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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
31

Insights into the nature of retroviral replication and infection analyses of minus-strand DNA transfer, double infection, and virion and RNA dimer maturation /

Dang, Que. January 1900 (has links)
Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains ix, 172 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.
32

Quantificação e seqüênciamento do gene da transcriptase reversa em gatos naturalmente infectados com vírus da imunodeficiência felina tratado com AZT

Figueiredo, Andreza Soriano [UNESP] 22 June 2007 (has links) (PDF)
Made available in DSpace on 2014-06-11T19:29:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-06-22Bitstream added on 2014-06-13T18:39:11Z : No. of bitstreams: 1 figueiredo_as_me_botfmvz.pdf: 290159 bytes, checksum: 38ae46f390705b8d387a7ef9c78d6040 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O Vírus da Imunodeficiência Felina (FIV) é um lentivírus que causa uma síndrome de imunodeficiência em gatos domésticos. O FIV tem sido particularmente utilizado em estudos de resistência viral aos análogos de nucleosídeos devido a Transcriptase Reversa (TR) apresentar propriedades físicas, catalíticas e sensibilidade às drogas semelhantes à TR do HIV. Os objetivos desse trabalho foram tratar com AZT gatos naturalmente infectados com o FIV, fazer o monitoramento da carga viral e DNA proviral por PCR em tempo real e monitoramento genético por seqüenciamento. Dos 12 animais infectados, 6 receberam o AZT na dose de 10mg/kg/dia e 6 receberam placebo. Durante 96 dias de tratamento, o plasma e sangue destes animais foram analisado com relação à carga viral e concentração relativa de DNA proviral utilizando-se a técnica de quantificação relativa por PCR em tempo real com SYBR Green, desenvolvida por nossa equipe. Além disso, foi realizado o sequenciamento genético da região que codifica a TR de 3 dos animais. Foi realizada com sucesso a padronização da PCR em tempo real para quantificação relativa do FIV. Não houve diferença estatisticamente significativa da carga viral ou do DNA proviral entre os grupos tratado e controle. O seqüenciamento genético revelou a presença de lisina na posição 41 do sítio ativo da TR. A presença deste aminoácido confere até 4 vezes menor sensibilidade ao AZT em mutantes do HIV. Por possuir alta estabilidade genética, supomos que os vírus dos demais animais não sequenciados possuem também a 41-lisina A presença da 41-lisina pode ser uma das possíveis explicações para a falha do tratamento com AZT. Outra hipótese é a de que a dose fornecida não foi adequada. / Feline Immunodeficiency Virus (FIV) is a lentivirus which causes a progressive disruption of the host's immune functions. FIV has been particularly used as a model for studies in retroviral resistance to nucleoside analogs because its similarities in physical properties, catalytic and sensitivity in comparison with HIV/RT. The aims of this work were to treat cats naturally infected with FIV, quantify viral load and proviral DNA by real time quantitative PCR with SYBR Green and analyze the viral nucleotide sequence. From 12 animals naturally infected, 6 received AZT at a dose of 10mg/kg/day and 6 received placebo. During 96 days of treatment, viral load and concentration of proviral DNA were measured by relative quantitative real time PCR developed by our staff. The nucleotide sequence of the RT encoding region was also achieved for 3 animals. The real time PCR relative quantification was successfully standardized for FIV. There was no significant statistical difference between treated and control groups. The nucleotide sequence revealed a lysine at position 41 on the enzyme active site. This lysine confers 4-fold decreased sensitivity to AZT in HIV RT-mutants. FIV subtype B has high genetic stability and we purposed that the other virus not sequenced have the same amino acid and hypothesized that this mutations can be one of the reasons determining the failure of the treatment. The other hypothesis is that the dose was not adequate.
33

Caracterização dos padrões de expressão de glucosiltransferases B e C, da proteina ligante de glucano B e de possiveis genes reguladores em genotipos distintos de Streptococcus mutans / Expression analysis of glucosyltransferases B and C, glucan-binding protein B and their putative regulatory genes in distinct genotypes of Streptococcus mutans

Stipp, Rafael Nobrega, 1982- 23 February 2006 (has links)
Orientador: Renata de Oliveira Mattos-Graner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-06T06:24:36Z (GMT). No. of bitstreams: 1 Stipp_RafaelNobrega_M.pdf: 1453482 bytes, checksum: 75a8c7b14bc2c1994765a8fd89ae2d9f (MD5) Previous issue date: 2006 / Resumo: Streptococcus mutans são os principais patógenos da cárie dentária, pois são capazes de se acumular no biofilme dentário na presença de sacarose, e sob condições altamente acidogênicas, promovem a desmineralização dentária. As glucosiltransferases B (GtfB) e C (GtfC) produzidas por S. mutans são fundamentais neste processo, porque catalisam a síntese de glucanos extracelulares insolúveis, a partir da sacarose. A proteína ligante de glucano B (GbpB) parece também participar do acúmulo de S. mutans nos biofilmes, embora por processos ainda não compreendidos. Pouco se sabe sobre os mecanismos que regulam a expressão dos genes que codificam essas proteínas de virulência (gtfB, gtfC e gbpB). Neste sentido, objetivamos caracterizar o padrão de expressão dos genes gtfB, gtfC e gbpB e de seus possíveis genes regulatórios (vicR, comE, ciaR e rr11) em genótipos distintos de S. mutans. Para isso, foram realizadas análises de transcrição reversa-PCR semi-quantitativa (RT-PCR) dos genes alvo em diferentes fases de curvas de crescimento planctônico, em meio Brain Heart Infusion, a 37°C, em condições de anaerobiose. RNAs das células nas diferentes fases de crescimento foram extraídos e submetidos a reações de transcriptase reversa com primers arbitrários, para obtenção dos cDNAs totais. Análises de PCR semi-quantitativas dos cDNAs foram então realizadas com primers específicos e normalizados pela expressão do gene housekeeping 16SRNA, cuja expressão foi constante nas condições experimentais estudas. Os padrões de transcrição de gtfB e gtfC foram específicos ao background genético da cepa estudada. Os níveis de transcritos de gtfB e de gtfC foram coordenados durante fases específicas de crescimento, mas divergências nas curvas expressão dos mesmos ocorreram em grande parte dos genótipos. Os níveis de transcritos de gbpB foram também variáveis entre cepas, mas assumiram padrão independente de genes gtf e foi caracterizado por menores variações entre as diferentes fases de crescimento. Os genes regulatórios demonstraram picos de expressão nas fases log e/ou estacionária de crescimento, de acordo com o genótipo testado. Os resultados indicam que o padrões de expressão dos genes estruturais (gtfB, gtfC e gbpB) e regulatórios são cepa-específicos e que gtfB e gtfC são regulados por sistemas independentes, os quais parecem ser ativados em fases distintas de crescimento / Abstract: Streptococcus mutans, the main pathogen of dental caries, have the capacity to accumulate in the dental biofilm in the presence of sucrose, under highly acidic conditions that are responsible for teeth demineralization. The glucosyltransferases B (GtfB) and C (GtfC) produced by S. mutans are essential in this process, because catalyze the synthesis of water-insoluble from sucrose. The Glucan-binding protein B (GbpB) appears to also participate in S. mutans accumulation, but under processes that are still unclear. Little is known about the mechanisms that regulate expression of genes encoding these proteins (gtfB, gtfC and gbpB). In this sense, we aimed to characterize the patterns of transcription of gtfB, gtfC and gbpB in distinct S. mutans genotypes. For that purpose, semi-quantitative analysis of reverse transcription¿PCR (RT-PCR) were performed in strains at different phases of the growth curves in Brain Heart Infusion broth bath cultures at 37°C, under anaerobiosis. RNAs were extracted from cells at different growth phases, and applied in reactions of reverse transcription with arbitrary primers to yield total cDNAs. Semi-quantitative PCR reactions were then performed with the cDNAs using specific primers to each target gene, and normalized by the expression of the housekeeping gene 16SRNA, whose expression remained constant at the experimental conditions. Patterns of expression of gtfB and gtfC were specific to the strain background. Transcriptions of gtfB e de gtfC were coordinated in specific phases of growth, but the curves of transcription diverged at different phases in the majority of the genotypes studied. The levels of gbpB transcripts were variable between strains and assumed an independent pattern when compared with gtf genes. The levels of gbpB transcripts were characterized by lesser variations between the different phases of growth. The regulatory genes have shown peaks of expression at log and/or stationary phases of growth and the curves of transcript levels were strain-dependent. The results indicate that patterns of expression of gtfB, gtfC and gbpB and regulatory genes are strain-specific, and that gtfB and gtfC are regulated by distinct systems that may be activated at distinct phases of growth / Mestrado / Microbiologia e Imunologia / Mestre em Biologia Buco-Dental
34

Planejamento e síntese de novos candidatos a protótipos de fármacos anti-HIV, desenhados a partir da delavirdina, um inibidor não nucleosídico da transcriptase reversa / Planning and synthesis of new prototype candidates of anti-HIV drugs drawn from delavirdine an inhibitor non-nucleoside reverse transcriptase

Machado, Antônio Silva 10 March 2015 (has links)
Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-04-04T18:58:10Z No. of bitstreams: 2 Dissertação - Antônio Silva Machado - 2015.pdf: 1923171 bytes, checksum: 991e35e30785c0ac34b0f9290b2fc249 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-04-05T10:37:58Z (GMT) No. of bitstreams: 2 Dissertação - Antônio Silva Machado - 2015.pdf: 1923171 bytes, checksum: 991e35e30785c0ac34b0f9290b2fc249 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-04-05T10:37:58Z (GMT). No. of bitstreams: 2 Dissertação - Antônio Silva Machado - 2015.pdf: 1923171 bytes, checksum: 991e35e30785c0ac34b0f9290b2fc249 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-10 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / AIDS is considered an epidemic disease shaping up as a serious public health problem. Since its discovery in 1981, considerable efforts have been made to better understand the HIV infection mechanism thus, propelling the research and drug development process. According to their mechanism of action, HIV drugs can be classified into six mainly subgroups: nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PI), non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide reverse transcriptase inhibitors (NtRTI ), entry inhibitors and integrase inhibitors. Currently, searching for safer and more effective drugs showing less collateral effects remains an encouraging pace. In this context, our work describes a synthesis of a new family of heterocyclic compounds, chemically related to delavirdine, a NNRTIs currently used in AIDS treatment. Bioisosterism strategy was applied to obtain synthetic products (54a-54h) in four steps only. Conventional heating method (A) and optimized microwave reactor (B) methodology were both compared by means of their intermediate products yields. Results showed increased yields of partial products (20a-20h [91-98%]); (29a-29h [69-88%]); (37a-37h [82-92%]) for microwave reactor methodology, as well a gain in the time spent during the procedure. All compounds (20a-20h; 29a-29h; 37a-37h e 54a-54h) were characterized by Nuclear Magnetic Resonance of Hydrogen (1H NMR), Carbon (13 C NMR) and Infrared spectroscopy (IR). / A Aids é considerada uma epidemia de cunho global, se configurando como um grave problema de saúde pública. Desde de sua descoberta em 1981, foram realizados esforços consideráveis para a melhor compreensão do mecanismo pelo qual o HIV propicia a infecção, iniciando o processo de pesquisa e desenvolvimento de fármacos. Sendo estes classificados de acordo com os seus mecanismos de ação, apresentados em 6 grupos i.e inibidores nucleosídicos da transcriptase reversa (NRTI), inibidores da protease (PI), inibidores não nucleosídicos da transcriptase reversa (NNRTI), inibidores nucleotídeos da transcriptase reversa (NtRTI), inibidores de entrada e inibidores de integrase. Logo, a busca por novos fármacos que sejam mais efetivos, seguros e com menos efeitos coletareis é de grande relevância. Neste contexto, o trabalho descreve a síntese de uma nova família de compostos heterocíclicos, planejados estruturalmente a partir do fármaco delavirdina - atualmente empregada no tratamento da AIDS, atuando como inibidor não nucleosídico da transcriptase reversa. Os compostos finais sintetizados (54a-54h) são obtidos em quatro etapas sintéticas. Estas foram planejadas e seus rendimentos comparadas a partir da estratégia de bioisosterismo. Seus intermediários foram sintetizados tanto por metodologia de aquecimento convencional (A) assim como, por reator de micro-ondas (B). Os resultados obtidos demonstraram aumento do rendimento parcial (20a-20h [91-98%]); (29a-29h [69-88%]); (37a-37h [82-92%]) e ganho de tempo na síntese dos intermediários, pela metodologia otimizada por reator de microondas. Todos os compostos (20a-20h; 29a-29h; 37a-37h e 54a-54h) foram caracterizados por meio de ressonância magnética nuclear de Hidrogênio (RMN 1H), de Carbono (RMN 13C) e espectroscopia de infravermelho (IV).
35

Binding of the Nonnucleoside Reverse Transcriptase Inhibitor Efavirenz to HIV-1 Reverse Transcriptase Monomers and Dimers

Braz, Valerie Ann January 2010 (has links)
No description available.
36

Genotyping and antiretroviral resistance profile test from HIV-1 samples in patients with therapeutic failure from Cearà / Genotipagem e perfil de resistÃncia aos antiretrovirais do virus da imunodeficiÃncia tipo 1 em populaÃÃo com falha terapeutica no CearÃ, Brasil - 2002 a 2004

Melissa Soares Medeiros 03 February 2006 (has links)
Faculdade Christus / Introduction: Genotypic testing for HIV-1 drug resistance is useful for selecting antiretroviral drugs for patients developing treatment failure. O melhor entendimento da sua interpretaÃÃo facilitarà sua utilizaÃÃo como ferramenta mÃdica na terapÃutica do HIV. The optimal understanding of its interpretation will give an important tool for HIV treatment. Objective: To identify common combinations of resistance mutations and antiretroviral resistance profile. Methods: Between April 2002 and March 2004, 101 protease and reverse transcriptase (RT) sequences were determined for HIV-1 isolates from patients who were failing antiretroviral therapy. Resistance profile was obtained by Stanford program. Results: male were 76.2%, median age 38 years, CD4 media was 279.21 cells/mm3 and Viral load 4.49 log. Total of 31 mutational patterns were detected to protease inhibitor (IP), 49 to nucleoside RT inhibitor (NRTI), and 17 to nonnucleoside RT inhibitor (NNRTI). K65R was detected in 5.9% isolates. The most frequent mutations were L90M, M184V and K103N to IP, NRTI and NNRTI respectively. The main mutational patterns accounted for 49% of mutant sequences to IP, 38.5% to ITRN accounted and 40,9% to NNRTI. Patients with three or more therapeutic failure had worst resistance profile to all IP except for Lopinavir, and NRTI except for Tenofovir. High resistance to Lamivudine and NNRTI were independent of failure quantity. Conclusion: The best susceptibility was found to Lopinavir at IPâs class and to Tenofovir at ITRNâs. The main mutational patterns to IP, ITRN and NNRTI represented almost half of all patterns found. / A Genotipagem està sendo usada como mÃtodo para guiar a seleÃÃo de antiretrovirais em pacientes com falha terapÃutica. O melhor entendimento da sua interpretaÃÃo facilitarà sua utilizaÃÃo como ferramenta mÃdica na terapÃutica do HIV. Objetivo: Avaliar o perfil de resistÃncia aos antiretrovirais e identificar padrÃes mutacionais das seqÃÃncias de protease e TR do HIV-1. MÃtodos: Foram estudadas as sequÃncias de genes da protease e TR isoladas de 101 amostras de pacientes com HIV-1 em falha terapÃutica, entre abril/2002 a marÃo/2004, atravÃs de Genotipagem realizadas no CearÃ. O Banco de dados de Stanford foi utilizado para avaliaÃÃo de resistÃncia e SPSS versÃo 11 e Epi Info versÃo 6 para anÃlise estatÃstica. Resultados: Sexo masculino 76,2%, mediana de idade 38 anos, CD4 mÃdio de 279,21 cells/mm3 e Carga Viral 4.49 log. Na classe de Inibidores de Protease (IP) 31 padrÃes mutacionais foram encontrados, nos inibidores da transcriptase reversa anÃlogos de nucleosÃdeos (ITRN) 49 e para inibidores da transcriptase reversa nÃo anÃlogos de nucleosÃdeos (ITRNN) 17. As mutaÃÃes mais frequentes foram L90M, M184V e K103N para IP, ITRN e ITRNN espectivamente. A K65R foi detectada em 5,9% dos isolados. TrÃs ou mais falhas terapÃuticas apresentaram maior perfil de resistÃncia para todos os IPs exceto para Lopinavir, e para todos os ITRNs exceto para Tenofovir. Os seis principais padrÃes mutacionais para IPs equivaleram a 49% das sequÃncias, para ITRNs a 38,5%, e para ITRNNs os dois principais padrÃes corresponderam a 40,9%. Foram encontrados altos Ãndices de resistÃncia para ITRNNs independente da quantidade de falhas terapÃuticas. ConclusÃo: Nos IPs a menor resistÃncia encontrada foi ao Lopinavir e nos ITRNs ao Tenofovir. Os principais padrÃes mutacionais para IPs, ITRNs e ITRNNs representaram quase metade de todos os padrÃes de resistÃncia encontrados.
37

Zebrafish telomerase reverse transcriptase (TERT): molecularcloning, characterization and retinal expression

Lau, Wui-man., 劉匯文. January 2005 (has links)
published_or_final_version / abstract / Anatomy / Master / Master of Philosophy
38

Detection of positive selection resulting from Nevirapine treatment in longitudinal HIV-1 reverse transcriptase sequences.

Ketwaroo, Bibi Farahnaz K. January 2006 (has links)
<p>Nevirapine (NVP) is a cheap anti-retroviral drug used in poor countries worldwide, administered to pregnant women at the onset of labour to inhibit HIV enzyme reverse transcriptase. Viruses which may get transmitted to newborns are deficient in this enzyme, and HIV-1 infection cannot be established, thereby preventing mother to child transmission (MTCT). In some cases, babies get infected and positive selection for viruses resistant to nevirapine may be inferred. Positive selection can be inferred from sequence data, when the rate of nonsynonymous substitutions is significantly greater than the rate of synonymous substitutions.</p> <p>Unfortunately, it is found that available positive selection methods should not be used to analyse before- and after- NVP treatment sequence pairs associated with MTCT. Methods which use phylogenetic trees to infer positive selection trace synonymous and nonsynonymous substitutions further back in time than the short time duration during which selection for NVP occurred. The other group of methods for inferring positive selection, the pairwise methods, do not have appreciable power, because they average susbtituion rates over all codons in a sequence pair and not just at single codons. We introduce a simple counting method which we call the Pairwise Homologous Codons (PHoCs) method with which we have inferred positive selection resulting from NVP treatment in longitudinal HIV-1 reverse transcriptase sequences. The PHoCs method estimates rates of substitutions between before- and after- NVP treatment codons, using a simple pairwise method.</p>
39

Framework para classificação das mutações de vírus HIV / HIV mutation classification framework

Ozahata, Mina Cintho 15 May 2014 (has links)
Um grande número de medicamentos utilizados no tratamento contra o HIV agem procurando inibir a ação das proteínas transcriptase reversa e protease. Mutações existentes nas sequências dessas proteínas podem estar relacionadas à resistência aos medicamentos e podem prejudicar o desempenho de um tratamento. O estudo do genótipo dos vírus pode ajudar na tomada de escolhas específicas em tratamentos para cada indivíduo, tornando maiores a chance de sucesso. Com a maior acessibilidade a exames de genotipagem, uma grande quantidade de sequências do vírus está disponível, contendo um grande volume de informação. Padrões de ocorrência de mutações são exemplos de informações contidas nessas sequências e são importantes por estarem relacionados à resistência aos medicamentos. Um dos caminhos que pode ser capaz de nos levar ao entendimento desses padrões de mutações é a aplicação de técnicas de agrupamento e biclustering. Essas técnicas visam a geração de grupos ou biclusters que possuam dados com propriedades em comum. São empregadas em casos em que não há grande quantidade de informação prévia e existem poucas hipóteses sobre os dados. Assim, pode-se encontrar os padrões de mutações que ocorrem nessas sequências e tentar relacioná-los com a resistência aos medicamentos, utilizando métodos de agrupamento e bicluster em sequências de protease e transcriptase reversa. Existem alguns sistemas que tentam predizer a resistência ou susceptibilidade das sequências, porém, devido à grande complexidade dessa relação, ainda é necessário esclarecer o vínculo entre combinações de mutações e níveis de resistência fenotípica. Desta forma, a principal contribuição deste trabalho é o desenvolvimento de um framework baseado na aplicação dos algoritmos KMédias e Bimax às sequências de transcriptase reversa e protease de pacientes infectados com HIV, em uma codificação binária. O presente trabalho também introduz uma representação visual dos grupos e biclusters baseada em dados de microarranjos para casos em que se tem grandes volumes de dados, de forma a facilitar a visualização da informação extraída e a caracterização dos grupos e biclusters no domínio da doença. / Drugs used in HIV treatment intend to inhibit protease and reverse transcriptase. Mutations in the sequences of these proteins can be related to drug resistance and can reduce treatment efficacy. Studying virus genotype may help choosing specific treatments for each patient, increasing success probability. As genotyping tests become available, a great amount of virus sequences, which comprehend lots of information, are more accessible. Patterns of mutation are examples of information comprised in the sequences and are important since are related to drug resistance. One way that can lead to the understanding of these mutation patterns is the use of clustering and biclustering techniques. These techniques search for clusters or biclusters comprising data with similar attributes. They are used when there is not a lot of previous information and there are few hypothesis about the data. Therefore, it may be possible to find patterns of mutations in the sequences and to relate them to drug resistance using clustering and biclustering techniques with protease and reverse transcriptase sequences. There are a few systems that predict drug resistance according to the sequence of the virus, however, due to the complexity of the relationship, it is still necessary to elucidate the connection between mutation combinations and the level of phenotypic resistance. Accordingly, this work main contribution is the development of a framework based on Kmeans and Bimax algorithms with protease and reverse transcriptase sequences from HIV patients in a binary form. This work also presents a visual representation of the clusters and biclusters based on microarray data suitable for large data volumes, helping the visualization of information extracted from data and cluster and bicluster characterization in the disease domain.
40

Relationship Between RNase H and Excision Activities of HIV-1 Reverse Transcriptase (RT)

Acosta-Hoyos, Antonio J. 29 July 2010 (has links)
Replication of HIV-1 is inhibited by azidothymidine (AZT), which leads to chain termination and inhibition of DNA synthesis. Resistance to AZT is frequently the result of mutations that increase the ability of RT to remove the chain-terminating nucleotides after they have been incorporated. It has been proposed that RNase H cleavage of the RNA template can occur when RT is stalled near the site of chain termination and contributes to the inhibition by causing the dissociation of the primer-template before the chain terminator can be excised. Mutations in the connection and RNase H domains of RT have been shown to increase excision. It has long been known that resistance to thymidine analogs is conferred by the mutations M41L, D67N, K70R, L210W, T215F/Y and T219Q/E in RT and that this resistance is suppressed by the additional presence of the M184V mutation. Changes in excision activity on DNA templates have been observed with these mutant RTs, but effects on RNase H cleavage resulting in indirect effects on excision activity is also possible with RNA templates. We used a 5'-labeled -3'-chain-terminated DNA primer annealed to either a DNA or RNA template to evaluate primer rescue activities, a 5'-labeled RNA template to evaluate RNA cleavage activity and a biotin-tagged chain-terminated oligodeoxynucleotide to monitor primer-template dissociation. We first investigated differences between RNA and DNA templates when the primers were chain terminated and observed a correlation between RNase H activities and template/primer (T/P) dissociation. An inverse correlation was observed between excision rescue rates and RNase H cleavages leading to T/P dissociation. We observed that the chain terminator (i.e. AZTMP or ddAMP) affected RNase H cleavages and excision rates with RNA template and dNTPs. When we investigated mutations in the N-terminal domain of RT associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance we found that primer rescue was decreased when M184V was present in combination with thymidine analog mutations (TAMs) and the template was RNA with either ATP or PPi as excision substrate. RNase H cleavage at secondary cleavage sites (-7, -8) was substantially reduced with M41L/T215Y RT in comparison with wild type RT, and primer-template dissociation was decreased. In contrast, when M184V was present, RNase H cleavage at the secondary cleavage sites and dissociation of the primer-template occurred at higher levels and excision rescue was decreased. The ability of RT to rescue an AZT terminated primer in the presence of the 184V mutation was restored when the RNase H activity was inactivated by the RNase H negative mutation E478Q. Electromobility shift assay (EMSA) analysis of AZT-resistant mutant RT with M184V showed an increased Kd for formation of the ternary complex. These results suggest that RNase H-mediated RNA-DNA template-primer dissociation is influenced by mutations associated with thymidine analog resistance, and that suppression of resistance to nucleoside RT inhibitors by M184V may be partly explained by effects on RNase H cleavage that decrease the time available for excision to occur. This is the first time that mutations in the polymerase domain are shown to affect excision rescue through an RNase H-dependent mechanism.

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