Abstract Yellow head virus (YHV) has caused mass mortalities in Penaeus monodon shrimp farmed throughout Southeast Asia since it was first discovered in the early 1990’s. YHV possesses a positive-sense, single-stranded RNA genome and a rod-shaped enveloped virion. Together with the closely related gill-associated virus (GAV) identified in P. monodon shrimp in Australia, it is classified in the genus Okavirus, family Roniviridae within the order Nidovirales. YHV particles contain only three structural proteins, a nucleocapsid (N) protein (p20) protein and two envelope glycoproteins gp116 and gp64. In this study, the glycosylation status of gp116 and gp64 extracted from YHV virions was characterized in detail, including the identification of active N-linked glycosylation sites and the nature of the attached carbohydrates. This was achieved by optimizing and applying a combination of methods that included SDS-PAGE followed by carbohydrate-specific staining of gels or probing of membrane-bound proteins using lectins with different carbohydrate specificities, enzymatic removal of N-linked carbohydrates and a variety of mass spectrometry techniques. In these analyses, it was found that N-linked glycans are the major contributor to the higher estimated mass of gp116 and gp64 by SDS-PAGE compared to those estimated from their deduced amino acid sequences. Neither gp116 nor gp64 were found to posses O-linked glycans. Mannose residues were identified to be the major glycan component of carbohydrates linked to gp116 and gp64 and are possibly the sole component of carbohydrate linked to gp64. Unlike gp64, other glycans such as terminal N-acetyl--D-galactosamine and N-acetyl--D-glucosamine were identified to be attached to gp116. Assuming that glycosylation processes in shrimp mimic those of vertebrates that are known in more detail, the nature of the glycans attached to gp116 suggests that they might be added and modified during the transportation of the protein from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN). Mass spectrometry analyses of tryptic peptides derived from the native glycoproteins and following their enzymatic deglycosylation, generated approximately 81% (gp116) and 66% (gp64) coverage of their predicted amino acid sequences. Detailed mass spectrometry analyses of peptides derived from the deglycosylated proteins identified that most of the potential N-linked glycosylated site in the virion envelope glycoproteins, 6 of 7 present in gp116 and 3 of 4 present in gp64 were identified to be modified by glycans. In gp116, one site was not identified and in gp64 one site was not utilized. As phosphorylation has been shown to affect nucleocapsid protein (N) functioning in vertebrate nidoviruses, SDS-PAGE using two phosphoprotein-specific staining methods, as well as mass spectrometry methods, were employed to examine whether the YHV N protein present in virions is phosphorylated. The protein staining methods provided contradicting results and no phosphate-containing peptides were identified by mass spectrometry. The apparent absence of phosphate in the N protein was also supported by its isoelectric point (pI ~10) determined by isoelectric focusing and two-dimensional electrophoresis (2-DE) analysis, which was very similar to that predicted (pI = 9.98) from its deduced amino acid sequence. Taken together, the data suggest that the YHV N protein encapsulated within virions is not phosphorylated. The RNA-binding capability of the GAV N protein was assessed using an electrophoretic mobility shift assay (EMSA) technique. Full-length and variously truncated forms of the GAV N protein expressed in bacteria were assessed in the assays. It was found that the full-length recombinant N protein bound to RNA in a sequence non-specific manner. Analysis of the five truncated N protein constructs localized the RNA-binding domain to a 50 amino acid sequence in the N-terminal region residing between Met11 and Arg60. A motif rich in proline and arginine residues, which are commonly found in other RNA-binding proteins, occurred in first 18 amino acids of this region. Although RNA-binding was not sequence-specific, the data suggest that this region of the GAV N protein is the most likely site at which it interacts with and nucleates viral genomic RNA during nucleocapsid formation. A synthetic peptide spanning the 18 amino acid of the putative RNA-binding domain was shown to possess RNA-binding properties similar to the recombinant protein fragment. These results indicated that the 18 amino acid, proline and arginine rich motif (MPVRRPLPPQPPRNARLI) in the N-terminal region of the GAV N protein confers its RNA-binding function. Using an immuno-co-precipitation assay, a host protein was found to interact abundantly with the GAV N protein in infected lymphoid organ cells. Mass spectrometry analysis identified the protein as -actin. Immuno-histochemistical double-labeling methods in conjunction with observations made using confocal and electron microscopy revealed that actin and the N protein were co-located in cytoplasm of infected cells. Electron microscopy suggested that interaction of the two proteins occurs before nucleocapsid envelopment within virions, suggesting that -actin might be involved in transporting the N protein or the nucleocapsid from their sites of synthesis to the rough endoplasmic reticulum where the virion acquires its envelopes. In summary, the research described in this thesis has advanced understanding of the YHV/GAV proteome through the identification of the glycosylation sites in the envelope glycoproteins gp116 and gp64, and demonstrating that nucleocapsid protein encapsulated within virion is unlikely to be phosphorylated. Functional studies have also shown that the nucleocapsid protein binds RNA non-specifically through an 18 amino acid domain near its N-terminus and that it binds and co-localizes with -actin in infected cells, suggesting that -actin may play role in trafficking N protein in infected cells.

La myopathie myotubulaire est une maladie musculaire congénitale très sévère. Le laboratoire d’accueil a démontré que les échantillons de muscle de patients atteints de cette maladie ainsi que le modèle murin présentent une surexpression de DNM2, alors que sa réduction par croisement génétique améliore les signes cliniques et histologiques de la maladie. Le but de ce travail consistait à développer, tester et valider des composés injectables qui ciblent DNM2 et diminuent son niveau. Deux approches thérapeutiques ont été développées l’une basée sur l’utilisation de virus adéno-associés (AAV) exprimant des shRNA, l’autre sur les oligonucleotides antisens (ASO). L’injection des vecteurs AAV-shDnm2 ou bien les ASO-Dnm2 pouvait corriger les défauts histologiques et fonctionnels des muscles des souris myopathes.Les résultats obtenus montrent le potentiel thérapeutique de la réduction de DNM2, et présente une nouvelle approche pour le traitement de la myopathie myotubulaire. / Myotubular myopathy is a severe muscle disease. We previously have shown that muscle specimens of both patients and the mouse model presented an overexpression of DNM2, while its genetic reduction prevents the development of the muscle phenotypes. The aim of this work was to develop, test and validate deliverable compounds. Two therapeutic approaches were used. Injection of antisense oligonucleotide or adeno-associated virus expressing shRNA restores a normal lifespan with improved muscle structure and function of the myopathic mice. These results demonstrate that therapeutic potential of reduction of DNM2 level and provides an attractive therapeutic strategy that could be applied to treat myotubular myopathy.

L’expression d’un transgène grâce au vecteur AAV offre une perspective thérapeutique très prometteuse dans le traitement de maladies monogéniques. Malheureusement, il apparait souvent que des réponses immunes contre le vecteur et le transgène conduisent à un rejet des cellules transduites ainsi qu’à la mise en place d’une mémoire immunitaire spécifique empêchant un nouveau traitement ultérieur. Avec la perspective d’éviter tout rejet immun des cellules transduites, j’ai développé deux approches distinctes. D’une part, nous avons développé un système dans lequel l’expression du transgène est déstabilisée dans les cellules présentatrices de l’antigène grâce à l’ajout de cibles du miRNA 142.3p qui est spécifiquement exprimé dans le système hématopoïétique. Nous avons ainsi montré que la réponse immunitaire contre le transgène était favorisée par la transduction des cellules présentatrices de l’antigène par le vecteur, conduisant à la présentation directe du produit du transgène. En comparant l’initiation des réponses immunes contre plusieurs transgènes modèles, nous avons montré que la réponse immune dirigée contre le transgène pouvait être contrôlée mais que celle-ci dépendait étroitement de l’immunogénicité intrinsèque du transgène en question, c'est-à-dire de la présence d’épitopes reconnus par des lymphocytes T CD4 auxiliaires ainsi que par les lymphocytes B. Une autre approche a concerné l’utilisation de lymphocytes T régulateurs exprimant le facteur de transcription Foxp3 (Treg) et plus particulièrement l’étude de leur mode d’induction in vivo. La présence de Treg conférant une tolérance immunitaire spécifique du transgène a été décrite dans diverses situations et les Treg induits à partir de CD4+ matures (pTreg/iTreg) semblent avoir un potentiel thérapeutique important. Cependant, la population précise de lymphocytes CD4+ à même d’être convertie en Treg n’avait pas été identifiée auparavant. Au cours de mes travaux, analysant la capacité de conversion de cellules naïves, mémoires ou de récents émigrants thymiques, j’ai mis en évidence que le potentiel de conversion des lymphocytes CD4 naïfs issus de souris âgées était diminué et que ceci était dû à une caractéristique intrinsèque des lymphocytes T CD4+ provenant de telles souris. Enfin, nous avons montré que cette faible capacité de conversion des lymphocytes CD4 naïfs en Treg était associée à un rejet de greffe accru dans un modèle de transplantation de peau, montrant que la sénescence peut impacter négativement des protocoles d’induction de tolérance faisant appel à l’induction de Treg en périphérie. / Transgene expression through AAVvectors offers a very promising therapeutic perspective in the treatment of monogenic disorders. Unfortunately, it often appears that the immune responses against the transgene and the vector lead to the rejection of transduced cells and to an establishment of a specific immune memory preventing further processing. With a view to avoid immune rejection of transduced cells, I developed two distinct approaches.First, we have developed a system where the transgene expression is destabilized in the antigen presenting cells by addition of the target miRNA 142.3p which is specifically expressed in the hematopoietic system. We have shown that the immune response against the transgene was favored by transduction of antigen presenting cells with the vector, leading to the direct presentation of the transgene product. Comparing the initiation of immune responses against more transgenes models, we showed that the immune response against the transgene could be controlled but it depended greatly on the intrinsic immunogenicity of the transgène: the presence of epitopes recognized by T helper cells and CD4 by B lymphocytes. Another approach has involved the use of regulatory T cells expressing the transcription factor Foxp3 ( Treg ) and more specifically the study of their mode of induction in vivo. The presence of Treg conferring transgene -specific immune tolerance has been described in various situations and induced Treg from CD4 + mature ( pTreg / iTreg ) appear to have a significant therapeutic potential . However, the precise population of CD4 + lymphocytes capable of being converted into Treg was not identified previously. During my work, analyzing the conversion capacity naive, memory cells or thymic recent emigrants, I highlighted that the potential of conversion of naive CD4 lymphocytes from old mice was decreased and this was due to an intrinsic defect of CD4 + T cells from such mice. Finally, we showed that low conversion ability of CD4 naive Treg was associated with an increased graft rejection in a model of skin transplantation, showing that senescence may negatively impact protocols of tolerance induction using induction of Treg in the periphery.

Aujourd’hui, plus de 80% des nouvelles infections par le virus de l’immunodéficience humaine (VIH) se produisent au cours d’un rapport sexuel, avec une transmission du virus par voie muqueuse. Le sperme constitue donc une source majeure de virus à l’échelle mondiale. Le sperme d’hommes infectés par le VIH contient le virus sous deux formes : des particules virales libres et des cellules infectées, principalement des leucocytes.Plusieurs hypothèses ont été proposées afin d’expliquer le passage du virus à travers la barrière muqueuse, qu’il s’agisse d’une muqueuse génitale (cervico-vaginale, pénienne ou urétrale) ou intestinale (muqueuse anale ou rectale). Toutefois, une grande majorité des études qui ont été menées jusqu’à présent se sont concentrées sur le rôle des particules virales libres, et celui des cellules infectées demeure mal compris. Une étude menée dans notre laboratoire a montré que des leucocytes infectés par le virus de l’immunodéficience simienne (VIS) sont capables de transmettre l’infection après inoculation vaginale.Le projet de cette thèse est d’étudier le rôle des leucocytes infectés présents dans le sperme de macaque dans la transmission muqueuse du VIS/VIH. Ainsi, trois axes d’étude principaux ont été définis: 1) l’étude des leucocytes présents dans le sperme de macaque cynomolgus, et de l’influence que peut avoir l’infection par le VIS sur eux ; 2) l’identification des cellules immunitaires infectées présentes dans le sperme de macaque, et l’étude de leur dynamique au cours de l’infection par le VIS. ; 3) l’étude du pouvoir infectieux des deux principales cellules cibles pour le VIS/VIH : les lymphocytes CD4+ (LT CD4+) et les macrophages, in vitro et in vivo, après inoculation rectale et vaginale à des macaques cynomolgus.Le sperme de macaque contient toutes les cellules cibles du VIS/VIH : des lymphocytes T CD4+ (LTCD4+), des macrophages et des cellules dendritiques dans une moindre proportion). Les LTCD4+ et les macrophages du sperme présentent un phénotype d’activation, de différenciation et d’expression de marqueurs de migration typique des leucocytes résidant dans les tissus muqueux. L’infection par le VIS induit des changements significatifs dans leur phénotype et leur dynamique. Ces deux types cellulaires peuvent être infectés de façon productive et sont présents dans le sperme à tous les stades de l’infection. Ces données suggèrent que les LTCD4+ et les macrophages du sperme seraient capables de transmettre l’infection par voie muqueuse.Si le rôle des leucocytes infectés du sperme est confirmé in vivo, il sera important à l’avenir de prendre en compte ce mécanisme de transmission dans le développement de nouvelles stratégies préventives de l’infection par le VIH, notamment les microbicides. / Human Immunodeficiency Virus (HIV) infection mostly spreads by the mucosal route: sexual transmission is the dominant mode of transmission, responsible for between 85% and 90% of cases of infection worldwide. These epidemiological data indicate that semen is one of the major sources of HIV-1 transmission. Semen, like other bodily secretions involved in HIV sexual transmission, contains the virus as two forms: cell-free viral particles and cell-associated virus, mostly in infected leukocytes. Although cell-to-cell HIV transmission has been extensively described as more efficient, rapid and resistant to host immune responses, very few studies have investigated the role in vivo of infected leukocytes in virus mucosal transmission. One such study has been recently conducted in our lab, and demonstrated that SIV-infected splenocytes are able to transmit infection to female macaques after vaginal exposure. However, all these studies used immune cells from peripheral blood or lymphoid tissues, such as spleen, and none have investigated the capacity of infected leukocytes in semen to transmit the infection in vivo. Indeed, nature, phenotype and infectivity of HIV associated with semen leukocytes may be different from that of HIV from other sources.Therefore, the objectives of this work are, first, to study of semen leukocytes and their dynamics during SIVmac251 infection in detail, then to investigate seminal factors that may influence semen infectiousness, and finally to test semen leukocyte infectivity in vitro and in vivo, using a model of mucosal exposure in cynomolgus macaques.Macaque semen contains all the target cells for HIV/SIV: CD4+ T cells, macrophages and dendritic cells in lower proportions. Semen CD4+ T cells and macrophages display an activation, differenciation and expression of migration markers profile which is typical of mucosal leucocytes. SIV infection induces significant changes in their phenotype and dynamics. Both cell types can be productively infected and are found in the semen at all stages of infection. These observations suggest that semen CD4+ T cells and macrophages may be able to transmit infection after mucosal exposure.If the role of semen infected leukocytes in HIV/SIV mucosal transmission is confirmed in vivo, this mechanism will be important to consider for further preventive strategies design, like microbicides.

One of the major challenges facing medicine and drug discovery is the large number of genetic diseases caused by inherited mutations leading to a toxic gain-of-function, or loss-of-function of the disease protein. Microbiology offered a new glimpse of hope to address those disorders with the adaptation of the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as a genome editing tool. Cas9 is a unique CRISPR-associated endonuclease protein that can be easily programmed with an RNA [a single-guide RNA (sgRNA)] that is complementary to nearly any DNA locus. Cas9 creates a double-stranded break (DSB) that can be exploited to knock out toxic genes or replenish therapeutic expression levels of essential proteins. In addition to a matching sgRNA sequence, Cas9 requires the presence of a short signature sequence [a protospacer adjacent motif (PAM)] flanking the target locus. Over the past few years, several Cas9-based therapeutic platforms have emerged to correct DNA mutations in a wide range of mammalian cell lines, ex vivo, and in vivo by adapting recombinant adeno-associated virus (rAAV). However, most of the applications of Cas9 in the field have been limited to Streptococcus pyogenes (SpyCas9), which, in its wild-type form, suffers from inaccurate editing at off-target sites. It is also difficult to deliver via an all-in-one (sgRNA+Cas9) rAAV approach due to its large size. In this thesis, I describe other Cas9 nucleases and their development as new AAV-based genome editing platforms for therapeutic editing in vivo in mouse disease models. In the first part of this thesis, I develop the all-in-one AAV strategy to deliver a Neisseria meningitidis Cas9 ortholog (Nme1Cas9) in mice to reduce the level of circulating cholesterol in blood. I also help characterize an enhanced Cas9 from another meningococcus strain (Nme2Cas9) and show that it is effective in performing editing not only in mammalian cell culture, but also in vivo by all-in-one AAV delivery. Additionally, I describe two AAV platforms that enable advanced editing modalities in vivo: 1) segmental DNA deletion by delivering two sgRNAs (along with Nme2Cas9) in one AAV, and 2) precise HDR-based repair by fitting Nme2Cas9, sgRNA and donor DNA within a single AAV capsid. Using these tools, we successfully treat two genetic disorders in mice, underscoring the importance of this powerful duo of AAV and Cas9 in gene therapy to advance novel treatment. Finally, I present preliminary data on how to use these AAV.Nme2Cas9 vectors to treat Alexander Disease, a rare progressive neurological disorder. These findings provide a platform for future application of gene editing in therapeutics.

Indiana University-Purdue University Indianapolis (IUPUI) / High-efficiency transduction of hematopoietic stem cells (HSCs) by recombinant adeno-associated virus serotype 2 (AAV2) vectors is limited by (i) inadequate expression of cellular receptor/co-receptors for AAV2; (ii) impaired intracellular trafficking and uncoating in the nucleus; (iii) failure of the genome to undergo second-strand DNA synthesis; and (iv) use of sub-optimal promoters. Systematic studies were undertaken to develop alternative strategies to achieve high-efficiency transduction of primary murine HSCs and lineage-restricted transgene expression in a bone marrow transplant model in vivo. These included the use of: (i) additional AAV serotype (AAV1, AAV7, AAV8, AAV10) vectors; (ii) self-complementary AAV (scAAV) vectors; and (iii) erythroid cell-specific promoters. scAAV1 and scAAV7 vectors containing an enhanced green-fluorescent protein (EGFP) reporter gene under the control of hematopoietic cell-specific enhancers/promoters allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice. Self complementary AAV vectors containing an anti-sickling human beta-globin gene under the control of either the beta-globin gene promoter/enhancer, or the human parvovirus B19 promoter at map-unit 6 (B19p6) were tested for their efficacy in a human erythroid cell line (K562), and in primary murine hematopoietic progenitor cells (c-kit+, lin-). These studies revealed that (i) scAAV2-beta-globin vectors containing only the HS2 enhancer are more efficient than ssAAV2-beta-globin vectors containing the HS2+HS3+HS4 enhancers; (ii) scAAV-beta-globin vectors containing only the B19p6 promoter are more efficient than their counterparts containing the HS2 enhancer/beta-globin promoter; and (iii) scAAV2-B19p6-beta-globin vectors in K562 cells, and scAAV1-B19p6-beta-globin vectors in murine c-kit+, lin- cells, yield efficient expression of the beta-globin protein. These studies suggest that the combined use of scAAV serotype vectors and the B19p6 promoter may lead to expression of therapeutic levels of beta-globin gene in human erythroid cells, which has implications in the potential gene therapy of beta-thalassemia and sickle cell disease.

Mitochondria are dynamic organelles that function in a complex interconnected network within the cell. Neurons are sensitive and highly energy demanding cells in the brain which require a functioning mitochondrial network that is able to provide ATP and modulate calcium. Mitochondrial networks have yet to be explored which gives rise to the need for specific and efficient molecular tools. In this project, I designed and produced adeno-associated virus vectors carrying a fluorescent reporter gene for imaging mitochondrial networks under human synapsin 1 promoter to target neurons specifically. The design of each vector was conducted with careful consideration of the different components in the plasmid design that are important for optimal expression, which resulted in two constructs; one self-complementary adeno-associated virus vector that marks the mitochondria and one single-stranded that marks mitochondria and the membrane of neurons.  The modularity of viral vectors allows the usage of different serotypes which adapt the vector to the cell type and the model. For this project I chose the serotypes 1 for neurons in vitro and PHP.eB which suits in vivo models since it has better permeability to the blood brain barrier. The production was conducted in human embryonic kidney cells using the triple-plasmid transfection method, followed by extraction and purification. The existence of viral particles was verified through transmission electron microscopy and the DNA titer of the vector through quantitative polymerase chain reaction. The produced adeno-associated virus vectors were delivered into young brain organoids which were not able to express the reporter gene, probably due to not fully developed neurons. The fluorescent protein expression targeting specifically mitochondria and the membrane was however verified in the human embryonic kidney cells during the packaging stages.

No description available.

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type strain for the genus Ampelovirus, family Closteroviridae. There has been only one report that claimed the complete nucleotide sequence of GLRaV-3 (isolate NY-1, AF037268). Here we report the complete sequence of the South African GLRaV-3, isolate GP18 (EU259806) and show a significantly extended 5’ end. We used RLM-RACE to determine the 5’ end of GP18 and found the 5’ UTR to be 737 nt compared to 158 nt in the NY-1 sequence. This extended UTR was found in all other South African isolates of GLRaV-3 that were tested. In two collaborative studies the existence of the extended 5’ UTR was confirmed and further investigated. In the first study (Coetzee et al., 2010), metagenomic data generated by next generation sequencing (Illumina Genome Analyzer II) was analysed for GLRaV-3 specific sequences. Sequences similar to the GP18 isolate confirmed the sequence of the extended 5’ UTR. In the second study (Jooste et al., 2010), three genetic variants were identified and their respective 5’ UTRs studied. Great diversity was observed between the 5’ UTRs of the different genetic variants, however within a variant the 5’ UTR was found to be highly conserved. Grapevine leafroll-associated virus 3 is a positive sense, single stranded RNA virus that has been shown, like other closteroviruses, to produce subgenomic (sg) RNAs during replication. These sgRNAs are deployed for the expression of the ORFs on the 3’ half of the genome. In this study a dsRNA blot confirmed the presence of three, 3’ coterminal sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in GLRaV-3-infected plant material when using a probe directed at the coat protein gene. The specific 5’ terminal nucleotides for these sgRNAs as well as four additional sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were determined by RLM-RACE for GLRaV-3 isolate GP18. The construction of a GLRaV-3 mini-replicon, analogous to RNA1 of Lettuce infectious yellows virus, for the evaluation of putative sg-promoters is also described. / AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ‘n lid van die Closteroviridae familie en die hooflid vir die genus Ampelovirus. Tot dusver was daar net een studie wat die volledige nukleïensuurvolgorde van GLRaV-3 gerapporteer het (isolaat NY-1, AF037268). In hierdie studie rapporteer ons die volledige volgorde van ‘n Suid-Afrikaanse GLRaV-3, isolaat nl. GP18 (EU259806) wat noemenswaardig langer is aan die 5’ kant. RLM-RACE is gebruik om die 5’ eindpunt van GP18 te bepaal en daar is gevind dat die 5’ ongetransleerde streek (UTR) 737 nt lank is in vergelyking met die 158 nt van die NY-1 volgorde. Die verlengde 5’ UTR is gevind in alle Suid-Afrikaanse monsters wat getoets is. Die verlengde 5’ UTR is bevestig en verder bestudeer tydens twee samewerkingsprojekte. In die eerste studie (Coetzee et al., 2010), is metagenomiese data gegenereer deur volgende-generasie volgordebepaling (Illumina Genome Analyzer II) en geanaliseer vir GLRaV-3 spesifieke volgordes. Volgordes soortgelyk aan die GP18 isolaat het die verlengde 5’ UTR volgorde bevestig. In die tweede studie (Jooste et al., 2010), is drie genetiese variante van GLRaV-3 geidentifiseer en hulle onderskeie 5’ UTR volgordes bepaal en bestudeer. Daar is groot diversiteit tussen die 5’ UTRs van die verskillende genetiese variante gevind, maar tussen isolate van dieselfde variant is die volgordes gekonserveerd. Grapevine leafroll-associated virus 3 is ‘n positiewe-sin, enkelstring RNA virus wat al voorheen bewys is om, soos ander closterovirusse, subgenomiese (sg) RNAs te produseer tydens replisering. Hierdie sgRNAs word ingespan vir die uitdrukking van die ORFs op die 3’ helfte van die virusgenoom. In hierdie studie is ‘n dsRNA klad gebruik om die voorkoms van 3’ ko-terminale sgRNAs [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] te bevestig in GLRaV-3 geinfekteerde plantmateriaal deur gebruik te maak van ‘n peiler teen die kapsiedproteïengeen. Die spesifieke 5’ terminale nukleotiedes vir hierdie sgRNAs sowel as vier additionele sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] is bepaal deur gebruik te maak van RLM-RACE op die GLRaV-3 isolaat GP18. Die konstruksie van ‘n GLRaV-3 mini-repliserings konstruk, analoog aan die RNA1 van Lettuce infectious yellows virus, vir die evaluasie van moontlike sg-promotors word ook beskryf.

No description available.