Gotowa bibliografia na temat „Viroplasm”

Rice ragged stunt virus (RRSV), an oryzavirus, is transmitted by brown planthopper in a persistent propagative manner. In this study, sequential infection of RRSV in the internal organs of its insect vector after ingestion of virus was investigated by immunofluorescence microscopy. RRSV was first detected in the epithelial cells of the midgut, from where it proceeded to the visceral muscles surrounding the midgut, then throughout the visceral muscles of the midgut and hindgut, and finally into the salivary glands. Viroplasms, the sites of virus replication and assembly of progeny virions, were formed in the midgut epithelium, visceral muscles and salivary glands of infected insects and contained the non-structural protein Pns10 of RRSV, which appeared to be the major constituent of the viroplasms. Viroplasm-like structures formed in non-host insect cells following expression of Pns10 in a baculovirus system, suggesting that the viroplasms observed in RRSV-infected cells were composed basically of Pns10. RNA interference induced by ingestion of dsRNA from the Pns10 gene of RRSV strongly inhibited such viroplasm formation, preventing efficient virus infection and spread in its insect vectors. These results show that Pns10 of RRSV is essential for viroplasm formation and virus replication in the vector insect.

ABSTRACT Viral inclusion bodies, or viroplasms, that form in rotavirus-infected cells direct replication and packaging of the segmented double-stranded RNA (dsRNA) genome. NSP2, one of two rotavirus proteins needed for viroplasm assembly, possesses NTPase, RNA-binding, and helix-unwinding activities. NSP2 of the rotavirus group causing endemic infantile diarrhea (group A) was shown to self-assemble into large doughnut-shaped octamers with circumferential grooves and deep clefts containing nucleotide-binding histidine triad (HIT)-like motifs. Here, we demonstrate that NSP2 of group C rotavirus, a group that fails to reassort with group A viruses, retains the unique architecture of the group A octamer but differs in surface charge distribution. By using an NSP2-dependent complementation system, we show that the HIT-dependent NTPase activity of NSP2 is necessary for dsRNA synthesis, but not for viroplasm formation. The complementation system also showed that despite the retention of the octamer structure and the HIT-like fold, group C NSP2 failed to rescue replication and viroplasm formation in NSP2-deficient cells infected with group A rotavirus. The distinct differences in the surface charges on the Bristol and SA11 NSP2 octamers suggest that charge complementarity of the viroplasm-forming proteins guides the specificity of viroplasm formation and, possibly, reassortment restriction between rotavirus groups.

Rotaviruses (RVs) cause acute gastroenteritis in infants and young children, and are globally distributed. Within the infected host cell, RVs establish replication complexes in viroplasms (‘viral factories’) to which lipid droplet organelles are recruited. To further understand this recently discovered phenomenon, the lipidomes of RV-infected and uninfected MA104 cells were investigated. Cell lysates were subjected to equilibrium ultracentrifugation through iodixanol gradients. Fourteen different classes of lipids were differentiated by mass spectrometry. The concentrations of virtually all lipids were elevated in RV-infected cells. Fractions of low density (1.11–1.15 g ml−1), in which peaks of the RV dsRNA genome and lipid droplet- and viroplasm-associated proteins were observed, contained increased amounts of lipids typically found concentrated in the cellular organelle lipid droplets, confirming the close interaction of lipid droplets with viroplasms. A decrease in the ratio of the amounts of surface to internal components of lipid droplets upon RV infection suggested that the lipid droplet–viroplasm complexes became enlarged.

Like other members of the family Reoviridae, rice black-streaked dwarf virus (RBSDV, genus Fijivirus) is thought to replicate and assemble within cytoplasmic viral inclusion bodies, commonly called viroplasms. RBSDV P9-1 is the key protein for the formation of viroplasms, but little is known about the other proteins of the viroplasm or the molecular interactions amongst its components. RBSDV non-structural proteins were screened for their association with P9-1 using a co-immunoprecipitation assay. Only P6 was found to directly interact with P9-1, an interaction that was confirmed by bimolecular fluorescence complementation assay in Spodoptera frugiperda (Sf9) cells. Immunoelectron microscopy showed that P6 and P9-1 co-localized in electron-dense inclusion bodies, indicating that P6 is a constituent of the viroplasm. In addition, non-structural protein P5 also localized to viroplasms and interacted with P6. In Sf9 cells, P6 was diffusely distributed throughout the cytoplasm when expressed alone, but localized to inclusions when co-expressed with P9-1, suggesting that P6 is recruited to viral inclusion bodies by binding to P9-1. P5 localized to the inclusions formed by P9-1 when co-expressed with P6 but did not when P6 was absent, suggesting that P5 is recruited to viroplasms by binding to P6. This study provides a model by which viral non-structural proteins are recruited to RBSDV viroplasms.

Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. The structures (as far as is known) and functions of these proteins are described. Recent studies using plasmid-only-based reverse genetics have significantly contributed to elucidation of the crucial roles of these proteins in RV replication. Thus, it has been recognized that viroplasms resemble liquid-like protein–RNA condensates that may be formed via liquid–liquid phase separation (LLPS) of NSP2 and NSP5 at the early stages of infection. Interactions between the RNA chaperone NSP2 and the multivalent, intrinsically disordered protein NSP5 result in their condensation (protein droplet formation), which plays a central role in viroplasm assembly. These droplets may provide a unique molecular environment for the establishment of inter-molecular contacts between the RV (+)ssRNA transcripts, followed by their assortment and equimolar packaging. Future efforts to improve our understanding of RV replication and genome assortment in viroplasms should focus on their complex molecular composition, which changes dynamically throughout the RV replication cycle, to support distinct stages of virion assembly.

Replication and assembly of viruses from the family Reoviridae are thought to take place in discrete cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. Rice black streaked dwarf virus (RBSDV) P9-1, a non-structural protein, has been confirmed to accumulate in these intracellular viroplasms in infected plants and insects. However, little is known about its exact function. In this study, P9-1 of RBSDV-Baoding was expressed in Escherichia coli as a His-tagged fusion protein and analysed using biochemical and biophysical techniques. Mass spectrometry and circular dichroism spectroscopy studies showed that P9-1 was a thermostable, α-helical protein with a molecular mass of 41.804 kDa. A combination of gel-filtration chromatography, chemical cross-linking and a yeast two-hybrid assay was used to demonstrate that P9-1 had the intrinsic ability to self-interact and form homodimers in vitro and in vivo. Furthermore, when transiently expressed in Arabidopsis protoplasts, P9-1 formed large, discrete viroplasm-like structures in the absence of infection or other RBSDV proteins. Taken together, these results suggest that P9-1 is the minimal viral component required for viroplasm formation and that it plays an important role in the early stages of the virus life cycle by forming intracellular viroplasms that serve as the sites of virus replication and assembly.

Zika virus (ZIKV) became a global health concern in 2016 due to its links to congenital microcephaly and other birth defects. Flaviviruses, including ZIKV, reorganize the endoplasmic reticulum (ER) to form a viroplasm, a compartment where virus particles are assembled. Microtubules (MTs) and microtubule-organizing centers (MTOCs) coordinate structural and trafficking functions in the cell, and MTs also support replication of flaviviruses. Here we investigated the roles of MTs and the cell’s MTOCs on ZIKV viroplasm organization and virus production. We show that a toroidal-shaped viroplasm forms upon ZIKV infection, and MTs are organized at the viroplasm core and surrounding the viroplasm. We show that MTs are necessary for viroplasm organization and impact infectious virus production. In addition, the centrosome and the Golgi MTOC are closely associated with the viroplasm, and the centrosome coordinates the organization of the ZIKV viroplasm toroidal structure. Surprisingly, viroplasm formation and virus production are not significantly impaired when infected cells have no centrosomes and impaired Golgi MTOC, and we show that MTs are anchored to the viroplasm surface in these cells. We propose that the viroplasm is a site of MT organization, and the MTs organized at the viroplasm are sufficient for efficient virus production.

The rotavirus (RV) genome is replicated and packaged into virus progeny in cytoplasmic inclusions called viroplasms, which require interactions between RV nonstructural proteins NSP2 and NSP5. How viroplasms form remains unknown. We previously found two forms of NSP2 in RV-infected cells: a cytoplasmically dispersed dNSP2, which interacts with hypophosphorylated NSP5; and a viroplasm-specific vNSP2, which interacts with hyperphosphorylated NSP5. Other studies report that CK1α, a ubiquitous cellular kinase, hyperphosphorylates NSP5, but requires NSP2 for reasons that are unclear. Here we show that silencing CK1α in cells before RV infection resulted in (i) >90% decrease in RV replication, (ii) disrupted vNSP2 and NSP5 interaction, (iii) dispersion of vNSP2 throughout the cytoplasm, and (iv) reduced vNSP2 protein levels. Together, these data indicate that CK1α directly affects NSP2. Accordingly, an in vitro kinase assay showed that CK1α phosphorylates serine 313 of NSP2 and triggers NSP2 octamers to form a lattice structure as demonstrated by crystallographic analysis. Additionally, a dual-specificity autokinase activity for NSP2 was identified and confirmed by mass spectrometry. Together, our studies show that phosphorylation of NSP2 involving CK1α controls viroplasm assembly. Considering that CK1α plays a role in the replication of other RNA viruses, similar phosphorylation-dependent mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication.

The badnavirus replication cycle is poorly understood and most knowledge is based on extrapolations from model viruses such as Cauliflower mosaic virus (CaMV). However, in contrast to CaMV, badnaviruses are thought not to produce viroplasms and therefore it has been a mystery as to where virion assembly occurs. In this study, ultrathin sections of a banana leaf infected with a badnavirus, banana streak MY virus (BSMYV), were examined by transmission electron microscopy. Electron-dense inclusion bodies (EDIBs) were sporadically distributed in parenchymatous tissues of the leaf, most commonly in the palisade and spongy mesophyll cells. These EDIBs had a characteristic structure, comprising an electron-dense core, a single, encircling lacuna and an outer ring of electron-dense material. However, much less frequently, EDIBs with two or three lacunae were observed. In the outer ring, densely packed virions were visible with a shape and size consistent with that expected for badnaviruses. Immunogold labelling was done with primary antibodies that detected the N-terminus of the capsid protein and strong labelling of the outer ring but not the central core or lacuna was observed. It is concluded that the EDIBs that were observed are equivalent in function to the viroplasms of CaMV, although obviously different in composition as there is not a paralogue of the transactivation/viroplasm protein in the badnavirus genome. It is postulated that production of a viroplasm could be a conserved characteristic of all members of the Caulimoviridae.

ABSTRACT Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.

The processes that regulate Rotavirus replication are not fully understood and the lack of a reverse genetic approach represent an obstacle for the investigations in Rotavirus biology. Viroplasms are cytoplasmic structures that form soon after infection, and constitute the site of virus replication. Structural proteins like the viral RNA-dependent RNA -polymerase VP1, the capping enzyme VP3, the scaffolding protein VP2,and the middle layer VP6 localize in viroplasms; in addition, also the non-structural proteins NSP5 and NSP2 have been demonstrated to be essential components for viroplasm formation. Following the characterization of the interaction between NSP5 and VP1, we characterized the relationships between NSP5 and the structural protein VP2. In this work, interaction of NSP5 with VP2 was investigated by coexpression of the two proteins in uninfected cells, which resulted in a strong hyperphosphorylation of NSP5 and in the formation of viroplasm like structures (VLS). The behaviour of NSP5 in the presence of VP2 is very similar to that induced by NSP2 and already described (1), (60). Therefore, a comparison between the phosphorylation degree of NSP5 and VLS formation induced either by VP2 or by NSP2 was conducted. In both cases VLS formation was shown to assemble independently of the phosphorylation degree of NSP5, and to recruit the viroplasm-resident proteins VP1. However, VP6 (the protein forming the middle layer of the virion) was shown to be recruited only into VLS induced by VP2 (VLS(VP2i)), while it remains organized in tubular structures when VLSinduced by NSP2 (VLS(NSP2i)) were formed. Attempts to coimmunoprecipitate NSP5 and VP2 failed both from infected and co-transfected cells. However, promising preliminary results were obtained with a recently isolated monoclonal Ab specific for NSP5. Altogether, these data showed that two different viral proteins induced the same kind of modifications in NSP5, suggesting that these modifications may have a fundamental role for virus replication. Moreover, these data suggest that NSP5 plays a key role in architectural assembly of viroplasms and in recruitment of the other viroplasmic proteins.

Rotaviruses are a leading cause of acute gastroenteritis in infants and young children worldwide and possess a genome of 11 double-stranded (ds) RNA segments. Early morphogenesis of RV particles and viral RNA replication occur in cytoplasmic inclusion bodies called ‘viroplasms’, of which the viral non-structural proteins NSP2 and NSP5 are essential components. Using confocal microscopy (CM), we demonstrated association of viroplasms with lipids and lipid droplet (LD)-associated proteins (perilipin A, ADRP). LD-associated proteins were found to co-localise with viroplasm-like structures (VLS) in cells co-transfected with NSP2- and NSP5- expressing plasmids in the absence of rotavirus infection, as well as viroplasms containing NSP5-EGFP. Close spatial proximity between NSP5-EGFP and perilipin A was demonstrated by Fluorescence Resonance Energy Transfer (FRET). Time course CM studies showed increasing recruitment of perilipin A to viroplasms during the progression of rotavirus infection. Separation of RV-infected cell extracts on iodixanol gradients demonstrated co-localisation of rotavirus dsRNA, NSP5 and perilipin A in low density fractions. Chemical compounds interfering with LD formation or homeostasis (isoproterenol + IBMX, triacsin C, C75) decreased the number and size of viroplasms in rotavirus-infected cells, dsRNA replication and the production of infectious progeny virus, whilst significantly protecting cells from cytopathicity caused by rotavirus infection. These data contribute to a novel understanding of the role of LDs in rotavirus replication. Using a cell line stably expressing NSP5-EGFP, the dynamics of viroplasm formation in relation to dsRNA replication and production of infectious virus was investigated.

L’infection de cellules par le rotavirus implique la formation de nombreux assemblages macromoléculaires qui recrutent, en plus des protéines virales, de nombreuses protéines cellulaires nécessaires à la morphogenèse virale, à la signalisation et à la mise en place de défenses anti-virales. Cette stratégie est commune à de nombreux virus. La connaissance de la composition, de la structure et du fonctionnement de ces complexes, dont certains ont été baptisés “usines virales“, est d’un intérêt majeur pour la biologie cellulaire fondamentale et pour la conception de stratégies thérapeutiques innovantes contre le rotavirus. Ce court travail de thèse a été consacré à l’isolement et à la caractérisation de différents complexes viraux correspondant à des étapes précoces de la morphogenèse virale. Un protocole de purification permettant le fractionnement du cytoplasme de cellules infectées par le rotavirus a été mis au point afin (i) d’isoler des complexes macromoléculaires contenant la protéine de la capside intermédiaire, VP6, (ii) d’en analyser le contenu protéique par électrophorèse bidimensionnelle avec une première dimension en conditions non dénaturantes (Blue Native PAGE) suivi d’une deuxième dimension en conditions dénaturantes, (iii) de réaliser une étude protéomique fine pour identifier les protéines impliquées dans ces complexes. Cette approche a permis d’identifier quatre familles de protéines : (a) des chaperonnes cytoplasmiques, parmi lesquelles Hsc70, Hsp70, Hsp90, (b) des chaperonnes réticulaires, telles que la calréticuline, Grp78 et la protéine disulfide isomérase (PDI), (c) des protéines associées à l’actine dont l’Ezrine, l’α actinine 4, la GAPDH et (d) un groupe plus hétérogène contenant des protéines ayant des fonctions métaboliques et des co-chaperonnes. Des études d’immunofluorescence en microscopie optique et des expériences de co-immunoprécipitation ont permis de confirmer l’interaction, au sein de ces complexes macromoléculaires, de la protéine VP6 du rotavirus avec Hsp70, Hsp90, PDI et la calréticuline. Ce travail démontre ainsi la faisabilité d’une approche originale qui doit permettre d’avancer dans la compréhension des mécanismes précoces de l’assemblage et du trafic du rotavirus.

Rotavirus is a major cause of acute gastroenteritis in infants and young children and responsible for approximately 453,000 infantile deaths per year. Rotaviruses are non-enveloped RNA viruses belonging to the Reoviridae family. The rotavirus genome is composed of 11 segments of double-stranded RNA (dsRNA), enclosed in an icosahedral triple-layered protein capsid, and it encodes six structural proteins (VP) and six non-structural proteins (NSPs). Removal of the outer capsid from the triple-layered particle (TLP) during virus entry into the cell activates the synthesis and extrusion of the viral mRNAs from the double-layered particle (DLP) into the cytoplasm. The viral genome replication and assembly of immature DLPs occurs in specialized virus-induced electron-dense cytoplasmic inclusions called viroplasms (VMs), nucleated by two essential non-structural proteins NSP2 and NSP5, and the inner capsid protein VP2. NSP5 is crucial for recruitment of the viroplasmic proteins and the architectural assembly of VMs. VMs are dynamic entities that undergo fusion and localize at the perinuclear region in the infected cells. This study aims to identify the host factors interacting with the VM proteins NSP5 and NSP2, their cytoplasmic relocalization, sequestration in VMs, and the assembly of viral and cellular proteins in the viroplasmic structures. Part I: Identification of the cellular proteins interacting with viroplasmic proteins NSP5 and NSP2 NSP5 and NSP2-interacting proteins have been affinity purified in a pull-down assay and the protein complexes were analysed using LC-MS/MS. Mass spectrometry data revealed the presence of several cellular proteins including hnRNPs, ARE-BPs and others. These results were further validated by both immunoblotting of the pull down complexes, and co-immunoprecipitation. Part II: Cytoplasmic relocalization of nuclear proteins, their sequestration by the viroplasmic proteins and their biological significance in virus infection Rotavirus replication occurs in the cytoplasm, and none of the viral proteins are known to selectively translocate to the nucleus in infected cells. The finding in this study that a large number of hnRNPs and other proteins interact with NSP5 and NSP2 suggested the likely cytoplasmic relocalization of the host nuclear proteins and their interaction with viroplasmic proteins. The cytoplasmic redistribution of some nuclear proteins has been reported in several other viruses such as Poliovirus, HIV, JEV, MHV, and Enteroviruses, but their large-scale relocalization has not been reported. Confocal microscopy studies revealed that several hnRNPs and ARE-BPs relocalized to the cytoplasm and colocalized with VMs in the infected cells to form viroplasm structures (VSs). The basis for this large-scale cytoplasmic relocalization and sequestration of majority of the nuclear proteins and nuclear transport proteins in the viroplasm was explored. The results suggest that selective inhibition of nuclear import pathways occurring during rotavirus infection primarily contributes to the cytoplasmic accumulation of nuclear proteins, but inhibition of the importin α/β pathway, affecting the nuclear accumulation of PABPC1, severely affects rotavirus growth. Knockdown and overexpression studies of some of the relocalized cellular proteins revealed their differential influence on viral infection. Altogether, this study redefines the existing concept that ‘only virus components are present in the VMs’. Part III: Modulation of stress granules (SGs) and processing bodies (PBs) during rotaviral infection In common with many viruses, rotaviruses have been reported to block SG assembly (Montero et al., 2008) and cause dispersion of PBs (Bhowmick et al., 2015), however these results are based on the analysis of only two or three markers of SGs and PBs. Careful examination of these results suggest that in both studies, erroneous conclusions were drawn by the authors despite perfect colocalization of TIA1 and Dcp1a with the viroplasms, probably due to the prevailing concept that VMs consist only of viral components. The present study employing multiple methods, and analysing a large number of SG and PB proteins, unequivocally demonstrated that rotavirus remodels SGs and PBs by inducing selective dissociation of a few components and sequestration of the remodelled granules containing the majority of the SG and PB components into the viroplasms, forming the viroplasm structures (VSs). Part IV: Studies on the structural assembly of viroplasm structures (VSs) VMs are dynamic structures and considered to contain only viral components. In the previous part, it was demonstrated that VMs, consisting of NSP5 and NSP2, associate with other viral viroplasmic proteins and several host proteins to form VSs. Using High-resolution confocal microscopy, the structural organization the viral and cellular proteins in the VS, and the sizes of different VSs inside the infected cells was investigated. These studies revealed that the VSs are more complex than that is currently perceived with viral and cellular proteins being organised in a specific order with the cellular proteins localized in the VS based on their direct or indirect interaction with the viroplasmic proteins. This study provides the first insights into the spatial organization of viral and cellular proteins in the VS. Overall, the present study conclusively demonstrates that the composition of the VS is more complex than that is currently perceived, and provides the first insights into the unknown spatial organization of the viral proteins NSP5, NSP2, VP1 and VP6 as well as cellular proteins within the VS. These results lay the foundation for future studies in understanding the detailed structural organization of different proteins in the VS and the spatial and temporal assembly of viral and cellular proteins.

Overall, the present study demonstrates the dynamic nature of rotavirus viroplasm and how it manipulates the intracellular environment by intracellular relocalization of host proteins and their sequestration in viroplasms for its survival and to manifest the infection. This work demonstrates the mechanism by which the host proteins are recruited into the viral replication structures called viroplasms

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

Cellular ultrastructure micrographs revealed striking changes resulting from the Barley Yellow Dwarf Virus (BYDV-PAV) infection in Electron microscopy. In the cytoplasm, the Gold nanoparticles (AuNPs) may bind with different cytoplasmic organelles and interfere with the treated site’s metabolic processes. The micrographs of the treated plant leave with AuNPs showing; Endosomes, amorphous bodies, slender filaments fibers, myelin bodies with a high concentration of virus particles, and Gold Nanoparticles distributed in a circulated shape in the cytoplasm with virus particles.