Philosophiae Doctor - PhD / Hepatitis C was first recognized as a transfusion-associated liver disease not caused by hepatitis A or hepatitis B virus after serological tests were developed to screen for their presence in the blood. The infectious agent was finally identified with the cloning of the cDNA of hepatitis C virus (HCV) using random polymerase chain reaction (PCR) screening of nucleic acids extracted from plasma of a large pool of chimpanzee infected with non-A non-B hepatitis. NS5B, a membrane-associated RNA-dependent RNA polymerase essential in the replication of HCV, initiates the synthesis of a complementary negative-strand RNA from the genomic positive-strand RNA so that more positive-strand HCV RNA can then be generated from the newly synthesised negative-strand template. The crystal structure of NS5B presented typical fingers, palm and thumb sub-domains encircling the GDD active site, which is also seen in other RNA-dependent RNA polymerases, and is similar to the structure of reverse transcriptase of HIV-1 and murine Moloney leukaemia virus. The last 21 amino acids in the C-terminus of NS5B anchor the protein to the endoplasmic reticulum (ER)-derived membranous web. NS5B has been shown to interact with the core, NS3/NS4A, NS4B and NS5A proteins, either directly or indirectly. Numerous interactions with cellular proteins have also been reported. These proteins are mainly associated with genome replication, vesicular transport, protein kinase C-related kinase 2, P68 (DDX5), α-actinin, nucleolin, human eukaryotic initiation factor 4AII, and human VAMP-associated protein. Previous studies have confirmed that NS5B binds to full-length DDX5. By constructing deletion mutants of DDX5, we proceeded to characterize this interaction between DDX5 and HCV NS5B. We report here the identification of two exclusive HCV NS5B binding sites in DDX5, one in the N-terminal region of amino acids 1 to 384 and the other in the C-terminal region of amino acids 387 to 614. Proteins spanning different regions of DDX5 were expressed and purified for crystallization trials. The N-terminal region of DDX5 from amino acids 1 to 305 which contains the conserved domain I of the DEAD-box helicase was also cloned and expressed in Escherichia coli. The cloning, expression, purification and crystallization conditions are presented in this work. Subsequently, the crystal structure of DDX5 1-305 was solved and the high resolution three-dimensional structure shows that in front of domain I is the highly variable and disordered N terminal region (NTR) of which amino acids 51-78 is observable, but whose function is unknown. This region forms an extensive loop and supplements the core with an additional α-helix. Co-immunoprecipitation experiments demonstrated that the NTR of DDX5 1-305 auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-60 residues in NTR and NS5B binding site in DDX5 1-305, presumably located within residues 79-305. Furthermore, co-immunoprecipitation experiments revealed that DDX5 can also interact with other HCV proteins, besides NS5B.

Bovine viral diarrhea virus (BVDV) is an important viral pathogen affecting all ages of cattle, resulting in significant economic losses worldwide. BVDV infection is associated with a diverse array of symptoms including gastrointestinal disorder, respiratory distress, fetal malformation, stillbirth, abortions, and mucosal disease (MD). Transplacental infections of fetuses between 42 and 125 days of gestation can result in immune-tolerance and the surviving fetuses become persistently infected (PI). PI animals are major reservoir of BVDV and it becomes problematic to control the disease. The objectives of this dissertation were to: 1) develop a cost-effective testing scheme to detect BVDV PI animals from exposed herds, 2) characterize two virulent BVDV-2 Mississippi isolates associated with severe hemorrhagic diseases, and 3) perform phylogenetic analysis based on sequences of 5'UTR, E2, and NS5B regions. First, we developed a BVDV testing scheme by combining pooled real-time RT-PCR with antigen capture enzyme-linked immunosorbent assay (ACE) to screen cattle herds. From positive pools individual positives were identified using ACE. Data from a three year period indicated that 92.94% PI animals were infected with BVDV-1, 3.53% with BVDV-2, and 3.53% with both BVDV-1 and BVDV-2. Analysis of the 5'UTR of 22 isolates revealed the predominance of BVDV-1b followed by BVDV-2a. Second, two virulent BVDV isolates, M10-3432 and M10-5347, were successfully recovered from an adult beef breeding cow and feedlot calf respectively. When compared to the reference strain BVDV-2 125c, five and three unique amino acids in E2 regions were different from M10-5347 and M10-3432 respectively. Phylogenetic analysis of E2 region grouped both Mississippi isolates in BVDV-2a, a subtype containing high virulent strains. M10-3432 was clustered with high virulent strain 890 while M10-5347 was clustered with high virulent strain CD87. Third, we compared the phylogenetic analyses of BVDV based on the sequences of 5'UTR, E2, and NS5B at either nucleotides or amino acids level. Although slight differences were observed, the virulent BVDV isolates were consistently classified into BVDV-2a cluster regardless of region of sequences used. Furthermore, phylogenetic tree constructed using combined two or more regions had higher posterior probability and bootstrap value than phylogenetic trees constructed using a single region

RESUMO Cavalheiro, NP. Hepatite C: transmissão entre casais. São Paulo, 2004. 111p. Tese (Doutorado) - Faculdade de Medicina, Universidade de São Paulo. Introdução: A ocorrência e eficiência da transmissão sexual do VHC na ausência de outros fatores de risco ainda é muito controversa. Foram investigados e analisados 24 casais, ambos cônjuges infectados com o VHC. Destes, 22 apresentaram o mesmo subtipo viral e a análise filogenética de parte da região NS5b mostrou altos índices de homologia nas seqüências entre os vírus dos casais infectados. Objetivo: Análise da transmissão da Hepatite C entre casais heterossexuais. Método: O estudo recrutou 45 casais. Destes, 24 foram selecionados e incluídos na pesquisa, com diagnóstico clínico e laboratorial para a Hepatite C crônica. A infecção foi diagnosticada por testes imunoenzimáticos de terceira geração e pela presença da partícula viral circulante detectada pela PCR. As amostras de sangue foram coletadas entre os anos de 1999 e 2002. Foram seqüenciadas partes das regiões 5’NC e NS5b do VHC, para determinação dos subtipos virais. Os testes utilizados para as PCRs estão disponíveis para pesquisa e foram respectivamente TRUGENE 5’NC Test (Bayer Health Care Diagnostics, Tarrytown, NY, USA) e Titan One Tube RT-PCR Kits (Roche Molecular, Mannheim, Germany). Para as PCRs dos seqüenciamentos foi utilizado o kit CLIP sequencing test (Bayer Health Care Diagnostics, Tarrytown, NY, USA). As seqüências foram analisadas com o sistema de seqüenciamento Open Gene DNA, software na Versão 3.1, biblioteca específica (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Para o alinhamento das seqüências, referentes a região NS5b, foi utilizado o programa Clustal W (Clustal W Multiple Sequence Alignment Program, v1.7, June 1997) e a árvore filogenética foi gerada pelo método Neighbor Joining. Um questionário padrão e entrevistas foram usados para coleta de dados sobre fatores de risco para aquisição da doença e comportamento sexual. Os pacientes desta pesquisa foram recrutados no Ambulatório de Hepatite do Hospital das Clínicas da Universidade de São Paulo e Ambulatório de Hepatite do Hospital Guilherme Álvaro, da cidade de Santos. Resultados: Entre os 24 casais selecionados, 22 apresentaram o mesmo subtipo viral e altas porcentagens de homologia (região NS5b) entre 93,0% e 99,4%. Os subtipos HCV apresentados foram dois (9,1%) casais infectados por 1a, nove (40,9%) com subtipo 1b, um (4,6%) com subtipo 2b e dez (45,5%) dos casais pelo subtipo HCV 3a. Os dois casais discordantes apresentaram índices de 70,1% e 82,2% e foram infectados pelos subtipos 2b e 1b, e 1b e 1a respectivamente. A média de tempo de convivência foi de 22,4 anos, variando de 2 a 45 anos e a renda per capta anual foi em média US$2,270/ano. Com base nos questionários e entrevistas os fatores de risco apresentados pelos casais foram: 9 (37,5%) transfusão de sangue, 17 (70,8%) U.D. endovenosa e 15 (62,5%) U.D. inalatória, 4 (16,7%) acupuntura e 5 (20,8%) tatuagem. O compartilhar de utensílios de higiene pessoal relatado pelos casais apresentou altos índices e 6 (25,0%) dos casais assumiram o uso comum de escova de dente, 16 (66,7%) lâmina de barbear, 21 (87,5%) cortador de unhas e 14 (58,3%) alicate de manicure. Os dois casais discordantes relataram fatores de risco como transfusão de sangue e U.D. Conclusão: A alta similaridade encontrada entre as cadeias genômicas do VHC pode dar suporte a hipótese de transmissão do VHC entre esses casais. O uso compartilhado de utensílios de higiene pessoal e o tempo de convivência tornam difícil a interpretação dos dados. O uso compartilhado de utensílios de higiene pessoal pode dificultar a interpretação dos dados em relação à transmissão sexual do VHC. A hipótese do sentido mais provável de transmissão do VHC, de homem para mulher, foi reforçada neste trabalho. / ABSTRACT Cavalheiro, NP. Hepatitis C: transmission between couples. São Paulo, 2004. 111p. Thesis (Doctoral) - Faculdade de Medicina, Universidade de São Paulo. Introduction: The occurrence and the efficiency of HCV sexual transmission in the absence of other risk factors are still very controversial. I investigated and analyzed 24 couples, both infected with HCV, of whom 22 shared the same viral subtype. A phylogenetic analysis of NS5b region showed high sequence homology among the infected couples. Objective: Analysis of the Hepatitis C transmission between heterosexuals couples. Methods: The study recruited 45 couples, 24 were included, with anti-HCV positive and clinical diagnosis of active chronic hepatitis. HCV infection was diagnosed by positivity of serum samples for anti HCV (third-version enzyme immunoassay) and by circulating HCV-RNA detected by Polymerase Chain Reaction (PCR). All blood samples were collected between 1999 and 2002. Sequencing of the 5’NC region was performed utilizing the research available TRUGENE HCV 5’NC Test (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Sequencing of the NS5B region was performed by RT-PCR amplification with Titan One Tube RT-PCR Kits (Roche Molecular, Mannheim, Germany) and CLIP sequencing using a prototype NS5B genotyping assay (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Sequence analysis was completed using the Open Gene DNA Sequencing System, Gene Objects software package (Version 3.1), and Gene Librarian module (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Multiple sequence alignments of the NS5B region were performed with Clustal W (Clustal W Multiple Sequence Alignment Program, v1.7, June 1997), and phylogenetic trees were generated using the Neighbor Joining Method. A standardized questionnaire and interview was used to collect data concerning risk factors and sexual behaviors. Follow up of all subjects was conducted at the hepatitis clinic of the Clinical Hospital of the University of Sao Paulo and at the Hospital Guilherme Alvaro in the city of Santos, in the state of Sao Paulo, Brazil. Results: Among the 24 couples, 22 had matching viral subtypes with homology scores (NS5b) ranging from 93.0% to 99.4%. Of the 22 couples with matching subtype, two (9.1%) where infected with subtype 1a, nine (40.9%) with subtype 1b, one (4.6%) with subtype 2b and ten (45.5%) with subtype 3a. The two couples that did not show matching viral subtypes had scores of 70.1% and 82.2%, and were infected with subtypes 2b and 1b, and 1b and 1a, respectively. The average of duration of marriage was 22.4 years (range 2-45 years) and the per capita income was an average of US$2,270/year. Based on the questionnaire and interviews, cause of infection of the 24 couples could be attributed to: blood transfusions 9 (37.5%), drug use, I.V. 17(70.8%) and inhalation 15 (62.5%), acupuncture 4 (16.7%) and tattooing 5 (20.8%). Shared hygienic utensils showed a much higher correlation of possible route of transmission, and are better explained by the sequence homology data than by the other associated risk factors. A total of 6 (25.0%) couples shared tooth brushes, 16 (66.7%) shared shaving blades, 21 (87.5%) shared nail clippers and 14 (58.3%) shared manicure cutters. The two couples that had different subtypes, both of them related transfusion blood and I.V. drug use. Conclusions: The high similarity found among the genome chains of HCV supports the hypothesis of transmission between these couples. The shared use of personal hygiene utensils and the amount of time spent living together made it difficult to interpret the data. Also, the shared use of personal hygiene utensils can make it difficult to interpret the data in relation to the sexual transmission of HCV. The hypothesis in relation to the direction of the HCV transmission, from man to woman, was reinforced in this work.

RESUMO Cavalheiro, NP. Hepatite C: transmissão entre casais. São Paulo, 2004. 111p. Tese (Doutorado) - Faculdade de Medicina, Universidade de São Paulo. Introdução: A ocorrência e eficiência da transmissão sexual do VHC na ausência de outros fatores de risco ainda é muito controversa. Foram investigados e analisados 24 casais, ambos cônjuges infectados com o VHC. Destes, 22 apresentaram o mesmo subtipo viral e a análise filogenética de parte da região NS5b mostrou altos índices de homologia nas seqüências entre os vírus dos casais infectados. Objetivo: Análise da transmissão da Hepatite C entre casais heterossexuais. Método: O estudo recrutou 45 casais. Destes, 24 foram selecionados e incluídos na pesquisa, com diagnóstico clínico e laboratorial para a Hepatite C crônica. A infecção foi diagnosticada por testes imunoenzimáticos de terceira geração e pela presença da partícula viral circulante detectada pela PCR. As amostras de sangue foram coletadas entre os anos de 1999 e 2002. Foram seqüenciadas partes das regiões 5’NC e NS5b do VHC, para determinação dos subtipos virais. Os testes utilizados para as PCRs estão disponíveis para pesquisa e foram respectivamente TRUGENE 5’NC Test (Bayer Health Care Diagnostics, Tarrytown, NY, USA) e Titan One Tube RT-PCR Kits (Roche Molecular, Mannheim, Germany). Para as PCRs dos seqüenciamentos foi utilizado o kit CLIP sequencing test (Bayer Health Care Diagnostics, Tarrytown, NY, USA). As seqüências foram analisadas com o sistema de seqüenciamento Open Gene DNA, software na Versão 3.1, biblioteca específica (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Para o alinhamento das seqüências, referentes a região NS5b, foi utilizado o programa Clustal W (Clustal W Multiple Sequence Alignment Program, v1.7, June 1997) e a árvore filogenética foi gerada pelo método Neighbor Joining. Um questionário padrão e entrevistas foram usados para coleta de dados sobre fatores de risco para aquisição da doença e comportamento sexual. Os pacientes desta pesquisa foram recrutados no Ambulatório de Hepatite do Hospital das Clínicas da Universidade de São Paulo e Ambulatório de Hepatite do Hospital Guilherme Álvaro, da cidade de Santos. Resultados: Entre os 24 casais selecionados, 22 apresentaram o mesmo subtipo viral e altas porcentagens de homologia (região NS5b) entre 93,0% e 99,4%. Os subtipos HCV apresentados foram dois (9,1%) casais infectados por 1a, nove (40,9%) com subtipo 1b, um (4,6%) com subtipo 2b e dez (45,5%) dos casais pelo subtipo HCV 3a. Os dois casais discordantes apresentaram índices de 70,1% e 82,2% e foram infectados pelos subtipos 2b e 1b, e 1b e 1a respectivamente. A média de tempo de convivência foi de 22,4 anos, variando de 2 a 45 anos e a renda per capta anual foi em média US$2,270/ano. Com base nos questionários e entrevistas os fatores de risco apresentados pelos casais foram: 9 (37,5%) transfusão de sangue, 17 (70,8%) U.D. endovenosa e 15 (62,5%) U.D. inalatória, 4 (16,7%) acupuntura e 5 (20,8%) tatuagem. O compartilhar de utensílios de higiene pessoal relatado pelos casais apresentou altos índices e 6 (25,0%) dos casais assumiram o uso comum de escova de dente, 16 (66,7%) lâmina de barbear, 21 (87,5%) cortador de unhas e 14 (58,3%) alicate de manicure. Os dois casais discordantes relataram fatores de risco como transfusão de sangue e U.D. Conclusão: A alta similaridade encontrada entre as cadeias genômicas do VHC pode dar suporte a hipótese de transmissão do VHC entre esses casais. O uso compartilhado de utensílios de higiene pessoal e o tempo de convivência tornam difícil a interpretação dos dados. O uso compartilhado de utensílios de higiene pessoal pode dificultar a interpretação dos dados em relação à transmissão sexual do VHC. A hipótese do sentido mais provável de transmissão do VHC, de homem para mulher, foi reforçada neste trabalho. / ABSTRACT Cavalheiro, NP. Hepatitis C: transmission between couples. São Paulo, 2004. 111p. Thesis (Doctoral) - Faculdade de Medicina, Universidade de São Paulo. Introduction: The occurrence and the efficiency of HCV sexual transmission in the absence of other risk factors are still very controversial. I investigated and analyzed 24 couples, both infected with HCV, of whom 22 shared the same viral subtype. A phylogenetic analysis of NS5b region showed high sequence homology among the infected couples. Objective: Analysis of the Hepatitis C transmission between heterosexuals couples. Methods: The study recruited 45 couples, 24 were included, with anti-HCV positive and clinical diagnosis of active chronic hepatitis. HCV infection was diagnosed by positivity of serum samples for anti HCV (third-version enzyme immunoassay) and by circulating HCV-RNA detected by Polymerase Chain Reaction (PCR). All blood samples were collected between 1999 and 2002. Sequencing of the 5’NC region was performed utilizing the research available TRUGENE HCV 5’NC Test (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Sequencing of the NS5B region was performed by RT-PCR amplification with Titan One Tube RT-PCR Kits (Roche Molecular, Mannheim, Germany) and CLIP sequencing using a prototype NS5B genotyping assay (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Sequence analysis was completed using the Open Gene DNA Sequencing System, Gene Objects software package (Version 3.1), and Gene Librarian module (Bayer Health Care Diagnostics, Tarrytown, NY, USA). Multiple sequence alignments of the NS5B region were performed with Clustal W (Clustal W Multiple Sequence Alignment Program, v1.7, June 1997), and phylogenetic trees were generated using the Neighbor Joining Method. A standardized questionnaire and interview was used to collect data concerning risk factors and sexual behaviors. Follow up of all subjects was conducted at the hepatitis clinic of the Clinical Hospital of the University of Sao Paulo and at the Hospital Guilherme Alvaro in the city of Santos, in the state of Sao Paulo, Brazil. Results: Among the 24 couples, 22 had matching viral subtypes with homology scores (NS5b) ranging from 93.0% to 99.4%. Of the 22 couples with matching subtype, two (9.1%) where infected with subtype 1a, nine (40.9%) with subtype 1b, one (4.6%) with subtype 2b and ten (45.5%) with subtype 3a. The two couples that did not show matching viral subtypes had scores of 70.1% and 82.2%, and were infected with subtypes 2b and 1b, and 1b and 1a, respectively. The average of duration of marriage was 22.4 years (range 2-45 years) and the per capita income was an average of US$2,270/year. Based on the questionnaire and interviews, cause of infection of the 24 couples could be attributed to: blood transfusions 9 (37.5%), drug use, I.V. 17(70.8%) and inhalation 15 (62.5%), acupuncture 4 (16.7%) and tattooing 5 (20.8%). Shared hygienic utensils showed a much higher correlation of possible route of transmission, and are better explained by the sequence homology data than by the other associated risk factors. A total of 6 (25.0%) couples shared tooth brushes, 16 (66.7%) shared shaving blades, 21 (87.5%) shared nail clippers and 14 (58.3%) shared manicure cutters. The two couples that had different subtypes, both of them related transfusion blood and I.V. drug use. Conclusions: The high similarity found among the genome chains of HCV supports the hypothesis of transmission between these couples. The shared use of personal hygiene utensils and the amount of time spent living together made it difficult to interpret the data. Also, the shared use of personal hygiene utensils can make it difficult to interpret the data in relation to the sexual transmission of HCV. The hypothesis in relation to the direction of the HCV transmission, from man to woman, was reinforced in this work.

Until recently, the standard therapy for hepatitis C treatment has been interferon and ribavirin. Such treatment has only 50% efficacy and is not well tolerated. The emergence of new drugs has increased the treatment efficacy to 90%. Despite such an achievement, the success is limited since the virus mutates rapidly, causing the emergence of drug resistant forms. In addition, most new drugs were developed to treat genotype 1 infections. Thus, development of new potent antivirals is needed and drug discovery against hepatitis C is continued. In this thesis, a FRET-based protease assay was used to evaluate new pyrazinone based NS3 protease inhibitors that are structurally different to the newly approved and currently developing drugs. Several compounds in this series showed good potencies in the nanomolar range against NS3 proteases from genotype 1, 3, and the drug resistance variant R155K. We assume that these compounds can be further developed into drug candidates that possess activity against above mentioned enzyme variants. By using SPR technology, we analyzed interaction mechanisms and characteristics of allosteric inhibitors targeting NS5B polymerases from genotypes 1 and 3. The compounds exhibited different binding mechanisms and displayed a low affinity against NS5B from genotype 3. In order to evaluate the activity and inhibitors of the NS5B polymerase, we established an SPR based assay, which enables the monitoring of polymerization and its inhibition in real time. This assay can readily be implemented for the discovery of inhibitors targeting HCV. An SPR based fragment screening approach has also been established. A screen of a fragment library has been performed in order to identify novel scaffolds that can be used as a starting point for development of new allosteric inhibitors against NS5B polymerase. Selected fragments will be further elaborated to generate a new potent allosteric drug candidate. Alternative approaches have successfully been developed and implemented to the discovery of potential lead compounds targeting two important HCV drug targets.

Mémoire en recherche subventionné par le Conseil de recherche en sciences naturelles et en génie du Canada (CRSNG) / Le génome du virus de l’hépatite C (VHC), membre des Flaviviridae, est constitué d’un ARN monocaténaire linéaire de polarité positive, et contient un seul cadre de lecture ouvert flanqué par deux régions non traduites désignées 5’UTR et 3’UTR. Ces régions contiennent des éléments structurés qui sont importants pour la régulation des processus de traduction et de réplication virale. Mon projet de recherche vise le 3’UTR. Celui-ci a une structure tripartite composée d’une région variable (VR), une région poly (U/UC) et une région X. Notre laboratoire a identifié une séquence VR qui forme une structure duplexe avec une séquence distale située dans le NS5B. Considérant que le gène NS5B code pour la polymérase virale, et que la 3’UTR est impliquée dans la réplication virale, nous postulons que ce duplex pourrait contrôler le niveau de synthèse de l'ARN du VHC. Afin de tester cette hypothèse, nous avons construit des mutants individuels des séquences VR et NS5B du VHC 1b afin d’empêcher la formation du duplex, ainsi qu’un mutant complémentaire aux deux sites permettant la reconstitution du duplex. L’ARN provenant de ces constructions a été synthétisé in vitro et transfecté dans les cellules Huh7.5. Au 9e jour de culture, l’ARN a été extrait des cultures, et la technique RT-qPCR a été ensuite utilisée afin de quantifier et comparer les niveaux d’ARN viral provenant des cultures transfectées avec les mutants versus celles transfectées avec le virus parental. Les résultats obtenus montrent une augmentation de 5.3X et 3.4X, respectivement, entre les niveaux d’ARN viral produits de cultures transfectées avec le virus muté dans les régions 3’UTR-VR et NS5B, comparé au virus parental. Cependant, l’ARN viral obtenu suite à la reconstitution des deux sites complémentaires montre une augmentation de 2.6X. Malgré que la différence entre les niveaux d’ARN des virus mutants ne soit pas statistiquement significative lorsqu’on les compare avec le niveau d’ARN généré par le virus parental, la tendance suggère que la fonction des séquences appariées serait de moduler à la baisse la synthèse de l’ARN viral. Il sera nécessaire de poursuivre davantage ce travail afin de mieux comprendre le rôle de la structure duplexe VR-NS5B sur la réplication du VHC. / The hepatitis C virus (HCV), a member of the Flaviviridae, contains a single (+) strand linear RNA genome which incorporates one long open reading frame flanked by two untranslated regions known as the 5’UTR and 3’UTR. These regions contain structures known to function in the regulation of HCV translation and replication. The 3’UTR is the focus of this research project. Its tripartite structure consists of a variable region (VR), a poly (U/UC) region and the X-tail. Our laboratory recently identified a VR sequence forming a duplex structure with a distal sequence located in the NS5B gene. Because NS5B encodes the viral polymerase, and seeing that the 3’UTR is involved in viral replication, we postulated that this duplex may function in controlling the level of HCV RNA replication. To test this hypothesis, we constructed HCV 1b mutants in the respective VR and NS5B annealing sequences to disrupt complementarity and prevent duplex formation. We also created a mutant with complementary mutations at both sites to reconstitute the duplex. RNA from these constructs was synthesized in vitro and transfected into Huh7.5 cells. Following a 9-day incubation period, RNA from all transfected cultures was harvested and HCV RNA was quantified by RT-qPCR. The data indicate an increase of 5.3X and 3.4X for HCV viral RNA harvested from cultures with disrupted 3’UTR-VR and NS5B, respectively, relative to wild-type HCV 1b, whereas the reconstituted HCV construct yielded a 2.6X increase in viral RNA. While these figures do not reach statistical significance, they suggest that the duplex structure modulates HCV replication downward. Further pursuit of this work is necessary to better elucidate the role of the VR-NS5B duplex structure on HCV replication.

The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV. By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided. Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease. This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays. Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor. Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen. The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design.   Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.