Heterologous expression of the helicoverpa armigera stunt virus in Saccharomyces cerevisiae
- Authors: Venter, Philip Arno
- Date: 2002
- Subjects: Helicoverpa armigera Saccharomyces cerevisiae
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3895 , http://hdl.handle.net/10962/d1003811
- Description: Lepidopteran insects like Helicoverpa armigera, more commonly known as the cotton bollworm, are economically important pests of a wide variety of crops throughout the world. The Helicoverpa armigera stunt virus (HaSV), a tetravirus with a bipartite single-stranded positive-sense RNA genome, has great potential as a biological pesticide against H. armigera. The larger genomic strand of this virus (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71 kDa capsid protein precursor (p71). 240 copies of p71 assemble into a procapsid with the concomitant encapsidation of the viral RNA. This is followed by a complex maturation event that is characterized by the autoproteolytic cleavage of p71 into the 64 kDa capsid protein (P64) and a 7 kDa peptide (p7). The rearrangements that occur during maturation results in the formation of mature HaSV capsids that can thereupon deliver RNA to other susceptible host cells. The principal objective of the research described in this study was to demonstrate that this virus could be assembled in Saccharomyces cerevisiae. S. cerevisiae expression vectors were constructed for the production of p71. This protein was detected in cell lysates from two different strains of S. cerevisiae, both containing either chromosomal or episomal copies of an expression cassette for P71. A number of factors relating to the expression of P71 (e.g. strains used, expression loci and expression rate) and the preparation of protein extracts from S. cerevisiae (e.g. the presence of various protease inhibitors and salt concentrations) were examined to attain optimal levels of soluble p71. A small fraction of the optimized soluble p71 was shown to be in the form of virus-like particles (VLPs), with a yield of ≤10⁷ VLPs from a 1.5l culture of P71⁺ cells. These particles were exclusively in the procapsid form, had a similar buoyant density to that of wild-type HaSV and could undergo maturation when the pH was reduced to 5. S. cerevisiae vectors were constructed for the episomal expression of the HaSV genomic RNAs. These vectors directed the transcription of RNA1 and RNA2 transcripts, which had similar sizes to those of the HaSV genomic RNAs. Mature HaSV particles were purified from cells, transgenic for P71, RNA1 and RNA2, by way of two different virus purification protocols that were developed during this study. RT-PCR analyses on RNA-extracts from these particles demonstrated that RNA transcripts, which were produced in trans with p71, could be encapsidated by HaSV capsids in S. cerevisiae. A droplet-feed bioassay on H. armigera larvae demonstrated that the S. cerevisiae-derived HaSV particles caused impaired larval development. This response was correlated with the detection of HaSV RNA2 in RNA extractions from larvae that were used in this bioassay. The results that were generated through the course of this study, provided proof for the concept of the non-host production of infectious HaSV particles from S. cerevisiae. This work could serve as a foundation for future research on the development of an expression system for the large-scale production of this virus as a biopesticide.
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- Authors: Venter, Philip Arno
- Date: 2002
- Subjects: Helicoverpa armigera Saccharomyces cerevisiae
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3895 , http://hdl.handle.net/10962/d1003811
- Description: Lepidopteran insects like Helicoverpa armigera, more commonly known as the cotton bollworm, are economically important pests of a wide variety of crops throughout the world. The Helicoverpa armigera stunt virus (HaSV), a tetravirus with a bipartite single-stranded positive-sense RNA genome, has great potential as a biological pesticide against H. armigera. The larger genomic strand of this virus (RNA1) encodes the viral replicase, while the other (RNA2) encodes the 71 kDa capsid protein precursor (p71). 240 copies of p71 assemble into a procapsid with the concomitant encapsidation of the viral RNA. This is followed by a complex maturation event that is characterized by the autoproteolytic cleavage of p71 into the 64 kDa capsid protein (P64) and a 7 kDa peptide (p7). The rearrangements that occur during maturation results in the formation of mature HaSV capsids that can thereupon deliver RNA to other susceptible host cells. The principal objective of the research described in this study was to demonstrate that this virus could be assembled in Saccharomyces cerevisiae. S. cerevisiae expression vectors were constructed for the production of p71. This protein was detected in cell lysates from two different strains of S. cerevisiae, both containing either chromosomal or episomal copies of an expression cassette for P71. A number of factors relating to the expression of P71 (e.g. strains used, expression loci and expression rate) and the preparation of protein extracts from S. cerevisiae (e.g. the presence of various protease inhibitors and salt concentrations) were examined to attain optimal levels of soluble p71. A small fraction of the optimized soluble p71 was shown to be in the form of virus-like particles (VLPs), with a yield of ≤10⁷ VLPs from a 1.5l culture of P71⁺ cells. These particles were exclusively in the procapsid form, had a similar buoyant density to that of wild-type HaSV and could undergo maturation when the pH was reduced to 5. S. cerevisiae vectors were constructed for the episomal expression of the HaSV genomic RNAs. These vectors directed the transcription of RNA1 and RNA2 transcripts, which had similar sizes to those of the HaSV genomic RNAs. Mature HaSV particles were purified from cells, transgenic for P71, RNA1 and RNA2, by way of two different virus purification protocols that were developed during this study. RT-PCR analyses on RNA-extracts from these particles demonstrated that RNA transcripts, which were produced in trans with p71, could be encapsidated by HaSV capsids in S. cerevisiae. A droplet-feed bioassay on H. armigera larvae demonstrated that the S. cerevisiae-derived HaSV particles caused impaired larval development. This response was correlated with the detection of HaSV RNA2 in RNA extractions from larvae that were used in this bioassay. The results that were generated through the course of this study, provided proof for the concept of the non-host production of infectious HaSV particles from S. cerevisiae. This work could serve as a foundation for future research on the development of an expression system for the large-scale production of this virus as a biopesticide.
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Assembly of full-length cDNA, and heterologous expression, of Nudaurelia B virus RNA
- Authors: Luke, Gary Joseph
- Date: 2001
- Subjects: Imbrasia cytherea , RNA , Viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3913 , http://hdl.handle.net/10962/d1003972 , Imbrasia cytherea , RNA , Viruses , DNA
- Description: Nudaurelia beta virus (NβV) is a monopartite genome virus belonging to the family Tetraviridae. Its host range has been found to be limited to a single insect order, the Lepidoptera (moths and butterflies). The single-stranded positive-sense RNA genome consists of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerase. The 3' proximal ORF, of 1836 nucleotides, encodes the 66 kDa capsid precursor protein and overlaps the replicase gene by more than 99% and is in the +1 reading frame relative to the replicase reading frame. The full-length cDNA construct of the NβV genome was assembled using a homologous overlapping PCR linking method. The starting material consisted of seven overlapping pieces that were constructed for sequencing. Due to the degradation of the full-length RNA obtained from virus extracted from field-collected Nudaurelia cytherea capensis larvae other alternative methods needed to be applied. Sub-cloning using restriction enzyme sites also required an alternative method being used, due to the abundance of restriction sites of the same type in the NβV genome. This led to the use of a method similar to "DNA Shuffling" where overlapping pieces were connected using a modified PCR protocol. After the construction of the NβV genome, the full-length PCR product was cloned and checked for large insertion and deletions that could have resulted from the PCR amplification. The heterologous expression of the NβV capsid protein linked to a fusion protein (Glutathione S-transferase) in E.coli, confirmed the authenticity of the prescribed capsid gene ORF. The expression showed that the virus protein was subjected to protease digestion in DH5α E.coli, suggesting that the protein was insoluble in the cell cytoplasm. The capsid gene expression in a modified E.coli strain, Epicurian Coli BL21-CodonPlus (DE3)-RIL, resulted in high levels of the correct molecular weight protein with minimal degradation. The modified strain was designed for over-expression of eukaryotic protein with lowered protease activity. The above results have opened the way for further research that would yield valuable insight into the molecular biology and replication strategy of the NβV in cell cultures.
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- Authors: Luke, Gary Joseph
- Date: 2001
- Subjects: Imbrasia cytherea , RNA , Viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3913 , http://hdl.handle.net/10962/d1003972 , Imbrasia cytherea , RNA , Viruses , DNA
- Description: Nudaurelia beta virus (NβV) is a monopartite genome virus belonging to the family Tetraviridae. Its host range has been found to be limited to a single insect order, the Lepidoptera (moths and butterflies). The single-stranded positive-sense RNA genome consists of 6625 nucleotides containing two open reading frames (ORFs). The 5' proximal ORF of 5778 nucleotides encodes a protein of 215 kDa containing three functional domains characteristic of RNA-dependent RNA polymerase. The 3' proximal ORF, of 1836 nucleotides, encodes the 66 kDa capsid precursor protein and overlaps the replicase gene by more than 99% and is in the +1 reading frame relative to the replicase reading frame. The full-length cDNA construct of the NβV genome was assembled using a homologous overlapping PCR linking method. The starting material consisted of seven overlapping pieces that were constructed for sequencing. Due to the degradation of the full-length RNA obtained from virus extracted from field-collected Nudaurelia cytherea capensis larvae other alternative methods needed to be applied. Sub-cloning using restriction enzyme sites also required an alternative method being used, due to the abundance of restriction sites of the same type in the NβV genome. This led to the use of a method similar to "DNA Shuffling" where overlapping pieces were connected using a modified PCR protocol. After the construction of the NβV genome, the full-length PCR product was cloned and checked for large insertion and deletions that could have resulted from the PCR amplification. The heterologous expression of the NβV capsid protein linked to a fusion protein (Glutathione S-transferase) in E.coli, confirmed the authenticity of the prescribed capsid gene ORF. The expression showed that the virus protein was subjected to protease digestion in DH5α E.coli, suggesting that the protein was insoluble in the cell cytoplasm. The capsid gene expression in a modified E.coli strain, Epicurian Coli BL21-CodonPlus (DE3)-RIL, resulted in high levels of the correct molecular weight protein with minimal degradation. The modified strain was designed for over-expression of eukaryotic protein with lowered protease activity. The above results have opened the way for further research that would yield valuable insight into the molecular biology and replication strategy of the NβV in cell cultures.
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Characterisation of the genome of Nudaurelia Omega Virus
- Authors: Cox, Dermot
- Date: 1995
- Subjects: Imbrasia cytherea , RNA , Insects -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4023 , http://hdl.handle.net/10962/d1004083 , Imbrasia cytherea , RNA , Insects -- Viruses
- Description: Nudaurelia co virus (Nco V) is a small RNA virus belonging to the Family Tetraviridae. Nco V was successfully isolated from field collected larvae of the pine emperor moth, Nudaurelia cytherea capensis. By polyacrylamide gel electrophoresis-it was possible Jo determine the size of the capsid proteins. Anti-NcoV antiserum was raised by inoculating a rabbit with purified virus. RNA was extracted from the purified virus using a phenol\chloroform extraction procedure. It was possible to separate the viral RNA into its constituent species using sucrose density gradient centrifugation. The sizes of both species of RNA was accurately determined by agarose gel electrophoresis. These sizes corresponded to the replicative form of the RNA which was extracted from infected host tissue. The absence of a poly(A) tract on the RNA was shown through poly(U) sepharose chromatography. Cell-free translation of the viral RNA elucidated the sizes of proteins encoded in vitro in a rabbit reticulocyte lysate system. Optimal conditions for in vitro translation of Nco V were determined for a range of conditions. Immunoprecipitaion of viral encoded proteins with anti-Nco V antiserum suggested that the putative coat protein of the virus was encoded by RNA 2, as a precursor polypeptide which underwent posttranslational cleavage. Reverse transcription - polymerase chain reaction (RT -PCR) was used to successfully produce a radiolabelled probe which could detect dot-blotted viral RNA. The efficacy of this probe in detecting the presence of Nco V RNA in infected tissue was also tested.
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- Authors: Cox, Dermot
- Date: 1995
- Subjects: Imbrasia cytherea , RNA , Insects -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4023 , http://hdl.handle.net/10962/d1004083 , Imbrasia cytherea , RNA , Insects -- Viruses
- Description: Nudaurelia co virus (Nco V) is a small RNA virus belonging to the Family Tetraviridae. Nco V was successfully isolated from field collected larvae of the pine emperor moth, Nudaurelia cytherea capensis. By polyacrylamide gel electrophoresis-it was possible Jo determine the size of the capsid proteins. Anti-NcoV antiserum was raised by inoculating a rabbit with purified virus. RNA was extracted from the purified virus using a phenol\chloroform extraction procedure. It was possible to separate the viral RNA into its constituent species using sucrose density gradient centrifugation. The sizes of both species of RNA was accurately determined by agarose gel electrophoresis. These sizes corresponded to the replicative form of the RNA which was extracted from infected host tissue. The absence of a poly(A) tract on the RNA was shown through poly(U) sepharose chromatography. Cell-free translation of the viral RNA elucidated the sizes of proteins encoded in vitro in a rabbit reticulocyte lysate system. Optimal conditions for in vitro translation of Nco V were determined for a range of conditions. Immunoprecipitaion of viral encoded proteins with anti-Nco V antiserum suggested that the putative coat protein of the virus was encoded by RNA 2, as a precursor polypeptide which underwent posttranslational cleavage. Reverse transcription - polymerase chain reaction (RT -PCR) was used to successfully produce a radiolabelled probe which could detect dot-blotted viral RNA. The efficacy of this probe in detecting the presence of Nco V RNA in infected tissue was also tested.
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Cleavage of the precursor coat protein of black beetle virus strain w17 in rabbit reticulocyte lysate
- Authors: Blackhurst, Diane Mary
- Date: 1988
- Subjects: Beetles , Insects -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3880 , http://hdl.handle.net/10962/d1001614
- Description: Black beetle virus (BBV) is a bipartite single-stranded RNA virus belonging to the family Nodaviridae. Its host range has been found to be limited to insects. RNA 1, the larger of the two RNA molecules, with a MW of 1,15 x 10⁶ and the smaller RNA 2 with a MW of 0,46 x 10⁶, are both packaged in the same virus particle. The two RNA molecules are translated separately, with RNA 1 coding for protein A of MW 105 x 10³ and RNA 2 coding for protein α of MW 47 x 10³. Protein α is the major capsid protein precursor, which during in vivo maturation is cleaved to form the coat protein β of MW 43 x 10³, and protein γ of MW 5 x 10³. Cell-free translation of BBV (strain W17) mRNA was carried out in rabbit reticulocyte lysates. Protein α was detectable between 0 and 30 minutes after RNA addition. A protein 'β', which was found to co-electrophorese on polyacrylamide gels with authentic β and which was immunoprecipitated by anti-BBV antiserum, was detectable after 30 minutes. Results of this work show that the formation of 'β' could be prevented by the addition of RNase to the lysate, indicating that intact RNA is necessary for α to β cleavage. Arresting protein synthesis by the addition of cycloheximide to the lysate mix did not inhibit the cleavage. The formation of β could also be prevented by cooling the lysate mix to 1°C. Cleavage of α to β still occurred when RNA 2, without the presence of RNA 1, was translated. Therefore the cleavage is not dependent on a translation product of RNA 1. Sedimentation of lysate on sucrose density gradients showed that α to β cleavage was not accompanied by assembly of BBV RNA and protein lnto a viral substructure as has been shown to occur with some viruses, for example certain picornaviruses. Serial dilution of lysate containing α showed that the level of β decreased with increasing dilution, indicating that the cleavage is not mediated by autocatalysis, but by some other unknown factor. Although much work has been carried out on black beetle virus, no work has been published to date concerning α to β cleavage as an indication of assembly in rabbit reticulocyte lysates. Results of these cell-free translation experiments thus indicate that BBV coat protein precursor α, in association with its messenger RNA 2, undergoes a maturation cleavage in the lysate to produce BBV coat protein β. In addition, this cleavage seems to occur without assembly into any intermediate viral structure
- Full Text:
- Authors: Blackhurst, Diane Mary
- Date: 1988
- Subjects: Beetles , Insects -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3880 , http://hdl.handle.net/10962/d1001614
- Description: Black beetle virus (BBV) is a bipartite single-stranded RNA virus belonging to the family Nodaviridae. Its host range has been found to be limited to insects. RNA 1, the larger of the two RNA molecules, with a MW of 1,15 x 10⁶ and the smaller RNA 2 with a MW of 0,46 x 10⁶, are both packaged in the same virus particle. The two RNA molecules are translated separately, with RNA 1 coding for protein A of MW 105 x 10³ and RNA 2 coding for protein α of MW 47 x 10³. Protein α is the major capsid protein precursor, which during in vivo maturation is cleaved to form the coat protein β of MW 43 x 10³, and protein γ of MW 5 x 10³. Cell-free translation of BBV (strain W17) mRNA was carried out in rabbit reticulocyte lysates. Protein α was detectable between 0 and 30 minutes after RNA addition. A protein 'β', which was found to co-electrophorese on polyacrylamide gels with authentic β and which was immunoprecipitated by anti-BBV antiserum, was detectable after 30 minutes. Results of this work show that the formation of 'β' could be prevented by the addition of RNase to the lysate, indicating that intact RNA is necessary for α to β cleavage. Arresting protein synthesis by the addition of cycloheximide to the lysate mix did not inhibit the cleavage. The formation of β could also be prevented by cooling the lysate mix to 1°C. Cleavage of α to β still occurred when RNA 2, without the presence of RNA 1, was translated. Therefore the cleavage is not dependent on a translation product of RNA 1. Sedimentation of lysate on sucrose density gradients showed that α to β cleavage was not accompanied by assembly of BBV RNA and protein lnto a viral substructure as has been shown to occur with some viruses, for example certain picornaviruses. Serial dilution of lysate containing α showed that the level of β decreased with increasing dilution, indicating that the cleavage is not mediated by autocatalysis, but by some other unknown factor. Although much work has been carried out on black beetle virus, no work has been published to date concerning α to β cleavage as an indication of assembly in rabbit reticulocyte lysates. Results of these cell-free translation experiments thus indicate that BBV coat protein precursor α, in association with its messenger RNA 2, undergoes a maturation cleavage in the lysate to produce BBV coat protein β. In addition, this cleavage seems to occur without assembly into any intermediate viral structure
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