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:
- 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:
Development of experimental systems for studying the biology of Nudaurelia capensis ß virus
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
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