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Foamy viruses (FVs), like other retroviruses, have gained interest due to their applications as viral vectors in gene therapy and various other fields. Despite their potential applications, FV stands out as a unique retrovirus, with distinct features in its replicative cycle that make it a compelling subject of study. However, it remains one of the least studied retroviruses. A crucial aspect of the retroviral replicative cycle, especially for its use as in gene transfer, is the selective packaging of its viral genomic RNA (vgRNA) out of the vast pool of RNAs found in virus producing host cells. This process, known as vgRNA enrichment, involves efficiently packaging vgRNA among the cellular and processed viral genetic material. Unlike other retroviruses, the selective packaging process of vgRNA in FV has not been explored, making it largely unknown. Previous studies on other retroviruses have indicated that vgRNA packaging selectivity is achieved through specific features within the vgRNA itself, such as specific sequences elements called packaging signals as well as dimerization of vgRNA resulting in the presence of two copies vgRNA in each retrovirus particle. Additionally, viral structural proteins, particularly Gag, play a significant role by interacting with vgRNA through specific protein regions. To shed light on the selective packaging process of FV vgRNA, the roles of structural proteins Env, Pol, and Gag were investigated, along with the vgRNA's dimerization capacity. To quantify the vgRNA selective packaging, an enrichment assay (E-assay) and a competitive assay (C-assay) were established. The E-assay allows to compare FV vgRNA packaging efficiency relative to non-viral RNAs, whereas the C-assay enables the determination of the preferential packaging of one vgRNA over another when both are present within a cell. The results obtained regarding the role of the structural proteins on the vgRNA selective packaging emphasized the delicate balance required between viral protein expression and vgRNA levels. Overexpression of Env and Gag severely disrupted selective packaging. Particularly the excess of Env protein amount led to an increased production of subviral particles that lack the capability to selectively package vgRNA. An interesting observation was the impact of the RNA template used for translating Gag on vgRNA enrichment. Expressing Gag from vgRNA (cis) enhanced vgRNA packaging selectivity, while expression from an RNA containing only expression-optimized gag ORF sequences (trans) reduced vgRNA enrichment. Nevertheless, the results from the C-assays suggest that non-Gag-translating vgRNA can still be selectively packaged over non-selectively packable dimerization deficient vgRNA. This indicates that while Gag might have a cis-acting mechanism in FV vgRNA selective packaging, this role appears to be non-essential. As mentioned earlier, vgRNA dimerization appears to be a crucial factor in the selective packaging process of most retroviruses. In the case of FV, the dimerization process was previously reported to be facilitated by three specific regions on the vgRNA known as dimerization sites one to three (DS-I to -III). Among these sites, DS-II stands out as being indispensable for vgRNA dimerization due to its 10 nt palindromic sequence, a determinant reported to be essential for the interaction between the two strands of vgRNA for most retroviruses. To investigate the significance of FV vgRNA dimerization in its selective packaging, we conducted E- and C-assays to assess the vgRNA packaging efficiency and specificity in FV DS-II mutants, previously identified as non-dimerizing (DS-II-M6 and -M7) or exhibiting a low dimerization rate (DS-II-M2). Intriguingly, FV vgRNA packaging was significantly negatively affected in the non-dimerizing FV mutants (DS-II-M6 and -M7), and to a lesser extent in the DS-II-M2 mutant, where dimerization was reported of occur at lower rates. This reveals a direct correlation between vgRNA packaging efficiency and the reported vgRNA dimerization potential of these DS-II mutants. These results suggest that, similar to other retroviruses, vgRNA dimerization plays a pivotal role in FV's selective packaging. Furthermore, it is well-documented that secondary structures within vgRNA in some retroviruses facilitate dimerization, thereby enhancing the selective packaging process. In-silico analyses of the FV vgRNA predicted the formation of a stem loop created by the palindromic sequence (SL-Pal). To gain insight into the role of these secondary structures within the DS-II region of FV vgRNA in its selective packaging, we designed a series of new dimerization mutants. These mutants were meticulously engineered to disrupt, modify, or restore the SL-Pal structure by introducing mutations inside or in proximity to the palindromic sequence based on computational secondary structure prediction. Notably, we observed that the palindrome's sequence could be mutated, as long as the SL-Pal structure and G-C proportion along all the stem-loop were preserved in a manner identical to the original structure. This preservation was crucial to ensuring the selective packaging of vgRNA and maintaining viral infectivity. Lastly, the evaluation encompassing protein analysis, vgDNA quantification, and infectivity assessment conducted on DS-I and DS-II mutants revealed a significant decrease not only on vgRNA selective packaging but also on viral infectivity, Pol packaging, cleaving, and the RTr process in non-dimerizing mutants. This underscores the intricate interrelation of these processes, emphasizing their collective importance for successful viral production. In summary, the findings presented in this project represent a significant advancement in understanding FV vgRNA selective packaging and dimerization. They offer valuable and novel insights that contribute to the expansion of our knowledge about FV molecular biology and its potential applications as a viral transfer vector.:I. ACKNOWLEDGEMENTS I II. TABLE OF CONTENT III III. INDEX OF FIGURES VI IV. INDEX OF TABLES VIII 1 INTRODUCTION 1 1.1 Retroviruses 1 1.1.1 Taxonomy of retroviruses 2 1.1.2 General features of retroviruses 3 1.2 Foamy viruses 6 1.2.1 PFV virion structure and genome organization 8 1.2.2 Viral proteins 11 1.2.2.1 Gag 11 1.2.2.2 Pol 13 1.2.2.3 Env 15 1.2.3 Replication cycle 17 1.2.3.1 Early phase 18 1.2.3.2 Late phase 19 1.3 Selective packaging of retroviral vgRNA 20 1.3.1 selective vgRNA packaging in orthoretroviruses 20 1.3.1.1 Role of the capsid protein Gag on the selective vgRNA packaging 20 1.3.1.2 vgRNA packaging signals 22 1.3.1.3 vgRNA dimerization and selective packaging 23 1.3.1.4 Factors that determine the vgRNA fate 24 1.3.2 Selective packaging in Hepadnaviruses 26 1.3.3 Selective packaging in PFV 26 2 THESIS AIM 29 3 MATERIALS AND METHODS 30 3.1 Buffers and solutions 30 3.2 Enzymes 34 3.3 Commercial kits 34 3.4 Nucleic acids 35 3.4.1 Oligonucleotides 35 3.4.1.1 Oligonucleotides for cloning 35 3.4.1.2 Oligonucleotides for qPCR analysis 37 3.4.2 Plasmids 39 3.4.2.1 Plasmid constructs used in this project: 39 3.4.2.2 New plasmid constructs 42 3.5 Bacteria strains 52 3.6 Cell lines 52 3.7 Antibodies 53 3.8 Software and Devices 54 3.9 Consumables 56 3.10 Molecular Biology methods 56 3.10.1 Bacteria culture 56 3.10.2 Transformation of competent bacteria 57 3.10.3 Plasmid extraction 57 3.10.4 Molecular cloning 58 3.10.4.1 Polymerase Chain Reaction (PCR) 58 3.10.4.2 Plasmid digest 59 3.10.4.3 Fragment purification 59 3.10.4.4 Ligation 60 3.10.4.5 Transformation of ligated plasmid constructs 60 3.10.4.6 Plasmid preparation small-scale 61 3.10.4.7 Plasmid Sequencing 61 3.10.4.8 Plasmid quantification 61 3.10.4.8.1 Photometric quantification 61 3.10.4.8.2 Fluorometric quantification 62 3.1 Biochemistry methods 62 3.1.1 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 62 3.1.2 Semi-Dry Western Blot 63 3.1.3 Immunodetection 63 3.1.4 Quantification of viral proteins by Western Blot 63 3.2 Cell Culture and Virological Methods 64 3.2.1 Cell lines 64 3.2.2 Cell passaging 64 3.2.3 Calcium phosphate transfection 65 3.2.4 Harvest of viral particles 65 3.2.5 Cell harvesting 66 3.2.6 Viral infectivity determination 66 3.3 Molecular virology methods 67 3.3.1 Viral RNA extraction 67 3.3.2 Cellular RNA extraction 67 3.3.3 DNase treatment 68 3.3.4 RNA quantification by RT-qPCR 68 3.3.5 DNA quantification by qPCR 69 3.3.6 Enrichment assay (E-assay) 70 3.3.7 Competitive assay 72 3.3.8 Secondary RNA structure prediction 74 4 RESULTS 75 4.1 Establishment of methodologies for quantification of selective vgRNA packaging efficiency 75 4.1.1 Establishment of the Enrichment (E)-assay 75 4.1.1.1 Reference mRNA 75 4.1.1.2 Background subtraction 77 4.1.2 Establishment of the Competition (C)-assay 80 4.1.2.1 Silent mutants characterization 81 4.1.2.2 Primer design 83 4.2 Selective packaging in foamy virus 88 4.2.1 Role of the viral structural proteins on the selective vgRNA packaging 92 4.2.1.1 Role of Env on the selective vgRNA packaging 92 4.2.1.2 Role of Pol on the selective vgRNA packaging 97 4.2.1.3 Role of Gag on the selective vgRNA packaging 100 4.2.1.3.1 Gag cis-acting mechanism on the selective vgRNA packaging 103 4.2.2 Role of dimerization on the selective packaging 109 4.2.2.1 Characterization of the dimerization mutants 111 4.2.2.2 Selective vgRNA packaging and dimerization 114 4.2.2.3 PFV vgRNA secondary structure (prediction) and dimerization potential 118 4.2.2.4 Dimerization and RTr 124 5 DISCUSSION 129 5.1 Establishment of methodologies for vgRNA selective packaging determination 129 5.1.1 E-assay 129 5.1.2 C-assay 130 5.2 Viral factors involved in the FV vgRNA selective packaging 132 5.2.1 Selective packaging in PFV 132 5.2.2 Role of the structural viral proteins on the vgRNA selective packaging 133 5.2.2.1 Env 133 5.2.2.2 Pol 136 5.2.2.3 Gag 136 5.2.3 Role of vgRNA dimerization on its selective packaging 139 5.2.3.1 vgRNA DS-II secondary structure and dimerization 141 5.2.3.2 vgRNA dimerization and RTr 142 6 CONCLUSION 145 7 REFERENCES 146 8 APPENDICES 162 8.1 Abbreviations list 162 8.2 Anlage 1 165 8.3 Anlage 2 166

Positive-strand RNA viruses associate with specific organelle membranes of host cells to establish viral replication complexes. The replication protein 1a of brome mosaic virus associates strongly with the nuclear endoplasmic reticulum (ER) membranes, invaginates membranes into the lumen, and recruits various host proteins to establish replication complexes termed spherules. 1a has a strong affinity towards the perinuclear ER membrane, however, the structural features in 1a that dictate its membrane associations and thereby membrane remodeling activities are unclear. This study examined the possible role of an amphipathic α-helix, helix B, in BMV 1a's membrane association. Deletion or single substitution of multiple amino acids of helix B abolished BMV 1a's localization to nuclear ER membranes. Additional reporter-based, gain-of-function assays showed that helix B is sufficient in targeting several soluble proteins to the nuclear ER membranes. Furthermore, we found that the helix B-mediated organelle targeting is a functionally conserved feature among positive-strand RNA viruses of the alphavirus-like superfamily that includes notable human viruses such as Hepatitis E virus and Rubella virus as well as plant viruses such as cucumber mosaic virus and cowpea chlorotic mottle virus. Our results demonstrate a critical role for helix B across members of the alphavirus-like superfamily in anchoring viral replication complexes to the organelle membranes. We anticipate our findings to be a starting point for the development of sophisticated models to use helix B as a novel target for the development of antivirals for positive-strand RNA viruses that belong to the alphavirus-like superfamily. / Doctor of Philosophy / Among the seven classes of viruses, the positive-strand RNA viruses dominate the domain of viral diseases of the world. Brome mosaic virus (BMV) is a positive-strand RNA virus that infects cereal crops such as wheat, barley, and rice. BMV has a simple genome organization and serves as a suitable model virus to study and characterize positive-strand RNA viruses. The replication of all positive-strand RNA viruses occurs at the organelle membranes of the host. Membrane association of the replication is one of the early steps and a crucial event in the life cycle of positive-strand RNA viruses. One of the proteins produced early on during BMV infection is the replication protein 1a, which is also the master regulator of viral replication; 1a recruits viral factors in addition to hijacking the necessary host factors at the membranous sites to initiate replication. Upon reaching the organelle membranes, 1a induces membrane rearrangements to form viral replication complexes that safeguard the recruited factors from the deleterious effects of the host cell. The structural determinants within 1a that are responsible for such membrane association are unknown. This study explored the potential roles of a short helical motif within the 1a protein for its ability to dictate such site-specific membrane associations. We show here that this helical region is necessary and sufficient for 1a's membrane-binding activity. We also discovered it to be a functionally conserved feature that is responsible for membrane associations in various viruses of the alphavirus-like superfamily that includes some of the notable human viruses such as Hepatitis E virus and Rubella virus in addition to plant viruses such as cucumber mosaic virus and cowpea chlorotic mottle virus.

Viruses have huge impact on controlling diseases and regulating many key ecosystem processes. As metagenomic data can contain many microbiomes including many viruses, by analyzing metagenomic data we can analyze many viruses at the same time. The first step towards analyzing metagenomic data is to identify and quantify viruses present in the data. In order to answer this question, we developed a computational pipeline, FastViromeExplorer. FastViromeExplorer leverages a pseudoalignment based approach, which is faster than the traditional alignment based approach to quickly align millions/billions of reads. Application of FastViromeExplorer on both human gut samples and environmental samples shows that our tool can successfully identify viruses and quantify the abundances of viruses quickly and accurately even for a large data set. As viruses are getting increased attention in recent times, most of the viruses are still unknown or uncategorized. To discover novel viruses from metagenomic data, we developed a computational pipeline named FVE-novel. FVE-novel leverages a hybrid of both reference based and de novo assembly approach to recover novel viruses from metagenomic data. By applying FVE-novel to an ocean metagenome sample, we successfully recovered two novel viruses and two different strains of known phages. Analysis of viral assemblies from metagenomic data reveals that viral assemblies often contain assembly errors like chimeric sequences which means more than one viral genomes are incorrectly assembled together. In order to identify and fix these types of assembly errors, we developed a computational tool called VirChecker. Our tool can identify and fix assembly errors due to chimeric assembly. VirChecker also extends the assembly as much as possible to complete it and then annotates the extended and improved assembly. Application of VirChecker to viral scaffolds collected from an ocean meatgenome sample shows that our tool successfully fixes the assembly errors and extends two novel virus genomes and two strains of known phage genomes. / Doctor of Philosophy / Virus, the most abundant micro-organism on earth has a profound impact on human health and environment. Analyzing metagenomic data for viruses has the beneFIt of analyzing many viruses at a time without the need of cultivating them in the lab environment. Here, in this dissertation, we addressed three research problems of analyzing viruses from metagenomic data. To analyze viruses in metagenomic data, the first question needs to answer is what viruses are there and at what quantity. To answer this question, we developed a computational pipeline, FastViromeExplorer. Our tool can identify viruses from metagenomic data and quantify the abundances of viruses present in the data quickly and accurately even for a large data set. To recover novel virus genomes from metagenomic data, we developed a computational pipeline named FVE-novel. By applying FVE-novel to an ocean metagenome sample, we successfully recovered two novel viruses and two strains of known phages. Examination of viral assemblies from metagenomic data reveals that due to the complex nature of metagenome data, viral assemblies often contain assembly errors and are incomplete. To solve this problem, we developed a computational pipeline, named VirChecker, to polish, extend and annotate viral assemblies. Application of VirChecker to virus genomes recovered from an ocean metagenome sample shows that our tool successfully extended and completed those virus genomes.

O gênero botânico Clerodendrum pertence à família Lamiaceae e compreende várias espécies ornamentais, geralmente trepadeiras, das quais as mais comumente cultivadas são coração-sangrento (C. x speciosum Tiejism. & Binn.) e lágrima-de-Cristo (C. thomsonae Balf.). Manchas cloróticas e necróticas em folhas de coração-sangrento foram observadas pela primeira vez em um jardim de Piracicaba, SP, associadas à infestação com Brevipalpus phoenicis Geijskes (Acari: Tenuipalpidae). Exames de secções de tecidos das lesões foliares ao microscópio eletrônico revelaram ocorrência de efeitos citopáticos do tipo nuclear e concluiu-se que os sintomas eram causados por um vírus transmitido por Brevipalpus (VTB), o qual foi tentativamente designado de mancha clorótica de Clerodendrum (Clerodendrum chlorotic spot virus- ClCSV). O ClCSV é transmitido mecanicamente de coração-sangrento para coração-sangrento e em ensaios preliminares foi transmitido mecanicamente e por Brevipalpus phoenicis para várias outras plantas, além da ocorrência de sua disseminação natural por esses ácaros para outras espécies. Ocorre a infecção sistêmica nas hospedeiras Chenopodium quinoa Will. e C. amaranticolor Coste & Reyn. infectadas com ClCSV caso as plantas sejam mantidas por cerca de 2 semanas entre 28-30 oC. Utilizando-se estas plantas realizou-se a purificação parcial do vírus. Este trabalho apresenta a caracterização biológica e molecular do ClCSV. Os resultados dos testes PTA-ELISA e RT-PCR demonstraram a detecção do ClCSV em diversas hospedeiras, além da análise da reação sorológica e molecular deste vírus com os outros VTBs do tipo nuclear. O seqüenciamento do produto de PCR revelou que as seqüências de nucleotídeos apresentaram similaridade com a polimerase de OFV (Orchid Fleck Virus), outro VTB do tipo nuclear. Além da identificação sorológica do vírus foram realizadas análises morfo-anatômicas para visualização das alterações causadas pelo ClCSV em tecidos de Clerodendrum x speciosum e em hospedeiras infectadas. / The botanical genus Clerodendrum belongs to the family Lamiaceae and includes several ornamental species, usually climbing, and heart-bloody (C. x speciosum Tiejism. & Binn.) and tear-in-Christ (C. thomsonae Balf. ) are among the most cultivated. Necrotic and chlorotic spots on leaves of heart-blood have been observed for the first time in a garden of Piracicaba, associated with an infestation of Brevipalpus phoenicis Geijskes (Acari: Tenuipalpidae). Sections of diseased tissues examined in the electron microscope revealed characteristic cytopathic effects of the nuclear type and concluded that the symptoms were caused by a virus transmitted by Brevipalpus (VTB), tentatively named Clerodendrum chlorotic spot virus (ClCSV). This virus is transmitted through mechanical inoculation from heart-bloody to heart-bloody and in preliminary tests mechanically and by mites for several other plants, in addition to the natural occurrence of its spread to other species. Systemic infection occurs in the host Chenopodium quinoa Will. and C. amaranticolor Coste & Reyn. infected with ClCSV if the plants are kept at 28-30°C for about 2 weeks. These plants were used for partial purification of vírus. This study presents the biological and molecular characterization of ClCSV. Through the PTA-ELISA and RT-PCR tests was possible to detect the ClCSV in different host, in addition to the analysis of serological and molecular reaction of this virus with other type of nuclear VTBs. From the sequencing of the PCR product was obtained from nucleotide sequences that showed similarity to the polymerase of OFV (Orchid Fleck Virus), another type of nuclear VTB. Morpho-anatomical analysis were performed to see the changes caused by ClCSV in tissues of Clerodendrum x speciosum and other infected hosts. It was possible to observe the occurrence of hypertrophy, cell plasmolisis and the reduction of starch grains in the áreas injured in all plants infected by ClCSV.

Made available in DSpace on 2015-04-22T22:12:45Z (GMT). No. of bitstreams: 1 Roberto Alexandre.pdf: 1763581 bytes, checksum: c0c0eb448d286d60be56fecd5eb291e0 (MD5) Previous issue date: 2010-08-03 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Human papillomavirus is responsible for lesions in the oral mucosa, anal and urogenital tract of male and female, transmitted by direct or indirect contact with infected skin or through sexual intercourse. In women these infections can progress to cervical cancer, which is estimated incidence for the Northern region in 2010 was the largest in Brazil. The nature of the infection depends on the degree of integration of viral DNA with host DNA linked primarily to genes of oncoproteins E6 and E7 of HPV. The determination of the viral types can be held from differences in the viral capsid L1 gene and the variants of a particular type of HPV can be identified through the study of viral non-coding region. Currently the development of prophylactic vaccines against HPV particles using "pseudo-viral" formed by the L1 protein of different subtypes of high risk, while a growing number of studies that use the oncoproteins E6 and E7 in the development of therapeutic vaccines. However, it is necessary for the development of such antiviral vaccines also consider the great diversity of variants of HPV types exist, since differences between the genomic regions of these variants may influence the degree of their infections. This paper describes the complete genome sequence of a variant of HPV 16, detected in Amazonian region, using techniques of genetic engineering and the analysis of this genome by bioinformatics tools. It was observed by analysis of genetic distance that the genome of this variant has a genetic proximity of those identified in the literature as "African variants, and phylogenetic analysis, performed from the non-coding region, support this hypothesis. In addition, several mutations were detected in the genome and obtained, resulting in changes in the positions and number of restriction sites in its sequence. The major differences between the genetic regions of the genome sequenced and the corresponding variants in Africa have been observed over E7. It is expected, with that work, look for future research projects involving protein expression and genomic analysis of HPV in the Amazon region to the regional peculiarities in variants and provide a concise and complete reference on the genome of HPV 16 in the region. / O Papillomavirus Humano é responsável por lesões na mucosa oral, anal e do trato urogenital masculino e feminino, transmitidas por contato direto ou indireto com a pele infectada ou através de relações sexuais. Na mulher essas infecções podem evoluir para um câncer de colo do útero, cuja estimativa de incidência para a região Norte no ano de 2010 foi a maior do Brasil. A natureza das infecções depende do grau de integração do DNA viral com o DNA do hospedeiro associada, principalmente, aos genes das oncoproteínas E6 e E7 do HPV. A determinação dos tipos virais pode ser realizada a partir de diferenças no gene L1 do capsídeo viral e as variantes de um determinado tipo de HPV podem ser identificadas por meio do estudo da Região Não Codificadora viral. Atualmente o desenvolvimento de vacinas profiláticas contra o HPV utiliza partículas pseudo-virais formadas pela proteína L1 de tipos virais de alto risco, enquanto cresce o número de estudos que utilizam as oncoproteínas E6 e E7 no desenvolvimento de vacinas terapêuticas. Contudo, é necessário que o desenvolvimento de tais vacinas antivirais também considere a grande diversidade das variantes dos tipos de HPV existentes, uma vez que diferenças entre as regiões genômicas dessas variantes podem influenciar o grau de suas infecções. Este trabalho descreve o sequenciamento completo do genoma de uma variante do HPV 16, detectado no Estado do Amazonas, utilizando técnicas de Engenharia Genética, bem como a análise desse genoma por ferramentas de Bioinformática. Observou-se, pela análise de distâncias genéticas, que o genoma dessa variante apresenta grande proximidade genética dos exemplares identificados na literatura como variantes africanas , e as análises filogenéticas, realizadas a partir da Região Não Codificadora, reforçam essa hipótese. Além disso, também foram detectadas várias mutações ao longo do genoma obtido, resultando em alterações nas posições e na quantidade de sítios de restrição de sua sequência. As maiores diferenças entre as regiões gênicas do genoma sequenciado e as correspondentes nas variantes africanas foram observadas ao longo de E7. Espera-se, com esse trabalho, atentar os futuros projetos de pesquisa que envolvam expressão de proteínas e análises genômicas de HPV na região amazônica para as peculiaridades existentes nas variantes regionais e fornecer uma referência concisa e completa sobre o genoma do HPV 16 na região.

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. Despite decades of research, it is still unclear how these viruses establish a lifelong, asymptomatic infection in their rodent hosts while infection of humans often results in severe disease. Unable to enter a state of bona fide latency, the transcription and replication of the viral genomic RNA is likely highly regulated in time and subcellular space. Moreover, we hypothesize that the viral nucleoprotein (NP), responsible for the encapsidation of the viral RNA and the most highly expressed viral gene product, plays a key role in the regulation of the viral gene expression program. Further, exploring host-virus interactions may elucidate the basic aspects of arenavirus biology and how they cause such severe disease in humans. To explore these questions in greater detail, this dissertation has pursued three main avenues. First, to better understand lymphocytic choriomeningitis mammarenavirus (LCMV) genome replication and transcription at the single-cell level, we established a high-throughput, single-molecule (sm)FISH image acquisition and analysis pipeline and followed viral RNA species from viral entry through the late stages of persistent infection in vitro. This work provided support for a cyclical model of persistence where individual cells are initially transiently infected, clear active infection, and become re-infected from neighboring reservoir cells within the population. Second, we used FISH to visualize viral genomic RNA to describe the subcellular sites where LCMV RNAs localize during infection. We observed that, viral RNA concentrates in large subcellular structures located near the cellular microtubule organizing center and colocalizes with the early endosomal marker Rab5c and the viral glycoprotein in a proportion of infected cells. We propose that the virus is using the surface of a cellular membrane bound organelle as a site for the pre-assembly of viral components including genomic RNA and viral glycoprotein prior to their transport to the plasma membrane where new particles will bud. Last, we used mass spectrometry to identify human proteins that interact with the NPs of LCMV and Junín mammareanavirus (JUNV) strain Candid #1. We provided a detailed map of the host machinery engaged by arenavirus NPs, and in particular, showed that NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. We demonstrated that JUNV antagonizes the antiviral activity of PKR completely, effectively abrogating the antiviral activity of this surveillance pathway. In sum, the work composing this dissertation has given us fresh insight into how arenaviruses establish and maintain persistence; the nature of the subcellular site where viral genomic RNA is transcribed, replicated, and assembled with other viral components; and a global view of the cellular machinery hijacked by the viral nucleoprotein. This work improves our basic understanding of the arenavirus life cycle and may suggest novel antiviral therapeutic targets that could be exploited in the future.

O gênero botânico Clerodendrum pertence à família Lamiaceae e compreende várias espécies ornamentais, geralmente trepadeiras, das quais as mais comumente cultivadas são coração-sangrento (C. x speciosum Tiejism. & Binn.) e lágrima-de-Cristo (C. thomsonae Balf.). Manchas cloróticas e necróticas em folhas de coração-sangrento foram observadas pela primeira vez em um jardim de Piracicaba, SP, associadas à infestação com Brevipalpus phoenicis Geijskes (Acari: Tenuipalpidae). Exames de secções de tecidos das lesões foliares ao microscópio eletrônico revelaram ocorrência de efeitos citopáticos do tipo nuclear e concluiu-se que os sintomas eram causados por um vírus transmitido por Brevipalpus (VTB), o qual foi tentativamente designado de mancha clorótica de Clerodendrum (Clerodendrum chlorotic spot virus- ClCSV). O ClCSV é transmitido mecanicamente de coração-sangrento para coração-sangrento e em ensaios preliminares foi transmitido mecanicamente e por Brevipalpus phoenicis para várias outras plantas, além da ocorrência de sua disseminação natural por esses ácaros para outras espécies. Ocorre a infecção sistêmica nas hospedeiras Chenopodium quinoa Will. e C. amaranticolor Coste & Reyn. infectadas com ClCSV caso as plantas sejam mantidas por cerca de 2 semanas entre 28-30 oC. Utilizando-se estas plantas realizou-se a purificação parcial do vírus. Este trabalho apresenta a caracterização biológica e molecular do ClCSV. Os resultados dos testes PTA-ELISA e RT-PCR demonstraram a detecção do ClCSV em diversas hospedeiras, além da análise da reação sorológica e molecular deste vírus com os outros VTBs do tipo nuclear. O seqüenciamento do produto de PCR revelou que as seqüências de nucleotídeos apresentaram similaridade com a polimerase de OFV (Orchid Fleck Virus), outro VTB do tipo nuclear. Além da identificação sorológica do vírus foram realizadas análises morfo-anatômicas para visualização das alterações causadas pelo ClCSV em tecidos de Clerodendrum x speciosum e em hospedeiras infectadas. / The botanical genus Clerodendrum belongs to the family Lamiaceae and includes several ornamental species, usually climbing, and heart-bloody (C. x speciosum Tiejism. & Binn.) and tear-in-Christ (C. thomsonae Balf. ) are among the most cultivated. Necrotic and chlorotic spots on leaves of heart-blood have been observed for the first time in a garden of Piracicaba, associated with an infestation of Brevipalpus phoenicis Geijskes (Acari: Tenuipalpidae). Sections of diseased tissues examined in the electron microscope revealed characteristic cytopathic effects of the nuclear type and concluded that the symptoms were caused by a virus transmitted by Brevipalpus (VTB), tentatively named Clerodendrum chlorotic spot virus (ClCSV). This virus is transmitted through mechanical inoculation from heart-bloody to heart-bloody and in preliminary tests mechanically and by mites for several other plants, in addition to the natural occurrence of its spread to other species. Systemic infection occurs in the host Chenopodium quinoa Will. and C. amaranticolor Coste & Reyn. infected with ClCSV if the plants are kept at 28-30°C for about 2 weeks. These plants were used for partial purification of vírus. This study presents the biological and molecular characterization of ClCSV. Through the PTA-ELISA and RT-PCR tests was possible to detect the ClCSV in different host, in addition to the analysis of serological and molecular reaction of this virus with other type of nuclear VTBs. From the sequencing of the PCR product was obtained from nucleotide sequences that showed similarity to the polymerase of OFV (Orchid Fleck Virus), another type of nuclear VTB. Morpho-anatomical analysis were performed to see the changes caused by ClCSV in tissues of Clerodendrum x speciosum and other infected hosts. It was possible to observe the occurrence of hypertrophy, cell plasmolisis and the reduction of starch grains in the áreas injured in all plants infected by ClCSV.

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Murine polyomavirus is an important member of Polyomaviridae family offering potential applications in gene therapy and immunotherapy. Viral mutant analysis is crucial for study of the virus, however, commonly used methods of its production are laborious and give low yields. This thesis involves development of the new experimental system that can produce intact viral genome from recombinant plasmid in vivo using Cre/loxP-mediated recombination. One loxP site is unavoidably introduced into newly generated viral genome during recombination. Two variants of production plasmids generating wild type viral genome with incorporation of loxP between the poly(A) signal sites of early and late genes or into the intronic region of early genes were prepared. LoxP insertion between the poly(A) signal sites has a dramatic effect on viral gene expression and leads to complete loss of virus infectivity. Conversely, the infectious virus was obtained from the viral genome containing loxP site in the early intronic region. To ensure expression of Cre recombinase I also prepared stably transfected cell lines which can simplify the virus production. This thesis shows that newly designed system gives satisfactory yield of the virus, solves restrictions connected with commonly used methods and can be used for low infectious viral...