Unravelling the replication biology of Providence virus in a cell culturebased model system
- Authors: Jarvie, Rachel Anne
- Date: 2020
- Subjects: Virology -- Research , RNA viruses , Viruses -- Reproduction , Providence virus
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142339 , vital:38071
- Description: There has been an increase in the number of viral outbreaks in the last decade; the majority of these are attributed to insect-human or animal-human transfer. Despite this awareness, there is limited understanding of the replication biology of the viruses causing the outbreaks and there are few model systems that are available to study RNA virus replication and viral persistence. In this study, we describe a Providence (PrV)-based model system to study virus replication biology. PrV is a single-stranded RNA virus that can cross Kingdom boundaries; it is capable of establishing a productive infection in insect and mammalian cell culture and it is also capable of replicating in plants. Only one other virus has been reported to infect a similar host range - the Nodavirus, Flock House virus (FHV). First, we performed a bioinformatic analysis of the PrV genome and validated the tools that were currently available to work with this model system in mammalian cells. Our data indicate that PrV infection of human cervical cancer (HeLa) cells results in the production of p130, p104/p40 and VCAP, albeit at low levels. While PrV replication in insect cells is associated with the Golgi apparatus and secretory vesicles, in HeLa cells, PrV replication is associated with the mitochondria. It is interesting to note that FHV replication factories are located on the outer mitochondrial membrane. In an attempt to study PrV virus replication in vitro, we adapted the BioID system reported by Roux et al. (2012). Here a promiscuous biotin ligase enzyme (BirA) was fused to a protein of interest and the expression of the fusion protein in mammalian cells resulted in the proximitybased biotinylation of proteins associated with the protein of interest. Using p40 as the protein of interest, we studied the fusion protein (BirA-p40) in transiently transfected HeLa cells and in a stable cell line, using western blot analysis and confocal microscopy. We faced challenges comparing the data collected using the two antibody-based detection techniques and the lack of BirA-p40 detection when using western analysis was attributed to the associated of p40 with detergent resistant membranes. BirA-p40 was subsequently expressed using in vitro coupled transcription/translation reactions, in the presence of excess biotin. While BirA-p40 was robustly expressed under these conditions, biotinylation of BirA-p40 was not detected. We attributed this to the conditions used in the experiments and given additional time, we would extend the duration of biotinylation, in vitro. PrV replication in mammalian cells was detectable using confocal microscopy however the levels of fluorescence were relatively low. The knowledge that p40 was associated with detergent resistant membranes led us to question the impact of detergent treatment of live cells on the detection of PrV replication. PrV-infected HeLa cells were treated with detergents with varying biochemical characteristics and the impact of these treatments on the detection of PrV replication were evaluated. We observed that linear and non-ionic detergents, namely NP-40 and Triton X-100, were most effective at enhancing the detection of viral replication in PrV-infected HeLa cells. Our data confirm that detergent treatment results in enhanced detection, and not enhanced PrV replication, in HeLa cells. Using the stable BirA-p40 expressing HeLa cell line, we showed that the protein is associated with membranes in vitro, and that the enhanced expression of BirA-p40 results in the formation of greater volumes of detergent-resistant membranes. In addition, detergent treatment of unfixed PrV-infected HeLa cells revealed the presence of the PrV p40 protein in the nucleoli of the cells. This is the first report of PrV proteins, which are translated in the cytosol of the mammalian cells, occurring in the nucleus. Our study has resulted in a deeper understanding of PrV replication in mammalian cell lines. A ‘simple RNA virus’ with only three predicted open reading frames has exhibited high levels of complexity within its elegant simplicity. This study has also highlighted the challenges associated with studying RNA virus replication biology in vitro. Looking forward, the identification of detergent-based enhancement for the detection of PrV replication provides the opportunity to perform more targeted PrV replication studies. The PrV-based model system can also be applied to the identification and analysis of potential broad-spectrum antiviral drugs in vitro. The latter application is particularly relevant considering the increase in the number of viral outbreaks over the last decade.
- Full Text:
- Date Issued: 2020
- Authors: Jarvie, Rachel Anne
- Date: 2020
- Subjects: Virology -- Research , RNA viruses , Viruses -- Reproduction , Providence virus
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/142339 , vital:38071
- Description: There has been an increase in the number of viral outbreaks in the last decade; the majority of these are attributed to insect-human or animal-human transfer. Despite this awareness, there is limited understanding of the replication biology of the viruses causing the outbreaks and there are few model systems that are available to study RNA virus replication and viral persistence. In this study, we describe a Providence (PrV)-based model system to study virus replication biology. PrV is a single-stranded RNA virus that can cross Kingdom boundaries; it is capable of establishing a productive infection in insect and mammalian cell culture and it is also capable of replicating in plants. Only one other virus has been reported to infect a similar host range - the Nodavirus, Flock House virus (FHV). First, we performed a bioinformatic analysis of the PrV genome and validated the tools that were currently available to work with this model system in mammalian cells. Our data indicate that PrV infection of human cervical cancer (HeLa) cells results in the production of p130, p104/p40 and VCAP, albeit at low levels. While PrV replication in insect cells is associated with the Golgi apparatus and secretory vesicles, in HeLa cells, PrV replication is associated with the mitochondria. It is interesting to note that FHV replication factories are located on the outer mitochondrial membrane. In an attempt to study PrV virus replication in vitro, we adapted the BioID system reported by Roux et al. (2012). Here a promiscuous biotin ligase enzyme (BirA) was fused to a protein of interest and the expression of the fusion protein in mammalian cells resulted in the proximitybased biotinylation of proteins associated with the protein of interest. Using p40 as the protein of interest, we studied the fusion protein (BirA-p40) in transiently transfected HeLa cells and in a stable cell line, using western blot analysis and confocal microscopy. We faced challenges comparing the data collected using the two antibody-based detection techniques and the lack of BirA-p40 detection when using western analysis was attributed to the associated of p40 with detergent resistant membranes. BirA-p40 was subsequently expressed using in vitro coupled transcription/translation reactions, in the presence of excess biotin. While BirA-p40 was robustly expressed under these conditions, biotinylation of BirA-p40 was not detected. We attributed this to the conditions used in the experiments and given additional time, we would extend the duration of biotinylation, in vitro. PrV replication in mammalian cells was detectable using confocal microscopy however the levels of fluorescence were relatively low. The knowledge that p40 was associated with detergent resistant membranes led us to question the impact of detergent treatment of live cells on the detection of PrV replication. PrV-infected HeLa cells were treated with detergents with varying biochemical characteristics and the impact of these treatments on the detection of PrV replication were evaluated. We observed that linear and non-ionic detergents, namely NP-40 and Triton X-100, were most effective at enhancing the detection of viral replication in PrV-infected HeLa cells. Our data confirm that detergent treatment results in enhanced detection, and not enhanced PrV replication, in HeLa cells. Using the stable BirA-p40 expressing HeLa cell line, we showed that the protein is associated with membranes in vitro, and that the enhanced expression of BirA-p40 results in the formation of greater volumes of detergent-resistant membranes. In addition, detergent treatment of unfixed PrV-infected HeLa cells revealed the presence of the PrV p40 protein in the nucleoli of the cells. This is the first report of PrV proteins, which are translated in the cytosol of the mammalian cells, occurring in the nucleus. Our study has resulted in a deeper understanding of PrV replication in mammalian cell lines. A ‘simple RNA virus’ with only three predicted open reading frames has exhibited high levels of complexity within its elegant simplicity. This study has also highlighted the challenges associated with studying RNA virus replication biology in vitro. Looking forward, the identification of detergent-based enhancement for the detection of PrV replication provides the opportunity to perform more targeted PrV replication studies. The PrV-based model system can also be applied to the identification and analysis of potential broad-spectrum antiviral drugs in vitro. The latter application is particularly relevant considering the increase in the number of viral outbreaks over the last decade.
- Full Text:
- Date Issued: 2020
Generation of polyclonal antibodies against Theiler's Murine Encephalomyelitis virus protein 2C, and their use in investigating localisation of the protein in infected cells
- Authors: Jauka, Tembisa Innocencia
- Date: 2010
- Subjects: Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3959 , http://hdl.handle.net/10962/d1004018 , Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Description: The Picornavirus family of positive sense RNA viruses includes some significant human and animal pathogens including Poliovirus (PV), Foot-and-Mouth disease virus (FMDV) and Human Rhinovirus (HRV). The genome is translated within the host cell into a polyprotein that is proteolytically cleaved into the structural and nonstructural proteins. The highly conserved, non-structural protein 2C has numerous roles during the virus life cycle and is essential for virus replication. Although the protein has been well studied in the case of PV, its interactions with the host cell during picornavirus infection is poorly understood. Theiler’s Encephalomyelitis virus (TMEV) is a picornavirus that infects mice, and is being used in our laboratory as a model in which to study the 2C protein. In this study, polyclonal antibodies against the TMEV 2C protein were generated and used to localise the protein in infected cells by indirect immunofluorescence. To produce antigen for immunisation purposes, the TMEV-2C protein sequence was analysed to identify hydrophilic and antigenic regions. An internal region of the 2C representing amino acid residues 31-210 was selected, expressed in bacteria and purified by nickel NTA affinity chromatography. Time course analysis of 2C (31-210) showed that the peptide was maximally expressed at 5 hours post induction. The peptide was solubilised using a mild detergent and 1.5 mg of purified antigen was used for immunisation of rabbits. Western blot analysis confirmed that the antibodies could detect both bacteriallyexpressed antigen, and virally-expressed 2C. Examination of virus-infected baby hamster kidney cells by immunofluorescence and confocal microscopy using the antiserum (anti-TMEV 2C antibodies) showed that the protein had a diffuse distribution upon early infection and at later stages it was located in a large perinuclear structure representing the viral replication complex. Furthermore, 2C localised to the Golgi apparatus as revealed by dual-label immunofluorescence using anti-TMEV 2C antibodies and wheat germ agglutinin (WGA). Furthermore, it was shown that TMEV infection results in changes in cell morphology and a redistribution of the cytoskeletal protein, β-actin. The successful production of antibodies that recognise TMEV 2C opens the way for further studies to investigate interactions between 2C and hostencoded factors.
- Full Text:
- Date Issued: 2010
- Authors: Jauka, Tembisa Innocencia
- Date: 2010
- Subjects: Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3959 , http://hdl.handle.net/10962/d1004018 , Picornaviruses , RNA viruses , Immunoglobulins , Encephalomyelitis
- Description: The Picornavirus family of positive sense RNA viruses includes some significant human and animal pathogens including Poliovirus (PV), Foot-and-Mouth disease virus (FMDV) and Human Rhinovirus (HRV). The genome is translated within the host cell into a polyprotein that is proteolytically cleaved into the structural and nonstructural proteins. The highly conserved, non-structural protein 2C has numerous roles during the virus life cycle and is essential for virus replication. Although the protein has been well studied in the case of PV, its interactions with the host cell during picornavirus infection is poorly understood. Theiler’s Encephalomyelitis virus (TMEV) is a picornavirus that infects mice, and is being used in our laboratory as a model in which to study the 2C protein. In this study, polyclonal antibodies against the TMEV 2C protein were generated and used to localise the protein in infected cells by indirect immunofluorescence. To produce antigen for immunisation purposes, the TMEV-2C protein sequence was analysed to identify hydrophilic and antigenic regions. An internal region of the 2C representing amino acid residues 31-210 was selected, expressed in bacteria and purified by nickel NTA affinity chromatography. Time course analysis of 2C (31-210) showed that the peptide was maximally expressed at 5 hours post induction. The peptide was solubilised using a mild detergent and 1.5 mg of purified antigen was used for immunisation of rabbits. Western blot analysis confirmed that the antibodies could detect both bacteriallyexpressed antigen, and virally-expressed 2C. Examination of virus-infected baby hamster kidney cells by immunofluorescence and confocal microscopy using the antiserum (anti-TMEV 2C antibodies) showed that the protein had a diffuse distribution upon early infection and at later stages it was located in a large perinuclear structure representing the viral replication complex. Furthermore, 2C localised to the Golgi apparatus as revealed by dual-label immunofluorescence using anti-TMEV 2C antibodies and wheat germ agglutinin (WGA). Furthermore, it was shown that TMEV infection results in changes in cell morphology and a redistribution of the cytoskeletal protein, β-actin. The successful production of antibodies that recognise TMEV 2C opens the way for further studies to investigate interactions between 2C and hostencoded factors.
- Full Text:
- Date Issued: 2010
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
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:
- Date Issued: 2005
- 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:
- Date Issued: 2005
- «
- ‹
- 1
- ›
- »