Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.

Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.

Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.

Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.

Retrovírus endógenos (ERVs) são vírus altamente difundidos no genoma de vertebrados. ERVs surgem quando retrovírus exógenos infectam células germinativas e se disseminam no genoma de seus hospedeiros, transmitindo seu material genético através das gerações por meio de herança mendeliana. ERVs são fundamentais na evolução dos genomas, sendo eles responsáveis por uma parte da diversidade genética de seus hospedeiros. O conhecimento sobre ERVs na família Felidae (Mammalia, Carnivora) estava praticamente restrito ao gato doméstico, e não se conhecia diversidade e padrões de evolução desses retroelementos em outras espécies. Este estudo teve como objetivo investigar diversidade, distribuição e padrões evolutivos de ERVs em espécies de gatos silvestres. Utilizando ferramentas de biologia molecular e bioinformática, foram identificadas e caracterizadas 85 sequências similares a retrovírus endógenos nos representantes das oito espécies brasileiras: Leopardus pardalis, L. wiedii, L. colocolo, L. geoffroyi, L. tigrinus, Puma concolor, P. yagouaroundi e Panthera onca. Encontrou-se uma predominância de ERVs similares a Gammaretrovirus, um padrão característico em muitas espécies de mamíferos. As análises filogenéticas evidenciaram três grupos principais de Gammaretrovirus, cada um evoluindo de maneira peculiar. Em uma visão geral, os ERVs provenientes de diferentes hospedeiros apresentaram-se distribuídos de forma heterogênea nas filogenias, dificultando a constatação de um padrão coevolutivo. No entanto, análises mais detalhadas de algumas sequências demonstraram peculiaridades, como no caso de um grupo de sequências similares a de um ERV oriundo do morcego Myotis lucifugus. Através de análises filogenéticas em comparação com dados obtidos na literatura, sugere-se que a infecção desse retrovírus ocorreu em uma espécie ancestral de felídeo, na segunda metade do Mioceno. Os resultados obtidos permitiram demonstrar que os felídeos neotropicais apresentam ERVs que seguem padrões semelhantes aos descritos a respeito de outros mamíferos, sugerindo também alguns casos de infecções de retrovírus muito similares entre diferentes ordens de mamíferos. / Endogenous retroviruses (ERVs) are widespread viruses in vertebrate genome. ERVs arise when exogenous retrovirus infects germinal cells and spread in the genome of their hosts, transmitting its genetic material throughout the generations by means of Mendelian inheritance. ERVs are fundamental for the evolution of genomes, being responsible for some part of the genetic diversity of their hosts. The knowledge on ERVs in felids (Mammalia, Carnivora, Felidae) was basically restricted to domestic cats, and the diversity and patterns of evolution of these retroviral elements in other species were not known. This study aimed to investigate diversity, distribution and evolutionary patterns of ERVs in wildcat species. Hence, by utilizing molecular biology and bioinformatics tools, 85 endogenous retrovirus-like sequences were identified and characterized in eight representative Brazilian species: Leopardus pardalis, L. wiedii, L. colocolo, L. geoffroyi, L. tigrinus, Puma concolor, P. yagouaroundi and Panthera onca. The analyses of these novel felid ERVs showed the predominance of Gammaretroviruslike sequences, which is a characteristic pattern present in many mammal species. Phylogenetic analyses have evidenced three major groups of Gammaretrovirus, each one evolving in a peculiar manner. ERVs from different hosts were distributed in a mixed way in the phylogenies, differently of a coevolutionary pattern. However, more detailed analyses of some sequences demonstrated peculiarities, as in the case of a group of sequences similar to an ERV from the bat Myotis lucifugus. Notably, through phylogenetic analyses, and in comparison to data obtained in the literature, it may be suggested that some infection by a retrovirus occurred in a felid ancestral species in the second half of the Miocene. Therefore, the results obtained demonstrate that ERVs from Neotropical felids follow patterns which are very similar to the ones described for other mammals, also suggesting some cases of similar retrovirus lineage infecting different mammal orders.

Retrovírus endógenos (ERVs) são vírus altamente difundidos no genoma de vertebrados. ERVs surgem quando retrovírus exógenos infectam células germinativas e se disseminam no genoma de seus hospedeiros, transmitindo seu material genético através das gerações por meio de herança mendeliana. ERVs são fundamentais na evolução dos genomas, sendo eles responsáveis por uma parte da diversidade genética de seus hospedeiros. O conhecimento sobre ERVs na família Felidae (Mammalia, Carnivora) estava praticamente restrito ao gato doméstico, e não se conhecia diversidade e padrões de evolução desses retroelementos em outras espécies. Este estudo teve como objetivo investigar diversidade, distribuição e padrões evolutivos de ERVs em espécies de gatos silvestres. Utilizando ferramentas de biologia molecular e bioinformática, foram identificadas e caracterizadas 85 sequências similares a retrovírus endógenos nos representantes das oito espécies brasileiras: Leopardus pardalis, L. wiedii, L. colocolo, L. geoffroyi, L. tigrinus, Puma concolor, P. yagouaroundi e Panthera onca. Encontrou-se uma predominância de ERVs similares a Gammaretrovirus, um padrão característico em muitas espécies de mamíferos. As análises filogenéticas evidenciaram três grupos principais de Gammaretrovirus, cada um evoluindo de maneira peculiar. Em uma visão geral, os ERVs provenientes de diferentes hospedeiros apresentaram-se distribuídos de forma heterogênea nas filogenias, dificultando a constatação de um padrão coevolutivo. No entanto, análises mais detalhadas de algumas sequências demonstraram peculiaridades, como no caso de um grupo de sequências similares a de um ERV oriundo do morcego Myotis lucifugus. Através de análises filogenéticas em comparação com dados obtidos na literatura, sugere-se que a infecção desse retrovírus ocorreu em uma espécie ancestral de felídeo, na segunda metade do Mioceno. Os resultados obtidos permitiram demonstrar que os felídeos neotropicais apresentam ERVs que seguem padrões semelhantes aos descritos a respeito de outros mamíferos, sugerindo também alguns casos de infecções de retrovírus muito similares entre diferentes ordens de mamíferos. / Endogenous retroviruses (ERVs) are widespread viruses in vertebrate genome. ERVs arise when exogenous retrovirus infects germinal cells and spread in the genome of their hosts, transmitting its genetic material throughout the generations by means of Mendelian inheritance. ERVs are fundamental for the evolution of genomes, being responsible for some part of the genetic diversity of their hosts. The knowledge on ERVs in felids (Mammalia, Carnivora, Felidae) was basically restricted to domestic cats, and the diversity and patterns of evolution of these retroviral elements in other species were not known. This study aimed to investigate diversity, distribution and evolutionary patterns of ERVs in wildcat species. Hence, by utilizing molecular biology and bioinformatics tools, 85 endogenous retrovirus-like sequences were identified and characterized in eight representative Brazilian species: Leopardus pardalis, L. wiedii, L. colocolo, L. geoffroyi, L. tigrinus, Puma concolor, P. yagouaroundi and Panthera onca. The analyses of these novel felid ERVs showed the predominance of Gammaretroviruslike sequences, which is a characteristic pattern present in many mammal species. Phylogenetic analyses have evidenced three major groups of Gammaretrovirus, each one evolving in a peculiar manner. ERVs from different hosts were distributed in a mixed way in the phylogenies, differently of a coevolutionary pattern. However, more detailed analyses of some sequences demonstrated peculiarities, as in the case of a group of sequences similar to an ERV from the bat Myotis lucifugus. Notably, through phylogenetic analyses, and in comparison to data obtained in the literature, it may be suggested that some infection by a retrovirus occurred in a felid ancestral species in the second half of the Miocene. Therefore, the results obtained demonstrate that ERVs from Neotropical felids follow patterns which are very similar to the ones described for other mammals, also suggesting some cases of similar retrovirus lineage infecting different mammal orders.

Retrovírus endógenos (ERVs) são vírus altamente difundidos no genoma de vertebrados. ERVs surgem quando retrovírus exógenos infectam células germinativas e se disseminam no genoma de seus hospedeiros, transmitindo seu material genético através das gerações por meio de herança mendeliana. ERVs são fundamentais na evolução dos genomas, sendo eles responsáveis por uma parte da diversidade genética de seus hospedeiros. O conhecimento sobre ERVs na família Felidae (Mammalia, Carnivora) estava praticamente restrito ao gato doméstico, e não se conhecia diversidade e padrões de evolução desses retroelementos em outras espécies. Este estudo teve como objetivo investigar diversidade, distribuição e padrões evolutivos de ERVs em espécies de gatos silvestres. Utilizando ferramentas de biologia molecular e bioinformática, foram identificadas e caracterizadas 85 sequências similares a retrovírus endógenos nos representantes das oito espécies brasileiras: Leopardus pardalis, L. wiedii, L. colocolo, L. geoffroyi, L. tigrinus, Puma concolor, P. yagouaroundi e Panthera onca. Encontrou-se uma predominância de ERVs similares a Gammaretrovirus, um padrão característico em muitas espécies de mamíferos. As análises filogenéticas evidenciaram três grupos principais de Gammaretrovirus, cada um evoluindo de maneira peculiar. Em uma visão geral, os ERVs provenientes de diferentes hospedeiros apresentaram-se distribuídos de forma heterogênea nas filogenias, dificultando a constatação de um padrão coevolutivo. No entanto, análises mais detalhadas de algumas sequências demonstraram peculiaridades, como no caso de um grupo de sequências similares a de um ERV oriundo do morcego Myotis lucifugus. Através de análises filogenéticas em comparação com dados obtidos na literatura, sugere-se que a infecção desse retrovírus ocorreu em uma espécie ancestral de felídeo, na segunda metade do Mioceno. Os resultados obtidos permitiram demonstrar que os felídeos neotropicais apresentam ERVs que seguem padrões semelhantes aos descritos a respeito de outros mamíferos, sugerindo também alguns casos de infecções de retrovírus muito similares entre diferentes ordens de mamíferos. / Endogenous retroviruses (ERVs) are widespread viruses in vertebrate genome. ERVs arise when exogenous retrovirus infects germinal cells and spread in the genome of their hosts, transmitting its genetic material throughout the generations by means of Mendelian inheritance. ERVs are fundamental for the evolution of genomes, being responsible for some part of the genetic diversity of their hosts. The knowledge on ERVs in felids (Mammalia, Carnivora, Felidae) was basically restricted to domestic cats, and the diversity and patterns of evolution of these retroviral elements in other species were not known. This study aimed to investigate diversity, distribution and evolutionary patterns of ERVs in wildcat species. Hence, by utilizing molecular biology and bioinformatics tools, 85 endogenous retrovirus-like sequences were identified and characterized in eight representative Brazilian species: Leopardus pardalis, L. wiedii, L. colocolo, L. geoffroyi, L. tigrinus, Puma concolor, P. yagouaroundi and Panthera onca. The analyses of these novel felid ERVs showed the predominance of Gammaretroviruslike sequences, which is a characteristic pattern present in many mammal species. Phylogenetic analyses have evidenced three major groups of Gammaretrovirus, each one evolving in a peculiar manner. ERVs from different hosts were distributed in a mixed way in the phylogenies, differently of a coevolutionary pattern. However, more detailed analyses of some sequences demonstrated peculiarities, as in the case of a group of sequences similar to an ERV from the bat Myotis lucifugus. Notably, through phylogenetic analyses, and in comparison to data obtained in the literature, it may be suggested that some infection by a retrovirus occurred in a felid ancestral species in the second half of the Miocene. Therefore, the results obtained demonstrate that ERVs from Neotropical felids follow patterns which are very similar to the ones described for other mammals, also suggesting some cases of similar retrovirus lineage infecting different mammal orders.

Gene therapy is a fast growing technology that offers treatments for genetic diseases. The method is based on introducing genetic material into a patient to replace the disease-causing gene, using a vector. This report examines the potential of some viral vectors for gene therapy, to give Bio-Works Technologies a recommendation on what the future market demands. Oncolytic viruses, vaccines and gene editing are not treated in the report as a delimitation.  Viral vectors have different biological properties and require different purification methods, making them suitable for different applications in gene therapy. In the purification of the viruses it can be challenging to obtain a high purity and large-scale manufacturing. One major drawback with most purification methods is that they are not specific to just one virus, which leads to contaminants in the solution and lower purity. The viral vectors handled in the report are the adenovirus, adeno-associated virus, gammaretrovirus, lentivirus, alpharetrovirus, foamy virus, herpes simplex virus and baculovirus. These were chosen as they are relevant vectors for gene therapy and stay within the scope of the report. Lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) will dominate the gene therapy field in the coming years. This is based on the information that the use of AAVs and LVs in clinical trials have increased in recent years, while the other vectors mentioned above have slightly decreased or show no apparent change. However, challenges still remain in the purification processes. Ligands used in affinity chromatography for purification of AAVs are effective at removing most contaminants, but cannot distinguish between empty and loaded capsids, which can induce immune response when used clinically. This is the main challenge when purifying AAVs. The empty capsids can be removed with ion exchange chromatography, which results in higher purity but also lower recovery. There is no specific purifying method for LVs, therefore a lentivirus-specific affinity ligand, such as an antibody ligand, would be beneficial for the purification and manufacturing procedure.  In addition to AAVs and LVs, baculoviral vectors and foamy viral vectors show great potential in a long-term perspective but they only have been researched in preclinical studies. Moreover, herpes simplex viral vectors and adenoviral vectors show potential in cancer treatments or as vaccines rather than in augmentation gene therapy.

Human endogenous retroviruses (HERVs) constitute about 8% of the human genome. There are around 4000 pol-containing retroviral integrations in the human genome, which makes it impractical to measure each of them separately. Therefore we developed a set of degenerate real time PCRs to detect major groups bearing sequence similarities to gammaretroviruses, one of the largest groups of human endogenous retrovirus, and betaretroviruses, some of which have integrated into the human genome most recently and which remain the most intact. It was found that, although both gammaretroviral and betaretroviral RNAs were broadly expressed in various healthy tissues including reproductive tissues and brain, a differential expression pattern was observed. My work further revealed that HERVE and HERVW, two gammaretroviral sequences, were ubiquitously and highly expressed in pathologic and normal female reproductive tissues with tissue specific patterns. Expression of HERVE was higher in endometriotic tissue than in normal endometrium. HERVE and HERVW RNAs were higher in normal ovarian tissue than in ovarian cancer. Besides these tissue- and neoplasia-related differences, there were wide differences in HERV expression among individuals. Next, a selective pattern of HERVW upregulation was demonstrated in SK-N-DZ, a neuroblastoma cell line, upon re-oxygenation after a period of hypoxia or with 5-azacytidine, a demethylating agent. Furthermore, broad and high expressions of gammaretrovirus-like transcripts in different brain areas analyzed were identified. The expression levels were variable among different donors. In conclusion a ubiquitous HERV expression was observed in tissues and cell lines, with various patterns. At this stage the data are not sufficient to conclude whether HERV has any physiological or pathological roles in humans. However, their differential expression patterns are compatible with functional roles of HERV in humans.

APOBEC3 (A3) proteins are a family of host-encoded cytidine deaminases that protect against retroviruses and other viral intruders. Retroviruses, unlike other viruses, are able to integrate their genomic proviral DNA within hours of entering host cells. A3 proteins hinder retroviral infectivity by editing retroviral replication intermediates, as well as by inhibiting retroviral replication and integration through deamination-independent methods. These proteins thus constitute the first line of immune defense against endogenous and exogenous retroviral pathogens. The overall goal of my Master's project was to better understand the critical role A3 proteins play in restricting inter- and intra-host transmission of retroviruses. There are two specific aspects that I focused on: first, investigating the role of mouse APOBEC3 (mA3) in limiting the zoonotic transmission of murine leukemia retroviruses (MLVs) in a rural environment; second, to identify the molecular features in MLVs that confer susceptibility or resistance to deamination by mA3. For the first part of my project, we collected blood samples from dairy and production cattle from four different geographical locations across Canada. We then designed a novel PCR screening strategy targeting conserved genetic regions in MLVs and Mouse Mammary Tumor Virus (MMTV) and MMTV-like betaretroviruses. Our results indicate that 4% of animals were positive for MLV and 2% were positive for MMTV. Despite crossing the species barrier by gaining entry into bovine cells, our study also demonstrates that the bovine A3 protein is able to potently inhibit the spread of these murine retroviruses in vitro. The next question we asked was whether mA3 could also mutate and restrict murine endogenous retroviruses and thereby partake in limiting zoonotic transmission. Moloney MLV and AKV MLV are two highly homologous murine gammaretroviruses with opposite sensitivities to restriction by mA3: MoMLV is resistant to restriction and deamination while AKV is sensitive to both. Design of MoMLV/AKV hybrid viruses enabled us to map the region of mA3 resistance to the region encoding the glyco-Gag accessory protein. Site-directed mutagenesis then allowed us to correlate the number of N-linked glycosylation sites with the level of resistance to deamination by mA3. Our results suggest that Gag glycosylation is a possible viral defence mechanism that arose to counteract the evolutionary pressure imposed by mA3. Overall, my projects show the important role A3 proteins play in intrinsic immunity, whether defending the host from foreign retroviral invaders or endogenous retroviral foes.