Viruses are the only organisms known to store their genetic information solely in the form of RNA, and have thus evolved unique machinery to replicate an RNA genome and initiate viral gene expression in the infected cell. The large polymerase protein (L) of negative-strand (NS) RNA viruses is a particularly intriguing model for viral replication, where all of the enzymatic activities required for mRNA transcription, RNA modification, and genomic RNA replication are contained within a single polypeptide. Whereas the host cell requires a suite of enzymes to accomplish these tasks, L alone is the catalytic engine driving NS RNA viral replication. Here we demonstrate purification of functional L protein from Machupo virus (MACV) and reconstitute arenavirus RNA synthesis initiation and gene expression regulation in vitro using purified recombinant components. Through single-molecule electron microscopy analysis of MACV L, we provide the first structural information of viral L proteins. Comparative analysis with nonsegmented NS RNA viral L proteins reveals how the various enzymatic domains are arranged into a conserved architecture shared by both polymerases. Our in vitro RNA synthesis data defines the basis of arenavirus sequence-specific polymerase recruitment and how inter-termini interactions regulate template recognition. Moreover, we discover a new role for the arenaviral matrix protein in regulating viral RNA synthesis by locking a polymerase-template complex. The inhibitory matrix-L-RNA assembly functionally links transcription regulation and polymerase packaging, and reveals a mechanism for NS RNA viruses to ensure polymerase incorporation during virion maturation. Reconstitution of RNA synthesis in vitro establishes a new framework to understand the arenaviral polymerase complex, and our structural and biochemical experiments provide a basis for mechanistic analysis of the NS RNA viral replication machinery.

Morbilliviruses belong to the family Paramyxoviridae of the Mononegavirale order of viruses. The Mononegavirale order contains viruses which contain negatively-polar, non-segmented and single stranded RNA genomes. This order contains some of most lethal pathogens known to the humankind. Ebola virus and Marburg virus are perhaps the most lethal human pathogens. Rinderpest virus, declared eradicated in 2011, was known to be the most significant cattle killer. Similarly the Canine distemper virus and Rabies virus, two topmost canine pathogens belong to this order. The L protein in the viruses of Morbillivirus genus harbours the viral RNA-dependent RNA polymerase that replicates and transcribes the viral genome and also all the mRNA capping enzymes, viz. RNA 5’ triphosphatase, guanylyltransferase, RNA (guanine-7-)methyltransferase and RNA 5’ cap-dependent (2’-oxo-)methyltransferase. Moreover this protein can act as a protein kinase that can regulate the function of P protein which serves as a switch between transcription and replication. mRNA capping is necessary for the virus for the purpose of exploiting host cellular machinery towards viral protein synthesis. The Rinderpest virus L protein serves as a model to study the capping enzymes of Morbillivirus. RNA triphosphatase (RTPase), the first enzyme of the capping cascade had earlier been located on the L protein. The RTPase minimal domain on the L protein was identified earlier by sequence homology studies done with RTPase proteins of Baculovirus and Vaccinia virus and cloned. The bacterially expressed recombinant domain was shown to possess RTPase activity. The enzymatic activity was characterized and the RTPase was found to be a metal-dependent enzyme which is highly specific to capping viral mRNA. Further characterization of the domain revealed that the domain also possesses nucleotide triphosphatase (NTPase), tripolyphosphatase and pyrophosphatase activities. Two site-directed mutants in motif-A of the domain: E1645A and E1647A were also tested and were found to be essential for the RTPase and NTPase activity. It was also recognized through these mutant studies that the active sites of RTPase and NTPase activities are partially overlapping. Earlier work done with Vesicular stomatitis virus capping enzymes showed that the Rhabdoviridae family of viruses follow unconventional capping pathway utilizing an enzyme polyribonucleotidyltransferase (PRNTase) which transfers GDP to 5’-monophosphated RNA. Characterization of the RTPase activity which converts 5’-triphosphated RNA into 5’-diphosphated RNA is an evidence for the morbilliviruses utilizing the conventional eukaryotic capping cascade. The results show that Paramyxoviridae do not follow unconventional capping pathway for the mRNA capping as has been the paradigm in the past decade.

Rinderpest virus (RPV) belongs to the order Mononegavirale which comprises non segmented negative sense RNA viruses including human pathogens such as Measles, Ebola and Marburg virus. RPV is the causative agent of Rinderpest disease in large ruminants, both domesticated and wild. The viral genome contains a non segmented negative sense RNA encapsidated by nucleocapsid protein (N-RNA). Viral transcription/replication is carried out by the virus encoded RNA dependent RNA polymerase represented by the large protein L and phosphoprotein P as (L-P) complex. Viral transcription begins at the 3’ end of the genome 3’le-N-P-M-F-H-N-tr-5’ with the synthesis of 55nt leader RNA followed by the synthesis of other viral mRNAs. A remarkable feature common to all members of Paramyxoviridae family is the gradient of transcription from 3’ end to the 5’ end of the genome due to attenuation of polymerase transcription at each gene junction. The present study aims at functional characterization of Rinderpest virus transcription and the associated activities required for viral mRNA capping. In addition, an attempt has been made to understand the novel role of a host factor, Ebp1, playing a key role in virus multiplication in infected cells. The specific aims of the study are presented in detail below. 1. Development of in vitro transcription system for RPV mRNA synthesis and role of phosphorylation of P protein in transcription. The transition of viral polymerase from transcription to replication in infected cells has been a long standing puzzle in all paramyxoviruses. Earlier work carried out using RPV minigenome with a CAT reporter gene and studies with phosphorylation null mutant P, has revealed the importance of P phosphorylation for viral transcription in vivo. However, the contribution of other cellular factors in the viral transcription/replication switch could not be ruled out in these assays. In order to understand the specific role of P protein in transcription/replication, it was necessary to develop a cell free transcription system for viral mRNA synthesis. Hence, viral genomic RNA (N-RNA) was purified from RPV infected cells using CsCl density gradient centrifugation. The viral RNA polymerase consisting of L-P complex was separately expressed in insect cells and partially purified by glycerol gradient centrifugation. Glycerol gradient fraction containing the L-P complex was found to be active in viral transcription. Notably, the gradient of transcription of viral mRNA was observed in vitro with the partially purified recombinant L-P complex similar to in vivo. However, the recombinant polymerase complex failed to synthesis the 55nt leader RNA, in agreement with the recent finding in VSV that the transcriptase complex was unable to synthesize leader RNA and viral transcription is initiated at the N gene start site unlike the conventional 3’ entry mode. The newly developed in vitro reconstituted transcription system was used to analyze the effect of P phosphorylation on viral transcription. The results presented in chapter 2, indicate that phosphorylated P supports transcription whereas unphosphorylated P transdominantly inhibits the transcription in vitro suggesting the possible role of the status of P protein phosphorylation in determining transcription/replication switch. 2. Enzymatic activities associated with RPV L protein- role in viral mRNA capping. Post transcriptional modification of mRNA such as capping and methylation determines the translatability of viral mRNA by cellular ribosome. In negative sense RNA viruses, synthesis of viral mRNA is carried out by the viral encoded RNA polymerase in the host cell cytoplasm. Since the host capping and methylation machinery is localized to the nucleus, viruses should either encode their own mRNA modification enzymes or adopt alternative methods as has been reported for orthomyxoviruses (cap snatching) and picornaviruses (presence of IRES element). In order to test, if RPV RNA polymerase possesses any of the capping and methylation activities, both virus as well as the RNP complex containing the viral N-RNA and RNA polymerase (L-P) were purified from infected cells. Using the purified virus and RNP complex, the first two activities required for mRNA capping vis-à-vis, RNA triphosphatase and guanylyltransferase were tested and the results are described in chapter 3 and 4. Purified virus as well as the RNP complex showed both RNA triphosphatase (RTPase) and Nucleotide triphosphatase activities. Neither purified N-RNA or recombinant P proteins show these activities suggesting that it is indeed mediated by viral L protein. By the metal dependency of the reaction and by the motif conservation with other reported RTPases, RPV L protein was assigned to the metal dependent RTPase tunnel family. Capping activity was also seen with the L protein present in RNP complex by its ability to form a covalent complex with GMP moiety of GTP. The specificity of the reaction with GTP, inhibition of Enzyme-GMP complex formation by the inorganic pyrophosphate and the susceptibility of Enzyme-GMP complex under acidic conditions clearly indicated that RPV L represents the viral guanylyl transferase. Further confirmation was obtained by the indirect capping assay in which Enzyme-GMP complex was formed when recombinant L protein was incubated with the cap labeled RNA due to the reversible nature of capping reaction. Owing to the large size of L protein (240 KDa), it is conceivable that the L protein functions in a modular fashion for different activities pertaining to RNA synthesis and modification. Sequence comparison of L proteins from different morbilliviruses revealed the presence of three conserved domains namely domain I (aa 1-606), domain II (aa 650-1694) and domain III (aa 1717-2183). Since domain II has already been assigned as the viral RNA dependent RNA polymerase, domain I and domain III were chosen for further characterization. Both domains were cloned, expressed and purified to homogeneity using recombinant baculovirus expression system. However, the recombinant domain III alone showed the NTPase activity where as neither domain I or III showed RTPase activity. This is expected since a part of the conserved RTPase motif was located in domain II in the multiple sequence alignment with other viral and yeast RTPases. In addition, the recombinant domain III also showed the characteristic enzyme-GMP complex formation but failed to be active in the indirect capping assay. Therefore, both domain II and domain III are likely to be involved in the co-transcriptional capping of viral mRNA. In support of this view, recent report in VSV suggests the presence of additional motif in domain II which is essential for viral mRNA capping. Preliminary evidence has been presented in the appendix section for the presence of N7 guanine methyl transferase activity with L protein although further experiments are needed to confirm this activity. 3. Role of host factor Ebp1 in negative sense RNA virus replication - a possible antagonist In recent years, many cellular factors such as actin, tubulin and profilin have been shown to be involved in viral transcription. Ebp1-ErbB3 binding protein was initially isolated as a cellular protein which binds to Influenza viral polymerase subunit PB1. Ebp1 selectively inhibits the influenza virus transcription in vitro whereas the cap binding and endonuclease activity of PB1 subunit of viral polymerase is unaffected. Till now there are no reports of the role of Ebp1 in non segmented negative sense RNA virus infection. The fifth chapter describes the role of Ebp1 in RPV infection and vice versa. RPV infection leads to down regulation of Ebp1 mRNA levels which in turn leads to decreased protein synthesis. Subsequently, it was found that Ebp1 interacts presumably with viral N protein, being a part of the viral RNP complex in both infected cells as well as in purified virion. Further, over expression of Ebp1 inhibits viral transcription and as a consequence the virus multiplication in vivo suggesting a mutual antagonism between virus and the host cell through Ebp1 protein.

Made available in DSpace on 2017-07-10T17:40:57Z (GMT). No. of bitstreams: 1 Tese_2012_Joao_Edson_Kaefer.pdf: 1393414 bytes, checksum: fdf2b5e5d88064ab53a8cf4b503d44bd (MD5) Previous issue date: 2012-06-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The canola term is an acronym for Canadian Oil Low Acid and was adopted as the standard to indicate low levels of erucic acid and glucosinolates. In general, tropical soils are poor in available nitrogen (N) because of the low stocks of organic matter, requiring the external supply of N to meet the demands of the culture. Among the main sources of N used in the culture of canola are nitrogen fertilizers urea and ammonium sulfate. Besides the choice of fertilizer nitrogen source related to N, to adjust the timing of nitrogen application at the time of greatest demand increased demand will increase crop production efficiency In view of this, six experiments were carried out from May 2009 to April 2010. These experiments were mainly aimed at evaluating the response of canola to nitrogen sources and application methods (seeding and / or coverage) of nitrogen fertilizer. All experiments were conducted in the experimental units at the Catholic University of Paraná - PUCPR - Toledo campus. To attend the responses of canola relative to rates and N sources were implanted three experiments in randomized blocks in a 7x2 factorial arrangement, consisting of seven levels of nitrogen at sowing (0, 20, 40, 60, 80, 100 and 120 kg ha -1) and two N sources (ammonium sulphate and urea), with four replications. To attend the responses of canola on the forms of nitrogen application and nitrogen sources were implanted over three experiments, which are conducted in randomized blocks in a 5x2 factorial arrangement consisting of five forms of nitrogen in the seeding and / or in coverage, respectively (0 and 0, 120 and 0, 0 and 120, 40 and 80, 80 and 40 kg N ha-1) and two nitrogen sources (ammonium sulphate and urea), with four replications. The coverage fertilization was performed in stage B4. The six experiments were carried out in three production cycles, two experiments in each cycle, corresponding to the following sowing dates: cycle 1: 12/05/2009; cycle 2: 20/06/2009 and cycle 3: 23/04 / 2010. All treatments received a fertilizer equivalent to 300 kg ha-1 00-25-25 formulation, each plot received the amount of nitrogen corresponding to the predetermined treatment. For the six experiments were evaluated the basal diameter, plant height, number of plants m-2, dry leaves, dry weight of stem + petioles, inflorescence dry mass, total plant dry mass, leaf area, area ratio of leaf, mass of pods per plant, weight per pod, weight of grains per pod, thousand grain weight, yield, leaf N content, protein and oil content in grain and oil yield per hectare. The results show that the variables were not influenced by sources of nitrogen fertilizer, ammonium sulfate and urea, for any of the six experiments conducted. The response to N rates influence these variables measured, and the higher productivity achieved with 88 kg ha-1 N. The increase of N doses promotes an increase in the protein reducing therefore the oil content in grains. The forms of nitrogen application also influenced the variables measured, and the best results achieved by the split of applying fertilizer at planting one-third and two-thirds coverage (40 and 80 kg N ha-1) / O termo canola é um acrônimo de CANadian Oil Low Acid e foi adotado como padrão para indicar baixos teores de ácido erúcico e glucosinolatos. De um modo geral, os solos tropicais apresentam baixa disponibilidade de nitrogênio (N) em função dos baixos estoques de matéria orgânica, exigindo o fornecimento externo de N para suprir as demandas da cultura. Dentre as principais fontes de N utilizados na cultura da canola estão os adubos nitrogenados uréia e sulfato de amônio. Além da escolha do adubo nitrogenado relacionado à fonte de N, ajustar o momento da aplicação do N ao momento de maior demanda da cultura aumenta a eficiência de produção. Visando instrumentar decisões relativas a estas alternativas de manejo, foram desenvolvidos quatro experimentos no período de maio de 2009 a abril de 2010. Estes experimentos tiveram como principal objetivo avaliar a resposta da canola a fontes, doses e momento de aplicação do N em Toledo Pr. Todos os experimentos foram conduzidos na unidade experimental da Pontifícia Universidade Católica do Paraná PUCPR - campus Toledo. Para quantificar as respostas da canola relativas a doses e fontes de N foram implantados dois experimentos de blocos casualizados, em esquema fatorial 7x2, constando de sete doses de N na semeadura (0; 20; 40; 60; 80; 100 e 120 kg ha-1 de N) e duas fontes de N (sulfato de amônio e uréia), com quatro repetições. Para quantificar as respostas da canola relativas ao momento de aplicação e às fontes de N foram implantados mais dois experimentos, sendo estes conduzidos em blocos casualizados, em esquema fatorial 5x2, constando de cinco combinações de momento de aplicação do N, na semeadura e/ou em cobertura, respectivamente (0 e 0; 120 e 0; 0 e 120; 40 e 80; 80 e 40 kg ha-1 de N) e duas fontes de N (sulfato de amônio e uréia), com quatro repetições. A adubação em cobertura foi realizada no estádio B4. Os quatro experimentos foram implantados em duas épocas de semeadura: 12/05 e 23/04. Todos os tratamentos receberam a adubação correspondente a 300 kg ha-1 de N, P2O5 e K2O na formulação 00-25-25 aplicado na semeadura, além da quantidade de N correspondente ao tratamento pré-estabelecido. Nos quatro experimentos foram avaliados o diâmetro basal, altura de planta, número de plantas m-2, massa seca de folhas, massa seca de caule+pecíolo, massa seca de inflorescência, massa seca da parte aérea, área foliar, razão de área foliar, massa de síliquas por planta, massa por síliqua, massa de grãos por síliqua, massa de mil grãos, produtividade, teor de N foliar, teor de proteína e óleo nos grãos e rendimento de óleo por hectare. Os resultados obtidos mostram que as variáveis avaliadas não foram influenciadas pelas fontes de N utilizadas, sulfato de amônio e uréia, para nenhum dos quatro experimentos conduzidos. Quanto à resposta às doses de N estas influenciaram as variáveis mensuradas, sendo a maior produtividade alcançada com 88 kg ha-1 de N. O aumento nas doses de N promove um incremento nos teores de proteína reduzindo, por consequência o teor de óleo nos grãos. O momento de aplicação do N também influenciou as variáveis mensuradas, sendo os melhores resultados alcançados pelo parcelamento da adubação aplicando-se um terço na semeadura e dois terços em cobertura (40 e 80 kg ha-1 de N)

Peste des petits ruminants virus (PPRV) belongs to the family paramyxoviridae which comprises non segmented negative sense RNA viruses including measles and rinderpest virus. PPRV is the causative agent of peste des petits rumaninats disease (also known as sheep or goat plague disease) in small ruminants. The viral genome contains a non segmented negative sense RNA encapsidated by viral encoded nucleocapsid protein (N-RNA). Viral transcription is carried out by the virus encoded RNA dependent RNA polymerase complex represented by the large protein L and phosphoprotein P. Viral transcription begins at the 3’ end of the genome synthesising all the viral transcripts (3’-N-P-M-F-HN-L-5’). A remarkable feature common to all members of Paramyxoviridae family is the gradient of transcription from 3’ end to the 5’ end due to attenuation of polymerase transcription at each gene junction. The objectives of the present study are characterization of peste des petits ruminants virus transcription and the associated activities required for post transcriptional modification of viral mRNA. In addition, an attempt has been made to develop in vitro transcription with heterologous combination of PPRV and RPV polymerase proteins. The first reaction in capping involves removal of γ-phosphate from triphosphate ended precursor mRNA by RNA triphosphatase. The domain having RNA triphosphatase activity within the L protein has been identified and expressed independently in E. coli. The details of the objectives are presented below. 1. Development of in vitro transcription system for PPRV mRNA synthesis In order to develop an in vitro transcription reconstitution system for PPRV, the viral RNP complex comprising large (L), phospho (P) and N protein encapsidating viral genomic RNA was purified from virus infected Vero cells. The in vitro transcription reconstituted system with RNP complex was able to synthesise all the viral mRNA as analysed by RT-PCR. As a control, total RNA from virus infected cells was isolated and analysed by RT-PCR. In order to refine the in vitro transcription system, separately expressed recombinant polymerase complex was used to reconstitute transcriptional activity in vitro. For this,viral genomic RNA (N-RNA) was purified from PPRV infected cells using CsCl density gradient centrifugation. The recombinant baculovirus for PPRV P protein was earlier generated in the lab. A recombinant baculovirus harbouring the L gene of PPRV was generated in the present study (described in part one). The viral RNA polymerase consisting of L-P complex was expressed in Sf21 insect cells and partially purified by ultra centrifugation on 5-20% glycerol gradient. Glycerol gradient fraction containing the L-P complex was found to be active in the in vitro transcription reconstitution system. Further quantitation of transcripts made in vitro and in infected cells has been carried out by real time PCR. Notably, the gradient of polarity of transcription of viral mRNA observed in vitro with the partially purified recombinant L-P complex was similar to the gradient observed in infected cells. Host proteins have been shown to modulate the transcription of many paramyxoviruses. In order to test the role of host factors, uninfected cell lysate of Vero cells was added to the in vitro transcription reaction and the transcript level was measured by real time PCR. The result showed an increase in the transcription by addition of host proteins suggesting the involvement of host factors in viral transcription. Further, the newly developed in vitro reconstitution system was used to test if recombinant L and P proteins of RPV can functionally replace PPRV L and P protein in the in vitro transcription complementation assay. The result presented in part one indicates that the L or P protein of PPRV can be replaced by RPV L and P protein in heterologous transcription reconstitution system ,with a reduced efficiency. However, the homologous polymerase complex of RPV failed to recognise the N-RNA genomic template of PPRV. 2. RNA triphosphatase activity of PPRV L protein and identification of RNA triphosphatase domain Post transcriptional modification of mRNA such as capping and methylation determines the translatability of viral mRNA by cellular ribosome. In negative sense RNA viruses, synthesis of viral mRNA is carried out by the viral encoded RNA polymerase in the host cell cytoplasm. Since the host capping and methylation machinery is localized to the nucleus, viruses should either encode their own mRNA modification enzymes or adopt alternative methods as has been reported for orthomyxoviruses (cap snatching) and picornaviruses (presence of IRES element). In order to test, if PPRV RNA polymerase possesses any of the capping activities, the RNP complex containing the viral N-RNA and RNA polymerase (L-P) were purified from virion. Using the purified RNP complex, the first activity required for mRNA capping, RNA triphosphatase was tested and the results are described in part two. RNP complex purified from virion showed both RNA triphosphatase (RTPase) activity. The RNA triphosphatase from viruses, fungi and other eukaryotes have been classified into two groups, metal dependent and metal independent. The cleavage of the γ-phosphate from triphosphate ended precursor mRNA by L protein of PPRV was found to be metal dependent. So, by the metal dependency of the RTPase reaction, PPRV L protein was assigned to the metal dependent RTPase tunnel family. One of the key features of metal dependent RTPase group members is the ability to hydrolyse γ-β phosphoanhydride bond of NTPs. PPRV L protein associated with RNP complex also was also able to cleave γ-β phosphoanhydride bond of NTPs. Owing to the large size of L protein (240 KDa), it is conceivable that the L protein functions in a modular fashion for different activities pertaining to mRNA synthesis and post transcriptional modification. Sequence comparison of L proteins from different morbilliviruses revealed the presence of three conserved domains namely domain I (aa 1-606), domain II (aa 650-1694) and domain III (aa 1717-2183). Domain II has the catalytic motif for viral RNA dependent RNA polymerase. Multiple sequence alignment of PPRV L protein with known RNA triphosphatases predicted a two hundred amino acid long region on L protein comprising the C terminus of domain II and N terminus of DIII as a possible candidate for RNA triphosphatase domain. The above predicted domain was cloned and expressed in E. coli. The ability of the purified recombinant RTPase domain to cleave γ-β phosphoanhydride bond of RNA was tested. The results described in part two suggest that the predicted RTPase domain has RNA triphosphatase activity. In addition to RNA triphosphatase, the RTPase domain also has the NTPase activity. The RNA triphosphatase of DNA viruses, yeasts and other fungi have three motifs essential for enzyme activity. Motif A and motif C are rich in glutamate and are involved in metal binding. Motif B is rich in basic amino acids and forms the centre for catalysis. The glutamate residue (E1647) of motif A of PPRV L protein RTPase domain was converted to alanine and the loss of RTPase activity was assessed. The results summarised in appendix 1 shows that the E1647A mutant has reduced RNA triphosphatase and NTPase activity.