Le ré-encodage aléatoire des codons à grande échelle est une nouvelle méthode d'atténuation virale qui consiste en l'insertion d'un grand nombre de mutations synonymes, individuellement peu délétères, de façon aléatoire dans une ou plusieurs régions codantes d'un virus. Cette approche permet de diminuer de façon significative et modulable le fitness réplicatif des virus in cellulo et in vivo, ainsi que la pathogénicité du virus chez la souris, tout en induisant une protection immunitaire spécifique et efficace lors d'une nouvelle infection par le virus sauvage. Les virus ré-encodés présentent également une grande stabilité et une absence de réversion ce qui en font des candidats vaccins très prometteurs en termes d'efficacité et de fiabilité pour la conception de candidats vaccins vivants atténués contre une grande variété de virus à ARN. La combinaison du ré-encodage aléatoire et d'une nouvelle méthode de génétique inverse permettant de générer de nouveaux virus en quelques jours: ISA (Amplicon Subgenomique Infectieux), est une approche prometteuse qui pourrait aider au développement de vaccins vivants atténués de nouvelle génération en un temps record. / Large-scale random codon re-encoding is a new method of viral attenuation consisting in the insertion of a high number of slightly deleterious synonymous mutations, randomly, in one or several coding regions of a virus. This approach significantly reduces the replicative fitness of re-encoded viruses in cellulo and in vivo, as viral pathogenicity, while inducing a specific and effective immune response in mice against a new infection with wild-type viruses. Re-encoded viruses also present a high stability and an absence of reversion, making them promising vaccine candidates in term of reliability and efficiency for the conception of new vaccine candidates against a wide variety of RNA viruses. Combination of random re-encoding with a new method of revers genetics allowing to generate new viruses in days : ISA (Infectious Subgenomic Amplicons) would be very helpful to develop new-generation vaccine candidates.

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

Etliche Membran Transport Proteine (MTP) sind essentiell in den Plasmodium Blutstadien, und geraten zunehmend in den Fokus der Wirkstoffentwicklung. Die physiologischen Rollen der Transporter sind jedoch oft ungeklärt. In dieser Arbeit wurden mittels experimenteller Genetik funktionelle Charakteristika der MTPs untersucht. Am Maus Parasiten Plasmodium berghei und der Plasmodium falciparum Blutstadien-Kultur wurden vier MTPs ausgewählt: ein konservierter Folat Transporter (FT2), sowie eine P. falciparum-spezifisches P-Typ ATPase und zwei essentielle MTPs (CRT und ATP4). Diese Auswahl verkörpert ein breites Spektrum an MTP Kandidaten und reflektieren zudem das Potenzial und die Grenzen funktioneller Analysen von Plasmodium MTPs mittels reverser Genetik. Für den Folat Transporter 2 (FT2) wurde eine Kombination von transgenen Strategien auf P. berghei angewandt. Durch ein endogenes tag von FT2 wurde die Lokalisierung im Apicoplast, sowie dessen Expression über fast den kompletten Zyklus hinweg gezeigt. Nach der Deletion von FT2, wiesen die Parasiten einen Defekt während der Sporulation auf. Demzufolge bilden sich nur nicht infektiöse Sporozoiten, was letztendlich zur Unterbrechung des Lebenszyklus der Parasiten führt. Eine Aminophospholipid P-Typ ATPase, wurde mittels CRISPR/Cas9 in P. falciparum genetisch deletiert und die Mutante analysiert. Im Gegensatz zu den meisten vitalen P-Typ ATPasen erweist sich das Gen in den asexuellen Blutstadien als entbehrlich. Des Weiteren bilden die MTPs ATP4 und CRT einen einflussreichen Faktor bei Malaria-Therapien. Eine umfassende Analyse von räumlichen und zeitlichen Expressionsmustern von transgenen Parasiten mit mCherry-getaggten Proteinen zeigt ein Expression der beiden MTPs über die Blutstadien hinaus, was auf zusätzliche Funktionen in den jeweiligen Stadien verweist. Diese Studie trägt, basierend auf Lokalisation, Expression und funktioneller Deletion, zur funktionellen Entschlüsselung der vier untersuchten MTPs bei. / Many membrane transport proteins (MTP) are essential for Plasmodium infection and gain importance as candidate drug targets in malaria therapy, whereas the physiological functions often remain enigmatic. In this thesis, we applied experimental genetics to determine key characteristics of four Plasmodium MTPs. We employed the murine malaria model parasite Plasmodium berghei and in vitro blood cultures of Plasmodium falciparum. We selected one conserved MTP called FT2, which was previously shown to transport folate, a P-type ATPase that is specific for P. falciparum as well as two essential MTPs, CRT and ATP4. These targets exemplify the range of druggable candidates and illustrate the potential and limitations of reverse genetics to decipher their physiological roles. A combination of transgenic and knockout strategies was applied to the P. berghei folate transporter 2 (FT2). We show that endogenously tagged FT2 localises to the apicoplast membranes, and is broadly expressed throughout the parasite’s life cycle. Analysis of FT2-deficient parasites revealed a severe sporulation defect in the vector; the vast majority of ft2– oocysts form large intracellular vesicles which displace the cytoplasm. Very few sporozoites are generated and these are non-infectious to the mammalian host, resulting in a complete arrest of Plasmodium transmission. A candidate aminophospholipid P-type ATPase, was assessed by a CRISPR/Cas9-mediated gene disruption. Compared to many vital P-type ATPases this gene is dispensable for asexual blood replication. Two MTPs, ATP4 and CRT are prime targets for antimalarial therapies. A comprehensive spatio-temporal expression analysis of transgenic parasites expressing mCherry-tagged proteins revealed expression beyond blood infection, indicative of functions in additional parasite stages. The findings of this study contribute towards a better understanding of the roles of the four MTPs based on localisation, expression and functional deletion.

Das Hämagglutinin (HA) des Influenzavirus ist post-translational durch S-Acylierung von drei Cysteinen modifiziert. Zwei davon befinden sich in seiner zytoplasmatischen Domäne (CD) und enthalten Palmitat und eines am Cytosol-zugewandten Ende der Transmembranregion (TMR) wird bevorzugt mit Stearat acyliert. Es wird vermutet, dass entweder die Aminosäureumgebung der Acylierungsstelle oder dessen Lage relativ zur Membran bestimmt welcher Fettsäuretyp angeheftet wird. Diese Acylierungstellen sind zudem essentiell für die Virusreplikation. Ob auch andere Aminosäuren der CD essentiell sind, ist nicht bekannt. Nach einem umfangreichen Sequenzvergleich zur Identifikation konservierter Aminosäuren wurden rekombinante Viren mit Aminosäureaustauschen in der Nähe der drei Acylierungstellen hergestellt. Diese Austausche enthielten Punktmutationen, Verschieben des TMR Cysteins in die CD sowie die Deletion der gesamten CD. Viren ohne CD und ein Austausch neben einem acylierten Cystein verhinderten die Virusreplikation. Eine konservative Substitution derselben Position, andere Austausche in TMR und CD sowie das Schieben des TMR-Cysteins in die CD dagegen beeinflussten das Viruswachstum nur schwach. Einige der mutierten Codons revertierten zur ursprünglichen oder einer neuen Aminosäure. Rekombinante Viren wurden in MDCK-Zellen und embryonierten Hühnereiern vermehrt und mittels Massenspektrometrie analysiert. Es wurden keine unteracylierten Peptide detektiert, und selbst die zwei Letalmutationen behielten die Acylierung. Punktmutationen beeinträchtigten nur mäßig den Stearat-Gehalt, wogegen die Verlagerung des TMR-Cysteins in die CD die Stearylierung praktisch eliminierte. Mehr Stearat wurde angeheftet, wenn humane Viren in Säugerzellen im Vergleich zu aviären Zellen angezüchtet wurden. Die Position einer Acylierungsstelle repräsentiert relativ zur TMR-Spanne das Hauptsignal der Stearylierung während der Sequenzkontext und der Zelltyp das Fettsäuremuster modulieren. / Influenza virus’s hemagglutinin (HA) is post-translationally modified by S-acylation of three cysteines. Two are located in its cytoplasmic tail (CT) and contain palmitate and one at the end of the transmembrane region (TMR) is acylated primarily with stearate. It is hypothesized that either the acylation site’s amino acid environment or its location relative to the membrane determines which type of fatty acid is attached. Additionally, these acylation sites are essential for virus replication. Whether other amino acids in the CT are required for virus replication, is not known. Based on a comprehensive sequence comparison to identify conserved amino acids, recombinant viruses with amino acid substitutions in the vicinity of HA’s acylation sites were created. These substitutions included point mutations, shifting of a TMR cysteine to the CT and the deletion of the entire tail. The truncated tail mutation and a substitution adjacent to an acylated cysteine disabled virus replication. In contrast, a conservative substitution at this position, other exchanges in TMR and CT and moving the TMR cysteine to the CT had only subtle effects on virus growth. Yet, some of the mutated codons reverted to the original or other amino acids. Recombinant viruses were propagated in MDCK cells and embryonated chicken eggs and analyzed by mass spectrometry. No under-acylated peptides were detected, even the two lethal mutations did not abolish acylation. Point mutations only moderately affected the stearate content, while relocating the TMR cysteine to the CT virtually eliminated attachment of stearate. More stearate was attached if human viruses were grown in mammalian compared to avian cells. Hence, the location of an acylation site relative to the TMR represents the principal signal for stearate attachment, while the sequence context and the cell type modulate the fatty acid pattern.

Die Assemblierung von Influenzaviren erfolgt an Rafts der apikalen Wirtszellplasmamembran mit denen das Hämagglutinin (HA) über Acylierungen im C-Terminus und hydrophobe Aminosäuren seiner Transmembrandomäne (TMD) interagiert. M2 besitzt eine cytoplasmatische amphiphile Helix (AH), die ebenso potenzielle Raft-Motive aufweist: Eine Acylierung und Cholesterol-Bindemotive. In dieser Arbeit wurde per Konfokalmikroskopie an polarisierten Zellen, die fluoreszenzmarkierte M2-Varianten exprimierten, gezeigt, dass diese M2-Motive nicht für den apikalen Transport, der vermutlich durch Raft-ähnliche Vesikel erfolgt, benötigt werden. Messungen des Förster-Resonanzenergietransfers über Fluoreszenz-Lebenszeit-Mikroskopie (FLIM-FRET) in der Plasmamembran lebender Zellen, die fluoreszenzmarkiertes HA und M2 koexprimierten, ergaben, dass diese Motive auch nicht für die Interaktion mit den durch HA, in Abhängigkeit von dessen Raft-Motiven, stabilisierten Raft-Domänen notwendig sind. Mittels reverser Genetik konnten infektiöse WSN-Viren mit fehlender Acylierung am Ende der HA-TMD, nicht jedoch Viren ohne die zwei cytoplasmatischen Acylierungen hergestellt werden. Weiterhin ergaben Wachstumsanalysen, dass die Acylierung von HA und M2 für den gleichen Schritt des viralen Replikationszyklus von Bedeutung sind. Für die M2-AH wurde postuliert, dass sie die Membrankrümmung detektiert und durch Insertion in die Wirtszellmembran die Virusabschnürung bewirkt. Infektiöse Viren ohne M2 oder ohne die AH konnten ebenso wie Viren mit M2 mit einer Helix mit reduzierter Amphiphilität in dieser Arbeit nicht hergestellt werden. Allerdings führte die Substitution der AH durch typische krümmungsdetektierende oder modulierende Helices zu Viren, deren Wachstum um zwei bis vier Titerstufen im Vergleich zum Wildtyp reduziert war. Die Helix-Amphiphilität scheint wichtig zu sein, aber auch die Sequenz oder bestimmte Aminosäuren sind offenbar für eine effiziente Virusreplikation notwendig. / The assembly of influenza virus particles occurs at the apical plasma membrane of the host cell at membrane rafts which the hemagglutinin (HA) interacts with via acylations in its C-terminal region and via hydrophobic amino acids in the transmembrane domain (TMD). M2 possesses a cytoplasmic amphiphilic helix (AH) that also contains potential raft motifs: an acylation and cholesterol-binding motifs. In this work, confocal microscopy of polarised cells, which were expressing fluorescently labelled M2-variants, demonstrated that these motifs of M2 are not required for apical transport, which is assumed to be mediated by raft-like vesicles. Furthermore, FLIM-FRET (Förster resonance energy transfer measured via fluorescence lifetime imaging microscopy) analyses, performed in the plasma membrane of living cells coexpressing fluorescently labelled HA and M2, revealed that these M2-motifs are not required for association with the large coalesced raft phase organised by HA. In contrast, deleting HA’s raft-targeting features clearly reduced clustering with M2. While the removal of the two cytoplasmic acylations prevented the rescue of infectious virus by reverse genetics, a mutant virus without acylation in the HA-TMD could be rescued. Moreover, growth analyses revealed that the acylations of HA and M2 are important for the same step in the viral replication cycle. It has been postulated that the M2-AH detects membrane curvature and accomplishes membrane scission by inserting into the host cell membrane. Viruses without M2, without the M2-AH or with M2 containing a helix with reduced amphiphilicity could not be produced in this work. However, substituting the AH by typical curvature-sensing or -generating helices led to viruses with two to four orders of magnitude reduced growth as compared to wildtype virus. The amphiphilicity of the helix seems to be important, but also the sequence or specific amino acids appear to be necessary for an efficient virus replication.

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

The nuclear pore complex (NPC) is perhaps the largest protein complex in the eukaryotic cell, and controls the movement of molecules across the nuclear envelope. The NPC is composed of up to 30 proteins termed nucleoporins (Nups), each grouped in different sub-complexes. The transmembrane ring sub-complex is composed of Nups responsible for anchoring the NPC to the nuclear envelope. Bioinformatic analysis has traced all major sub-complexes of the NPC back to the last eukaryotic common ancestor, meaning that the nuclear pore structure and function is conserved amongst all eukaryotes. In this study Arabidopsis T-DNA knockout lines for these genes were investigated to characterise gene function. Differences in plant growth and development were observed for the ndc1 knockout line compared to wild-type but gp210 plants showed no phenotypic differences. The double knockout line gp210 ndc1 was generated through crosses to observe plant response to the knockout of two anchoring-Nup genes. No synergistic affect from this double knockout was observed, suggesting that more, as yet unidentified Nups function the transmembrane ring in plants. The sensitivity to nuclear export inhibitor leptomycin B (LMB) was tested also for knockout lines, although growth sensitivity to the drug was not observed. Nucleocytoplasmic transport of knockout lines was measured in cells transformed by particle bombardment. To express fluorescent protein constructs actively transported through the NPC, localisation of protein determined the nucleocytoplasmic transport of the cell. The ndc1single knockout and the double knockout gp210 ndc1 exhibited decreased nuclear export. Further experiments in determining NDC1 localisation and identification of other Nups in the transmembrane ring sub-complex would bring a more comprehensive understanding to the plant NPC.