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
- 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
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
- 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
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