Investigating the expression of three small open reading frames encoded on Helicoverpa armigera stunt virus RNA 1
- Authors: De Bruyn, Mart-Mari
- Date: 2017
- Subjects: Helicoverpa armigera , RNA viruses , Insects Viruses , Proteins
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59168 , vital:27448
- Description: The Helicoverpa armigera stunt virus (HaSV), belonging to the Family Alphatetraviridae (Genus: Omegatetravirus), is a non-enveloped insect virus encapsidating a bi-partite, positive-sense single-stranded RNA genome. RNA1 encodes the replicase, as well as three small open reading frames (ORFs) arranged in tandem, and overlapping with the 3’ end of the replicase ORF. These ORFs, designated p11, p15 and p8, encode putative proteins of unknown function. The p11 and p15 ORFs are conserved in the genome of the related Omegatetravirus, Dendrolimus punctatus tetravirus. In HaSV, the stop codon of p11 is followed immediately by the start of p15, whereas the stop of p15 and start of p8 are separated by a glycine intercodon. Furthermore, only p11 is known to have a recognizable Kozak sequence. The aim of this study was to determine the expression and function of these three small proteins in the HaSV infectious lifecycle. The authenticity of the viral cDNA sequence, encoding the three small ORFs, was validated by sequencing multiple cDNA clones of the relevant region in viral RNA (vRNA), purified from infectious HaSV particles. The sequence of all three ORFs was conserved in seven cDNA clones, while point mutations were observed in each of two remaining cDNA clones, suggesting that the ORFs were conserved in infectious virus. Polyclonal antisera were raised against a p11 peptide, and a recombinant p15-p8 fusion protein (p23) expressed and purified from Escherichia coli. The affinity of the anti-p23 antiserum was confirmed by western blot analysis, while that of the anti-p11 antiserum was confirmed using immunofluorescence microscopy, as attempted expression of recombinant p11 in E. coli appeared to be toxic. The antisera were used to detect expression of the small proteins in HaSV-infected H. armigera larvae by western blot analysis. A band migrating at approximately 34 kDa was detected by both antisera in infected larvae, absent in uninfected larvae, suggesting the expression of a p11-p15-p8 polyprotein. Protein bands of 11 kDa and 8 kDa were also detected by the anti-p11 and anti-p23 antisera, respectively. Bioinformatic analysis revealed that the polyprotein would be produced by a novel type of stop codon read-through, however the mechanism required for individual expression could not be definitively determined. The mechanism by which these ORFs are translated was further investigated by expressing p11-p15, tagged with FLAG and enhanced green flourescent protein (EGFP) at its amino- and carboxyl-termini respectively (FLAG-p11-p15-EGFP), in Spodoptera frugiperda (Sf9) cells detected by flourescence microscopy. Punctate structures were observed throughout the cytoplasm that were also detected with antiFLAG, anti-p11 and anti-p23 antisera, complementing results obtained in previous studies. Since p15 does not exhibit a strong recognizable Kozak like p11, the dependency of p15 expression on that of p11 was investigated by mutating this construct such that p15 occurred in a +1 frame to p11. Both EGFP and anti-p23 fluorescence was detected with the same cytoplasmic distribution as the unmutated construct, whereas nothing was detected by anti-FLAG and anti-p11. Preliminary results therefore suggested p15 may also be expressed as a discrete protein, independent of p11. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2017
A baculovirus-mediated expression system for the analysis of HaSV RNA packaging
- Authors: Mendes, Adriano
- Date: 2012
- Subjects: RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4025 , http://hdl.handle.net/10962/d1004085 , RNA , Baculoviruses , Helicoverpa armigera , Plasmids
- Description: The Helicoverpa armigera stunt virus (HaSV) is a member of a family of small nonenveloped (+) ssRNA insect viruses currently known as the Tetraviridae. This family is unique in terms of the T=4 quasi-symmetry of its capsid particles and the unusually narrow host range and tissue tropism. Assembly of tetraviral particles has been well characterised and involves the combination of 240 copies of a single capsid precursor protein (VCap) into a procapsid followed by autoproteolytic cleavage to yield the major (β) and minor (γ) capsid subunits within the mature particle. HaSV has two genomic RNAs, RNA 1 encoding the replicase and RNA 2 encoding VCap and p17, the ORF of which lies upstream of and overlaping with the 5’ end of the VCap ORF. Prior to this study, Vlok (2009) used a plasmid expression system to study RNA packaging in HaSV VLPs assembled in Spodoptera frugiperda 9 (Sf9) cells co-expressing p17 and VCap. The study showed that the p17 ORF was required for the packaging of RNA 2 during capsid assembly but it was unclear whether p17 expression was required for packaging. In addition, expression from the transfected plasmids was sub-optimal affecting both the yield of VLPs and the detection of p17. The aim of this study was to use the plasmid system to test whether p17 expression was required for plasmid-derived VLP RNA packaging and then develop a baculovirus-mediated system to test this hypothesis. By using a plasmid in which the start codon of p17 was mutated, it was shown that p17 expression was required for RNA 2 packaging into plasmid-VLPs. For the baculovirus system, four recombinant baculoviruses based upon the pFastBac Dual expression system, were constructed. These included Bac20, expressing wild type RNA 2, Bac21, RNA 2 with p17 silenced, Bac23, RNA 2 and p17 expressed on a separate transcript and Bac24, RNA 2 with p17 silenced plus p17 expressed on a separate transcript. Assembly of VLPs was more efficient using the baculovirus expression system and p17 expression was observed in cells infected with Bac20, Bac23 and Bac24, but not Bac21. In contrast to the plasmid-VLPs, bac-VLPs did not require p17 for the encapsidation of RNA 2. In addition to RNA 2, Bac23 and Bac24 packaged the p17 mRNA transcribed separately from RNA 2. This insinuated that bac-VLPs may be packaging RNA non-selectively. It was proposed that p17 may play a role in packaging in an RNA-limiting environment (plasmid system) but functioned differently when viral RNA was in excess (baculovirus system). This data points to the importance of developing a replication system for the analysis of the packaging pathways of these viruses and this study has laid down the foundations for such a system in which RNA 1 and RNA 2 can be introduced into a single cell by means of a single recombinant virus.
- Full Text:
- Date Issued: 2012
Development of an experimental system to investigate the interaction between the Helicoverpa armigera stunt virus capsid protein and viral RNA
- Authors: Nel, Andrew James Mascré
- Date: 2005
- Subjects: Helicoverpa armigera , RNA viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3946 , http://hdl.handle.net/10962/d1004005 , Helicoverpa armigera , RNA viruses
- Description: Tetraviruses are entomopathogenic viruses that propagate solely in lepidopteran hosts. Viruses of this group possess non-enveloped 38- to 40-nm capsids arranged in T = 4 surface symmetry. The viral genome consists of one or two single stranded positive sense RNA strands, which define the two genera of this family, the monopartite betatetraviruses and the bipartite omegatetraviruses. Two extensively studied members of the tetraviruses are the omegatetraviruses, Helicoverpa armigera stunt virus (HaSV) and the closely related Nudaurelia capensis ω virus (NωV). The larger genomic strand of HaSV (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71-kDa capsid precursor protein (p71). The pro-capsid is assembled from 240 copies of p71, which undergo a maturation auto-catalytic cleavage into the 64-kDa (p64) capsid protein and a 7-kDa peptide (p7) forming the capsid shell. The mechanism for the recognition and packaging of the viral genome is poorly understood for these viruses. The principle objective of the research described in this study was to develop in vitro and in vivo experimental systems to investigate interactions between the N terminal domain of HaSV p71 and viral RNAs. More specifically, the two positively charged clusters of predominantly arginine residues that are conserved amongst tetraviruses and the structurally analologous nodaviruses capsid protomers’ N terminal domains were investigated. An in vitro RNA-protein “pull down” system was developed using the rapid protein purification technique of the IMPACTTM-CN system. The coding sequence of the N terminal domain of p71 was fused to that of a chitin binding affinity tag (intein). This fusion protein was used as protein bait for the viral RNA. It was proposed that if RNA interacted with the fusion protein, it would be pulled down by the mass of affinity matrix and be precipitated and fluoresce when analysed by agarose gel electrophoresis using ethidium bromide. Despite optimisation of the in vitro assay, results were affected by the interaction between the intein-tag and nucleic acids, the state of the expressed fusion protein (in particular self-cleavage) and the excessive fluorescence present on the gels. The ADH2-GAPDH yeast expression system was used to investigate the in vivo assembly of p71 containing deletions of either one or both clusters within N terminal domain. It was found that all p71 mutants were expressed with the exception of the mutant containing a deletion of the second cluster. The reasons for this still require further investigation. The expressed p71 mutants were not processed into p64 and were degraded in vivo. In addition, an experimental attempt to purify assembled p71 mutant VLPs was unsuccessful. The assembly defect of p71 mutants emphasised the significance of the clusters, which are possibly required for interaction with viral RNAs for efficient VLP assembly. The results of this study suggest that an alternative tag or in vitro RNA-protein interaction assay be used. In addition, further experiments are required to investigate whether the co-expression of full length viral RNAs are required to rescue the in vivo assembly defect of p71 mutants into VLPs.
- Full Text:
- Date Issued: 2005
Geographic susceptibility of Helicoverpa armigera (Lepidoptera: Noctuidae) to insecticidal proteins in Bt-cotton in South Africa
- Authors: Van Jaarsveld, Martha Johanna
- Date: 2004
- Subjects: Helicoverpa armigera , Noctuidae , Lepidoptera , Cotton -- Diseases and pests -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5701 , http://hdl.handle.net/10962/d1005387 , Helicoverpa armigera , Noctuidae , Lepidoptera , Cotton -- Diseases and pests -- South Africa
- Description: Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) (African bollworm) is a typical noctuid with a very catholic taste in food plants and whose larvae feed on a wide range of cultivated and wild plants. It has been identified as the most polyphagous and injurious pest in South Africa. Helicoverpa armigera is also a key pest of cotton in many parts of the world. This key pest requires extensive control as it adversely effects yield and has built up resistance to synthetic pyrethroid insecticides. Cotton is an important crop produced by commercial and small-scale farmers in South Africa. The local demand for cotton has not been exceeded yet, but to satisfy a demanding market, pest control costs play an important role in cotton production. The threat of an insect pest that has already shown resistance prompted the present study to investigate the possibility of resistance to Bt-cotton. Genetically engineered or Bt-cotton was introduced commercially in 1996 in South Africa. All Bt-cotton plants contain one or more foreign genes derived from the soil-dwelling bacterium, Bacillus thuringiensis (Berliner), which produces protein crystals. These crystals were isolated and transferred into the genome of a cotton plant resulting in the plant producing it’s own protein insecticide. In 1998, Monsanto (Pty) Ltd requested research into the geographic susceptibility of H. armigera to the insecticidal proteins in Bt-cotton in SA. Laboratory reared and field sampled populations of H. armigera were exposed to a diet mixed with various baseline concentrations of the Bt-gene Cry1Ac freeze dried protein. This study also determined the performance of H. armigera and Spodoptera littoralis (Boisduval) on different Bt-cotton field cultivars containing different Cry-protein genes. Results obtained indicated a significant difference in susceptibility in two field populations of H. armigera to the Bt-protein Cry1Ac, even though the LD50,s in the 2003 season did not indicate resistance. Bt-cotton cultivar 15985 BX controlled H. armigera and S. littoralis larvae, the best followed in descending order by cultivar 15985 X, 15985 B and DP50 B. Results on H. armigera also indicated that the Cry-proteins in the plant parts of the different cultivars did not diminish as the season progressed. The Bt-cotton cultivars induced retarded growth of larvae, due to either a repellent effect or lack of feeding by larvae. Widespread adoption of Bt-cotton by South African farmers led to regional declines in bollworm populations, reduced insecticide use, and increased yields. Genetically modified crops therefore contribute to a cost effective, sustainable, productive and efficient form of agriculture, with a resultant positive impact on the environment. As the market for commercial Bt-cotton in South Africa expands, it is recommended that a monitoring programme for potential resistant genes in H. armigera should be implemented at least every 2 - 3 years. This will ensure that effective resistance management strategies are utilised. Coupled with this are the Biosafety Risks regarding the effect of new proteins expressed in transgenic plants, which require further studies.
- Full Text:
- Date Issued: 2004
The development of a baculovirus expression system for the production of Helicoverpa armigera stunt virus capsids for use in the encapsidation of foreign molecules
- Authors: Mosisili, Kekeletso Mpho Thakane
- Date: 2003
- Subjects: Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4088 , http://hdl.handle.net/10962/d1007700 , Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Description: The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.
- Full Text:
- Date Issued: 2003
The status of the American bollworm, Heliothis armigera (Hubner) (Lepidoptera : Noctuidae), on sunflower in the central Transvaal
- Authors: Von Maltitz, Emil Friedrich
- Date: 1992
- Subjects: Helicoverpa armigera , Noctuidae -- South Africa -- Transvaal , Noctuidae , Sunflowers -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5773 , http://hdl.handle.net/10962/d1005461 , Helicoverpa armigera , Noctuidae -- South Africa -- Transvaal , Noctuidae , Sunflowers -- Diseases and pests
- Description: Sunflower production in South Africa has increased four fold since the 1970's. This study was done to elucidate the pest status of Heliothis armigera (Hiibner) on sunflower. Field studies were undertaken at Warmbaths, Brits and Delmas during the summer seasons of 1988/89 to 1990/91. The infestations at the latter two areas were negligible throughout the study period and their statistics have not been included in this thesis. Plant development and oviposition by natural H. armigera populations were found to be correlated as, regardless of planting date, oviposition started at six to seven weeks after planting; when the flowering stage began. A peak in egg numbers was reached by the tenth week with an average for the three seasons at two eggs per plant. The eggs were laid singly on the bracts and bases of the flower buds. A peak in larval numbers was reached at the thirteenth week after planting with the average for the three seasons of 0,4 larvae per plant. The preferred feeding sites were on, between and under the bracts from where the larvae burrow into the pithy tissue of the receptacle. Only six percent of the larvae were found feeding directly on the achenes. Eggs and larvae collected were reared to determine the degree of parasitism. Overall, 19% of the eggs were parasitised; 18% by Telenomus ullyetti Nixon (Scelionidae) and one percent by Trichogrammatoidea lutea (Trichogrammatidae). Larval parasitism at Warmbaths was 23% in 1988/89, 27% in 1989/90 and 34% in 1990/91. Of the parasitised larvae, 44% succumbed to Palexorista prob. laxa (Tachinidae). The remainder were unidentified Braconidae and Ichneumonidae. Predators, such as chrysopids, were observed during the study but their effect on egg and larvae numbers was not studied in detail. A polyhedral virus occurred late in the seasons and caused mortality of the larvae. The low numbers of H. armigera on sunflower, the slight damage to the crop and the reasonably high rate of parasitism, all seem to indicate that H. armigera is not an economica1ly important pest of sunflower and that additional control methods are not justified.
- Full Text:
- Date Issued: 1992
Feeding by larvae of the American bollworm, Heliothis armigera (Hübner) (Lepidoptera: Noctuidae) on cotton plants
- Authors: Van der Walt, Susanna Johanna
- Date: 1988
- Subjects: Cotton -- Diseases and pests , Helicoverpa armigera , Lepidoptera
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5622 , http://hdl.handle.net/10962/d1004386 , Cotton -- Diseases and pests , Helicoverpa armigera , Lepidoptera
- Description: H. armigera larvae are a key stage for pest management in conmercial irrigated cotton crops in South Africa. Effective survey methods for detecting larval populations in the field require an understanding of the biology of the larvae, particularly their feeding habits. Their feeding is central to the development of pest threshold levels for the implementation of integrated control programmes. This applies to routine surveys for the larvae as well as to the damage they cause. Biological characteristics of the larvae are described with the emphasis on the identification of the larval instars, which were consistently five in number in both field and laboratory populations. The distribution of H. armigera larvae on cotton plants in the field was examined, but was found to more or less random; had there been a clear preference for any height zones or compass direction this would have been an obvious avenue for improving the survey methods currently in use. Details of field and laboratory investigations of the selection of feeding sites by the larvae are given. The study confirmed a clear preference by the larvae for cotton buds, flowers and bolls (in the thesis collectively called "fruiting forms"), over leaves. There were indications that the larvae selected flowers more readily than buds or bolls. This "preference", however, is shown to be of no practical value for refining survey methods. Damage levels to cotton due to B. armigera are discussed. Both direct losses and indirect losses due to the abortion of fruiting forms are considered. These criteria are inadequate since they do not take into account the ability of cotton plants to compensate for these losses. It is concluded that this compensation by cotton plants should be taken into account in further studies of the pest status of B. armigera.
- Full Text:
- Date Issued: 1988