizando-se isolados obtidos dessas culturas. O vírus com partículas isométricas foi purificado quimicamente para produção de antissoro policlonal e submetido a estudos sorológicos, gama de hospedeiros e seqüenciamento de parte do genoma para caracterização. Concluiu-se que o potyvírus encontrado em lava-pratos é o CABMV e filogeneticamente se observou que ocorre adaptação hospedeira deste vírus em plantas das famílias Fabaceae e Passifloraceae. Para alguns isolados, a distância geográfica não foi o fator mais forte para agrupamento genético e os isolados brasileiros formam um grande grupo distinto aos de outros países. De acordo com os critérios de taxonomia, usando-se seqüência de aminoácidos da capa protéica e região conservada “tymobox”, foi determinada a presença em lava-pratos de um tymovírus, para o qual foi proposto ser uma nova espécie nomeada Cassia yellow mosaic-associated virus (CAYMaV).

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 3, 2010). Includes bibliographical references.

[ES] Las modificaciones químicas post-transcripcionales implican un nuevo nivel de modulación de la expresión génica. Al comienzo de esta Tesis, algunos componentes del complejo de metilación del nitrógeno en posición 6 de la adenosina (m6A) habían sido caracterizados en plantas. Sin embargo, a diferencia de mamíferos y levadura, ninguno de los 13 homólogos de AlkB (atALKBH1-10B) - potenciales desmetilasas (o erasers) - y las 13 proteínas de la familia YTH (ECT1- 11, AT4G11970 y CPSF30) - potenciales proteínas de reconocimiento de m6A (o readers) - identificadas en el genoma de Arabidopsis se habían caracterizado funcionalmente. Además, varios estudios describen la presencia de m6A en RNAs de virus de mamíferos y las diferentes funciones que desempeña esta modificación en la regulación de esas infecciones. No obstante, no se ha estudiado la posible implicación de este mecanismo molecular en las infecciones virales de plantas. El descubrimiento de la interacción entre la CP del virus del mosaico de la alfalfa (AMV) y una proteína de Arabidopsis (atALKBH9B) con homología a una eraser humana fue el punto de partida de esta Tesis. En este trabajo se confirma esta interacción, y se demuestra que atALKBH9B también puede reconocer los RNAs virales. Los resultados revelan que atALKBH9B tiene la capacidad de desmetilar m6A a partir de moléculas de RNA monocatenario in vitro. Esta proteína se acumula en gránulos citoplasmáticos que se colocalizan con siRNA bodies y se asocian a P-bodies, lo que sugiere que su actividad podría estar relacionada con el silenciamiento y/o degradación de mRNA. Por otro lado, ensayos preliminares muestran que los RNAs del AMV, el virus del mosaico del pepino (CMV), el virus de la arruga del nabo (TCV) y el virus del mosaico de la coliflor (CaMV) se metilan durante la infección en Arabidopsis. Además, para AMV y CMV, los resultados fueron corroborados por UPLC-PDA-Tof-MS y los sitios m6A a lo largo de los RNAs del AMV fueron identificados mediante MeRIP-seq. Los resultados presentados confirman que la relación m6A/A a lo largo de los RNAs virales aumenta en plantas atalkbh9b en comparación con las silvestres, mientras que la traducción y/o replicación se ven afectadas y el movimiento sistémico a los tallos florales está prácticamente bloqueado. A diferencia de la CP de AMV, la de CMV no interacciona con atALKBH9B por Y2H y, como ocurre con el resto de virus analizados (CMV, TCV y CaMV), su ciclo de infección no se ve afectado en plantas atalkbh9b. Además, la secuenciación de mRNA realizada en este trabajo revela que la infección por AMV induce algunos genes de Arabidopsis pertenecientes a la maquinaria m6A, MTA, MTB, VIR y ECT5. De acuerdo con el efecto antiviral dependiente de m6A para el AMV y teniendo en cuenta que ECT2, ECT3 y ECT4 fueron recientemente caracterizadas como readers citoplasmáticas, la supresión del módulo ECT2/ECT3/ECT5 aumenta significativamente los títulos sistémicos de AMV y CMV. El efecto antiviral de ECT2 sobre AMV parece estar modulado por su unión directa a los residuos de m6A presentes en los RNAs virales, ya que un mutante de ECT2 defectuoso en el reconocimiento de m6A pierde la actividad antiviral que sí presenta la proteína original y no es capaz de arrastrar RNAs virales in vivo. Por otro lado, acorde a la localización previamente descrita para ECT2 y ECT4 y la capacidad de ECT2 para experimentar una fase similar al gel in vitro, la expresión transitoria de ECT5 muestra un patrón citoplasmático con formación de agregados. Se propone que, como se ha descrito para las proteínas YTH de mamíferos, la interacción entre las ECTs y el RNA polimetilado (en este caso, RNA viral) promovería la formación de gránulos de estrés y, en consecuencia, reduciría las tasas de traducción y replicación viral. En resumen, en este trabajo se caracteriza la primera m6A eraser de plantas, atALKBH9B, y, por primera vez, se describe la influencia del mecanismo de metilación m6A en las infecciones virales de plantas. / [EN] Post-transcriptional chemical modifications entail a new level of gene expression modulation. At the beginning of this Thesis, some components of the N6-adenosine methylation (m6A) complex had been characterized in plants, whereas 13 homologs of AlkB (atALKBH1-10B) - putative demethylases (or erasers) - and 13 proteins of the YTH family (ECT1-11, AT4G11970 and 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, several reports have brought to light the presence of m6A residues in viral RNAs from mammalian viruses and the critical roles that this modification plays regulating viral infections. However, the potential relevance of this molecular mechanism on plant viral infections remained fully unexplored. The discovery of the interaction between the AMV CP and an Arabidopsis protein (atALKBH9B) with similarity to a human eraser was the starting point of this Thesis. Here, this interaction is confirmed and it is demonstrated that atALKBH9B can also recognize the viral RNAs. Furthermore, the obtained results prove that atALKBH9B has the capability of demethylating m6A from single-stranded RNA molecules in vitro. This protein was observed to accumulate in cytoplasmic granules that colocalize with siRNA-bodies and associate to P-bodies, suggesting that atALKBH9B activity could be related to mRNA silencing and/or decay processes. On the other hand, preliminary assays show that viral RNAs of AMV, Cucumber mosaic virus (CMV), Turnip crinkle virus (TCV) and Cauliflower mosaic virus (CaMV) become methylated during infection in Arabidopsis. Besides, for AMV and CMV, the results were corroborated by UPLC-PDA-Tof-MS and m6A sites along the RNAs of AMV were identified through MeRIP-seq approach. The results presented here confirm that m6A/A ratio along viral RNAs is increased in atalkbh9b plants compared to wild type, whereas translation and/or replication are impaired and systemic movement to the floral stems is practically blocked. In contrast to AMV, CMV CP does not interact with atALKBH9B by Y2H and, as it occurs with the rest of the assayed viruses (CMV, TCV and CaMV), its infection cycle is not affected in atalkbh9b plants. Furthermore, the mRNA-seq analysis performed in this Thesis reveals that some Arabidopsis factors belonging to the m6A machinery, MTA, MTB, VIR and ECT5 genes, are upregulated upon AMV infection. Consistent with the m6A-dependent antiviral effect for AMV and considering that ECT2, ECT3 and ECT4 were recently characterized as cytoplasmic m6A readers, mutations of ECT2/ECT3/ECT5 Arabidopsis module significantly increase AMV and CMV systemic titers. The antiviral effect of ECT2 on AMV seems to be modulated via its direct binding to the m6A residues presented in the viral RNAs, since an ECT2 mutant defective in m6A recognition loses wild type antiviral activity and is not able to pull down viral RNAs in vivo. On the other hand, according to the previous subcellular localization described for ECT2 and ECT4 and the ability of ECT2 to undergo gel-like phase in vitro, the transitory expression of ECT5 displays a cytoplasmic pattern with the formation of some aggregates. As found for mammal YTH proteins, the interaction between ECTs and poly-methylated RNA (in this case viral RNA) is proposed to promote the formation of stress granules and, consequently, reduce viral translation and replication rates. In summary, in this work, atALKBH9B is reported as the first m6A eraser identified in plants and, for the first time, it is described the influence of m6A methylation mechanism in plant viral infections. / [CA] Les modificacions químiques post-transcripcionals impliquen un nou nivell de modulació de l'expressió gènica. Al començament d'esta Tesi, s'havien caracteritzat alguns components del complex de metilació del nitrogen en posició 6 de la adenosina (m6A) en plantes. No obstant això, a diferència de mamífers i llevat, cap dels 13 homòlegs d'AlkB (atALKBH1-10B) - potencials desmetilases (o erasers) - i les 13 proteïnes de la família YTH (ECT1- 11, AT4G11970 i CPSF30) - potencials proteïnes de reconeixement de m6A (o readers) - identificades en el genoma d'Arabidopsis s'havien caracteritzat funcionalment. A més, diversos estudis han descrit la presència de residus m6A en RNAs de virus de mamífers i les diferents funcions que exercix esta modificació en la regulació de les infeccions virals. No obstant això, no s'ha estudiat la possible implicació d'este mecanisme molecular en les infeccions virals de plantes. El descobriment de la interacció entre la CP del virus del mosaic de l'alfals (AMV) i una proteïna d'Arabidopsis (atALKBH9B) amb homologia a una eraser humana va ser el punt de partida d'esta Tesi. En este treball es confirma esta interacció, i es demostra que atALKBH9B també pot reconéixer els RNAs virals. Els resultats revelen que atALKBH9B té la capacitat de desmetilar m6A a partir de molècules de RNA monocatenari in vitro. Esta proteïna s'acumula en grànuls citoplasmàtics que es colocalitzen amb siRNA bodies i s'associen a P-bodies, la qual cosa suggerix que l'activitat atALKBH9B podria estar relacionada amb els processos de silenciament i/o degradació de mRNA. D'altra banda, assajos preliminars mostren que els RNAs virals de l'AMV, el virus del mosaic del cogombre (CMV), el virus de l'arruga del nap (TCV) i el virus del mosaic de la coliflor (CaMV) es metilen durant la infecció en Arabidopsis. A més, per AMV i CMV els resultats van ser confirmats per UPLC-PDA-Tof-MS i els llocs m6A al llarg dels RNAs d'AMV s'identificaren mitjançant MeRIP-seq. Els resultats presentats confirmen que la relació m6A/A al llarg dels RNAs virals augmenta en les plantes atalkbh9b en comparació amb les silvestres, mentre que la traducció i/o replicació es veuen afectades i el moviment sistèmic a les tiges florals està pràcticament bloquejat. A diferència de la CP d'AMV, la de CMV no interacciona amb atALKBH9B per Y2H i, com ocorre amb la resta dels virus analitzats (CMV, TCV i CaMV), el seu cicle d'infecció no es veu afectat en plantes atalkbh9b. A més, la seqüenciació de mRNA realitzada en este treball revela que la infecció per AMV indueix alguns gens d'Arabidopsis pertanyents a la maquinària m6A, MTA, MTB, VIR i ECT5. D'acord amb l'efecte antiviral dependent de m6A per a l'AMV i tenint en compte que ECT2, ECT3 i ECT4 van ser recentment caracteritzades com readers citoplasmàtiques, la supressió del mòdul ECT2/ECT3/ECT5 augmenta significativament els títols sistèmics d'AMV i CMV. L'efecte antiviral d'ECT2 sobre AMV sembla estar modulat a través de la seua unió directa als nucleòtids m6A presents en els RNAs virals, ja que un mutant de la proteïna ECT2 defectuós en el reconeixement de m6A perd l'activitat antiviral que sí que presenta la proteïna original i no és capaç d'arrossegar RNAs virals in vivo. D'altra banda, d'acord amb la localització subcel·lular descrita prèviament per a ECT2 i ECT4 i la capacitat d'ECT2 per a experimentar una fase similar al gel in vitro, l'expressió transitòria d'ECT5 mostra un patró citoplasmàtic amb la formació d'agregats. Es proposa que, com s'ha descrit per a les proteïnes YTH de mamífers, la interacció entre les ECTs i el RNA polimetilat (en aquest cas, RNA viral) promouria la formació de grànuls d'estrès i, en conseqüència, reduiria les taxes de traducció i replicació viral. En resum, en este treball es caracteritza la primera m6A eraser de plantes, atALKBH9B, i, per primera vegada, es descriu la influència de l'mecanisme de metilació M6A en les infeccions virals de plantes. / Martínez Pérez, M. (2020). Involvement of the host RNA N6-adenosine methylation (m6A) pathway in the infection cycle of Alfalfa mosaic virus [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/155976

TCV (Turnip crinkle virus) coat protein is required for the resistance response in Arabidopsis thaliana Di-17 plants. An aspartate to asparagine mutation at amino acid four of the coat protein is sufficient to result in resistance-breaking. To determine the essential chemical properties responsible for the induction of resistance, a series of site-directed mutants were produced. Serine as well as asparagine at amino acid four induces systemic disease on both Di-3 and Di-17 plants; however, replacement of aspartate with glutamate retains the ability to induce the HR (hypersensitive response) and resist TCV infection with rapid and strong induction of PR-1 gene. These data suggest that the negative charge at the fourth amino acid of the coat protein is critical for the induction of resistance. Taken together with other mutagenesis research, the N-terminus of the coat protein appears to be the sole viral recognition element. The A. thaliana TIP protein is suggested to be involved in resistance, mainly through its C-terminus. Interestingly, one of the resistance-breaking mutants (D4N) produces a HR on Di-3 plants that are normally susceptible. The Di-3 TIP protein has several differences from the Di-17 TIP. To detect whether the delayed HR is related to interaction between Di-3 TIP and D4N mutation, a yeast two-hybrid assay was attempted. Interactions have not yet been detected. There are a number of possible explanations.

X-ray crystallography is a powerful method for determining the three-dimensional structures of biological macromolecules at atomic resolution. Crystallography can reliably provide the answer to many structure related questions, from global folds to atomic details of bonding. Crystallographic techniques find wide applications in understanding macromolecular assembly, enzyme mechanism, mode of activation of enzymes, substrate-specificity, ligand-binding properties, domain movement etc. The knowledge of accurate molecular structures is also a prerequisite for rational drug design and for structure based functional studies to aid the development of effective therapeutic agents. The current thesis can be broadly divided into two major parts. The first four chapters deal with assembly studies that have been carried out on Sesbania mosaic virus and the next two chapters describe the structure and function of a stationary phase survival protein, SurE from Salmonella typhimurium. In both studies X-ray crystallographic techniques have been used extensively for the structural studies. Viruses are obligate parasites with a proteinaceous capsid enclosing the genetic material. For genetic economy, several copies of capsid proteins self assemble to form complex virus capsids. Due to their intricate symmetric structures, viruses are considered as minute marvels of molecular architecture and study of virus structures serve as a paradigm for solutions to problems concerning macromolecular assembly and function in general. Crystallography provides a means of visualizing intact virus particles as well as their isolated constituent proteins and enzymes at near-atomic resolution, and is thus an extraordinarily powerful tool for understanding the function of these biological systems. Protein-protein interactions, protein-nucleic acid interactions, metal-ion mediated interactions, interactions between capsid proteins and auxillary or scaffolding proteins and particle maturation or post processing of capsid protein subunits are various elements that play a role in capsid assembly. Many structural and sequential motifs have been proposed as important conformational switches of capsid assembly. A functional analysis of these motifs by way of mutations in the capsid protein and structural studies of these mutants can provide further insight into capsid assembly pathways. Interaction between capsid protein subunits can determine the size and robustness of the capsid. Analysis of protein-protein interactions can help in understanding the principles of self-assembly. Arresting capsid assembly by disrupting intersubunit interactions and trapping the assembly intermediates will be helpful to delineate the changes that happen in capsid protein during the course of assembly and understand assembly pathways. Sesbania mosaic virus (SeMV) is a plant virus with a positive sense single-stranded RNA genome and belongs to the Sobemovirus genus. The protein and nucleic acids of SeMV can be separated and reassembled in vitro. Also, expression of the coat protein (CP) gene of SeMV in E. coli leads to the formation of virus like particles (VLPs). Therefore, SeMV is an excellent model system to study the assembly pathways that lead to the formation of complex virus shells. Earlier structural and functional studies on the native virus and the recombinant capsid protein and its various mutants have revealed the following: SeMV is a T=3 virus with chemically identical A-, B- and C-subunits occupying quasi equivalent positions in the icosahedral asymmetric unit of the virus particle. The A-type subunits form pentamers at the five-fold, and the B- and C- type subunits form hexamers at the icosahedral three-fold axes. The amino terminus of the polypeptide is ordered from residue 72 in the A- and B- subunits whereas it is ordered from residue 44 in the C-subunit. The disordered segment in all the subunits has an arginine rich motif (N-ARM). The segment ordered only in C-subunits has a -annulus structure that promotes intersubunit interactions at the quasi six-fold and a -segment (A). The virus is stabilized by protein-protein, protein–RNA and Ca2+ mediated protein-protein interactions. Virus like particles (VLPs) formed by the expression of full length CP encapsidate 23 S E. coli rRNA and CP mRNA. Expression of a deletion mutant lacking the N-terminal 65 residues (rCP∆N65) which results in the removal of the N-ARM, the -annulus and the A leads to the formation of stable T=1 particles. The -annulus, which was earlier believed to be an important molecular switch controlling the assembly of T=3 VLPs was found to be dispensable. The N-ARM, though important for RNA encapsidation, was not essential for capsid assembly . Depletion of Ca2+ ions led to slight swelling of virus particles and significantly reduced stability. Extensive studies on the VLPs suggested that the assembly is most likely initiated by the dimers of the capsid protein. Following a brief account of the historical highlights in the field of structural virology, a review of current literature on the available crystal structures of viruses and various assembly studies on viruses that have been carried out with emphasis on role of nucleic acid mediated interactions, protein-protein interactions and role of specific residues and ion-mediated interactions in assembly are presented in Chapter I of the thesis. A separate section in this chapter deals with the disassembly experiments that have led to the formation of smaller oligomers of spherical viruses. This chapter also gives an account of the earlier work that has been carried out on SeMV, which is the model system of study for the present thesis. Chapter II describes in detail the structural studies on the β-annulus deletion mutant of SeMV. A unique feature of several T = 3 icosahedral viruses is the presence of a structure called the β-annulus formed by extensive hydrogen bonding between protein subunits related by icosahedral three-fold axis of symmetry. This unique structure has been suggested as a molecular switch that determines the T = 3 capsid assembly. In order to examine the importance of the β-annulus, a deletion mutant of Sesbania mosaic virus coat protein in which residues 48–59 involved in the formation of the β-annulus were deleted retaining the rest of the residues in the amino terminal segment (rCP (Δ48–59)) was constructed. When expressed in Escherichia coli, the mutant protein assembled into virus like particles of size close to that of the wild type virus particles. The purified capsids were crystallized and their three dimensional structure was determined at 3.6Å resolution by X-ray crystallography. The mutant capsid structure closely resembled that of the native virus particles. However, surprisingly, the structure revealed that the assembly of the particles has proceeded without the formation of the β-annulus. Therefore, the β-annulus is not essential for T = 3 capsid assembly as speculated earlier and may be formed as a consequence of the particle assembly. This is the first structural demonstration that the virus particle morphology with and without the β-annulus could be closely similar. Chapter III begins with a detailed description of the interfacial residue mutations that have been carried out in SeMV with the aim of disrupting assembly and trapping an assembly intermediate. These mutations were performed in rCP as well as rCP∆N65 gene. Among these, a single point mutation of a Trp 170 to a charged residue (either Glu or Lys) arrested virus assembly and resulted in stable dimers of the capsid protein. The chapter also gives an account of the biophysical characterization of these mutants. rCP∆N65 dimer mutants showed a characteristic 230 nm peak in CD spectral studies which may be due to the interactions of a stretch of aromatic residues in the capsid protein. The isolated dimers were more susceptible to trypsin cleavage compared to the assembled capsids due to the exposed basic amino terminus. Thermal melting studies showed that the isolated dimer mutants were much less stable when compared to the assembled capsids, probably due to the loss of intersubunit interactions and Ca2+ mediated interactions. The structure of one of the isolated dimer mutant- rCP∆N65W170K was solved to a resolution of 2.65Å. Chapter IV describes the crystal structure analysis of the rCP∆N65W170K mutant dimer and compares its structure with the dimers of native virus, T=3 and T=1 VLPs. A number of structural changes occur especially in the loop and interfacial regions during the course of assembly. The dimer in solution was “more relaxed” than the dimer that initiates assembly. Ca2+ ion is not bound and consequently the C-terminal residues are disordered. The FG loop, which interacts with RNA, was found to be flexible and adopts a different conformation in the unassembled dimer. The present thesis also deals with the structural and functional studies of a phosphatase, SurE, the stationary phase survival protein from Salmonella typhimurium. Chapter V provides a general introduction on Salmonella, which is a mesophilic food borne pathogen, its general features, classification and stress responses. This chapter also gives an account of stationary phase in bacteria and stress responses. A brief description about phosphatases and their classification is also presented in this chapter. Following this, a review of the current literature on the structural, biochemical and functional role of stress related proteins and phylogenetic and enzymatic studies of various homologues of SurE are described in detail. Chapter VI deals with the detailed crystal structure analysis of SurE, the first stationary phase survival protein from a mesophilic organism. SurE, of Salmonella typhimurium forms part of a stress survival operon regulated by the stationary phase RNA polymerase alternative sigma factor. SurE is known to improve bacterial viability during stress conditions. It functions as a phosphatase specific to nucleoside monophosphates. Here we report the X-ray crystal structure of SurE from Salmonella typhimurium (St SurE). The protein crystallized in two forms- orthorhombic F222 and monoclinic C2. The two structures were determined to resolutions of 1.7Å and 2.7Å, respectively. The protein exists as a domain swapped dimer. The residue Asp 230 is involved in several interactions that are probably crucial for domain swapping. A divalent metal ion is found at the active site of the enzyme, which is consistent with the divalent metal-ion dependent activity of the enzyme. Interactions of the conserved DD motif present at the N-terminus with the phosphate and the Mg2+ present in the active site suggest that these residues play an important role in enzyme activity. The divalent metal ion specificity and the kinetic constants of SurE were determined using the generic phosphatase substrate- para- Nitro Phenyl Phosphate. The enzyme was inactive in the absence of divalent cations and was most active in the presence of Mg2+. Thermal denaturation studies showed that St SurE is much less stable compared to its homologues and an attempt was made to understand the molecular basis of the lower thermal stability based on solvation free energy. The thesis concludes with a brief summary of the entire work that have been presented and future prospects. The various crystallographic, biochemical and biophysical techniques employed in the investigations are described under the section experimental techniques in Appendix I and the NCS matrices used in the structure solution of the β-annulus deletion mutant are listed in Appendix II.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.