Exploring the structural integrity of a picornavirus capsid
- Authors: Upfold, Nicole Sarah
- Date: 2020
- Subjects: Picornaviruses , Immunoglobulins , Capsids (Virology) , Viruses Morphology , RNA viruses
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/131837 , vital:36758 , DOI https://doi.org/10.21504/10962/131837
- Description: Picornaviruses are a diverse family of small RNA viruses that cause a broad range of human and veterinary diseases. Despite decades of research into the molecular biology of these pathogens, no antivirals and few vaccines are commercially available for the treatment and prevention of picornavirus infections. The capsids of these non-enveloped viruses are involved in many important aspects of the picornavirus lifecycle, such as cell attachment and entry, uncoating, and protection of the viral RNA. Although the structures of many picornavirus capsids have been solved, a broader understanding of the molecular determinants that are required for structural integrity and stability is imperative for an improved understanding of the basic biology of these viruses, and for designing effective control strategies. Collectively, this thesis aims to elucidate the molecular determinants of structural stability and integrity in the Theiler’s murine encephalomyelitis virus capsid (TMEV). To study the TMEV GDVII capsid using biochemical techniques, neutralising polyclonal antibodies were generated against GDVII particles. The antibodies recognised linear epitopes in the C-terminus of the VP1 protein, but not those present in VP2 or VP3. The VP1 C-terminal residues were mapped to a loop above the putative receptor binding pit on the capsid surface, which prompted an investigation into the potential binding site of the TMEV co-receptor, heparan sulphate. Molecular docking revealed that heparan interacts with residues of the receptor binding pocket, as well as residues of the adjoining VP1 C-terminal loop. These findings suggest that the antibodies neutralise virus infection by preventing attachment of the virus to the co-receptor and possibly the unknown primary receptor. Few studies have identified the specific residues and interactions at subunit interfaces that significantly contribute to picornavirus capsid stability, assembly, and function. A novel in-silico screen was developed for the prediction of hotspot residues at protein-protein interfaces of a virus capsid. This screen can be applied to elucidate the residues that contribute significantly to the intraprotomer, interprotomer and interpentamer interfaces of any picornavirus capsid, on condition that the structure of the virus is available. The screen was applied to TMEV GDVII resulting in the identification of hotspots, several of which correspond to residues that are known to be important for aspects of the virus lifecycle, such as those that contribute to pH stability or form part of receptor binding sites. This observation suggests that residues involved in specific capsid functions may also play a role in capsid stability. Many of the residues identified as hotspots in TMEV corresponded to those required for assembly, uncoating, and virus growth in representative picornaviruses from various genera, suggesting that the residues that regulate capsid stability may be somewhat conserved across the family. Hotspots identified at the interpentamer interfaces of TMEV were individually substituted to alanine to further explore their importance to the TMEV lifecycle. All the amino acid substitutions prevented completion of the virus lifecycle as no CPE was observed following transfection of susceptible cells. Immunofluorescence experiments demonstrated that virus protein synthesis and RNA replication were not inhibited by substitution of the hotspot residues, but that infectivity was severely impeded. This confirmed that the residues were required for some aspect of the virus lifecycle, such as capsid assembly, or were critical for maintaining the conformational stability of the TMEV particles. Virus capsids become unstable and are prone to dissociation under certain conditions such as extreme pH and non-physiological temperatures. The thermostability of TMEV was explored by selecting GDVII virions with improved thermal tolerance through serial passage and heat exposure. Thermostable virions that could tolerate temperatures above 57 °C had reduced infective titres compared to the wild type TMEV suggesting that the virus adapted to thermal stress at the expense of viral fitness. Sequencing the capsid encoding regions of the mutant virions revealed a pair of amino acid substitutions that were present in all mutants. Additional substitutions that were unique to viruses selected at different temperatures were also identified. Most of the substitutions were located within the intraprotomer interfaces of the virus, unlike previous studies on enteroviruses where mutations were mostly localised to the receptor binding pocket. This thesis provides the first analysis of the structural determinants of TMEV capsid stability. The generation of tools to further explore the capsid structures of TMEV and other picornaviruses provides an opportunity for future studies which may contribute to the development of novel control strategies against this important family of viruses. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text: false
- Date Issued: 2020
- Authors: Upfold, Nicole Sarah
- Date: 2020
- Subjects: Picornaviruses , Immunoglobulins , Capsids (Virology) , Viruses Morphology , RNA viruses
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/131837 , vital:36758 , DOI https://doi.org/10.21504/10962/131837
- Description: Picornaviruses are a diverse family of small RNA viruses that cause a broad range of human and veterinary diseases. Despite decades of research into the molecular biology of these pathogens, no antivirals and few vaccines are commercially available for the treatment and prevention of picornavirus infections. The capsids of these non-enveloped viruses are involved in many important aspects of the picornavirus lifecycle, such as cell attachment and entry, uncoating, and protection of the viral RNA. Although the structures of many picornavirus capsids have been solved, a broader understanding of the molecular determinants that are required for structural integrity and stability is imperative for an improved understanding of the basic biology of these viruses, and for designing effective control strategies. Collectively, this thesis aims to elucidate the molecular determinants of structural stability and integrity in the Theiler’s murine encephalomyelitis virus capsid (TMEV). To study the TMEV GDVII capsid using biochemical techniques, neutralising polyclonal antibodies were generated against GDVII particles. The antibodies recognised linear epitopes in the C-terminus of the VP1 protein, but not those present in VP2 or VP3. The VP1 C-terminal residues were mapped to a loop above the putative receptor binding pit on the capsid surface, which prompted an investigation into the potential binding site of the TMEV co-receptor, heparan sulphate. Molecular docking revealed that heparan interacts with residues of the receptor binding pocket, as well as residues of the adjoining VP1 C-terminal loop. These findings suggest that the antibodies neutralise virus infection by preventing attachment of the virus to the co-receptor and possibly the unknown primary receptor. Few studies have identified the specific residues and interactions at subunit interfaces that significantly contribute to picornavirus capsid stability, assembly, and function. A novel in-silico screen was developed for the prediction of hotspot residues at protein-protein interfaces of a virus capsid. This screen can be applied to elucidate the residues that contribute significantly to the intraprotomer, interprotomer and interpentamer interfaces of any picornavirus capsid, on condition that the structure of the virus is available. The screen was applied to TMEV GDVII resulting in the identification of hotspots, several of which correspond to residues that are known to be important for aspects of the virus lifecycle, such as those that contribute to pH stability or form part of receptor binding sites. This observation suggests that residues involved in specific capsid functions may also play a role in capsid stability. Many of the residues identified as hotspots in TMEV corresponded to those required for assembly, uncoating, and virus growth in representative picornaviruses from various genera, suggesting that the residues that regulate capsid stability may be somewhat conserved across the family. Hotspots identified at the interpentamer interfaces of TMEV were individually substituted to alanine to further explore their importance to the TMEV lifecycle. All the amino acid substitutions prevented completion of the virus lifecycle as no CPE was observed following transfection of susceptible cells. Immunofluorescence experiments demonstrated that virus protein synthesis and RNA replication were not inhibited by substitution of the hotspot residues, but that infectivity was severely impeded. This confirmed that the residues were required for some aspect of the virus lifecycle, such as capsid assembly, or were critical for maintaining the conformational stability of the TMEV particles. Virus capsids become unstable and are prone to dissociation under certain conditions such as extreme pH and non-physiological temperatures. The thermostability of TMEV was explored by selecting GDVII virions with improved thermal tolerance through serial passage and heat exposure. Thermostable virions that could tolerate temperatures above 57 °C had reduced infective titres compared to the wild type TMEV suggesting that the virus adapted to thermal stress at the expense of viral fitness. Sequencing the capsid encoding regions of the mutant virions revealed a pair of amino acid substitutions that were present in all mutants. Additional substitutions that were unique to viruses selected at different temperatures were also identified. Most of the substitutions were located within the intraprotomer interfaces of the virus, unlike previous studies on enteroviruses where mutations were mostly localised to the receptor binding pocket. This thesis provides the first analysis of the structural determinants of TMEV capsid stability. The generation of tools to further explore the capsid structures of TMEV and other picornaviruses provides an opportunity for future studies which may contribute to the development of novel control strategies against this important family of viruses. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
- Full Text: false
- Date Issued: 2020
Market participation and welfare of smallholder farmers in the Eastern Cape Province South Africa
- Lesala, Mahali Elizabeth https://orcid.org/0000-0001-9921-2190
- Authors: Lesala, Mahali Elizabeth https://orcid.org/0000-0001-9921-2190
- Date: 2021-06
- Subjects: Farms, Small , Economic development -- South Africa , Agriculture -- Economic aspects -- South Africa
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/20916 , vital:46745
- Description: The low market participation of smallholder farmers in markets has received enormous attention from scholars, both in the country and the Eastern Cape Province. However, it is not clear how low their market participation is including its implications on farmer’s welfare. The purpose of the study was to determine the extent to which smallholder farmers in the homelands of the Eastern Cape participate in output markets and assess how their participation in markets has affected wellbeing of their households. This information will have important practical implications for policy regarding appropriate pathways for poverty alleviation and livelihoods improvements in the rural areas of the Eastern Cape Province. Three irrigation schemes; Qamata, Zanyokwe and Tyefu irrigation schemes were selected for this study. A sample of 210 smallholder irrigators were interviewed by means of a close-ended questionnaire. The data were analysed by means of descriptive statistical tools, the multiple-level choice models and the Propensity Score Matching (PSM) technique. SPSS and STATA computer programmes were used to carry out all the estimations. The analysis established that, although agriculture is the primary activity for rural livelihoods, it is not the main contributor to family income. Rather, remittances and social grants were the dominant sources of household income in the Qamata, Zanyokwe and Tyefu areas. From the standpoint of market, maize and potatoes are the most popular crops, but potatoes dominate the market. This result confirms that maize is the staple crop and therefore mostly grown for home consumption while production of potatoes is market-oriented. The Market Participation Index (MPI) revealed that farmers sell at least 55 percent of their farm produce, implying that farmers have made some transition from subsistence to semi-commercial farming. However, farmers’ priority still remains food self-sufficiency and market participation only takes place after satisfying their home food needs. The results revealed that the significant factors influencing the farmers’ decisions and their extent of participation in output markets were the age, gender, marital status of the household head, primary occupation of household head, size of farm cultivated, government financial support, access to extension services and farmer’s membership of cooperatives. Concerning the impact of output market participation on welfare of smallholders, the Average Treatment on the Treated (ATT) as the measure of change revealed that participation in output markets has a positive impact on welfare of the smallholder farmers through increased incomes. Farmers who participated in output market were at least R838.44 better off than those who did not participate in markets although social grants and remittances made significantly higher contribution to household welfare. The study suggests that despite some improvements in income of market participants, the standards of living of the rural households are still far from what would be considered optimal. Crop farming evidently contributes less than desired, hence the persistence of the widespread poverty. It is urgent to focus interventions on improving agricultural productivity while widening strategies for improving rural livelihoods beyond agriculture to diversify the choices open to rural dwellers. , Thesis (PhD) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-06
- Authors: Lesala, Mahali Elizabeth https://orcid.org/0000-0001-9921-2190
- Date: 2021-06
- Subjects: Farms, Small , Economic development -- South Africa , Agriculture -- Economic aspects -- South Africa
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/20916 , vital:46745
- Description: The low market participation of smallholder farmers in markets has received enormous attention from scholars, both in the country and the Eastern Cape Province. However, it is not clear how low their market participation is including its implications on farmer’s welfare. The purpose of the study was to determine the extent to which smallholder farmers in the homelands of the Eastern Cape participate in output markets and assess how their participation in markets has affected wellbeing of their households. This information will have important practical implications for policy regarding appropriate pathways for poverty alleviation and livelihoods improvements in the rural areas of the Eastern Cape Province. Three irrigation schemes; Qamata, Zanyokwe and Tyefu irrigation schemes were selected for this study. A sample of 210 smallholder irrigators were interviewed by means of a close-ended questionnaire. The data were analysed by means of descriptive statistical tools, the multiple-level choice models and the Propensity Score Matching (PSM) technique. SPSS and STATA computer programmes were used to carry out all the estimations. The analysis established that, although agriculture is the primary activity for rural livelihoods, it is not the main contributor to family income. Rather, remittances and social grants were the dominant sources of household income in the Qamata, Zanyokwe and Tyefu areas. From the standpoint of market, maize and potatoes are the most popular crops, but potatoes dominate the market. This result confirms that maize is the staple crop and therefore mostly grown for home consumption while production of potatoes is market-oriented. The Market Participation Index (MPI) revealed that farmers sell at least 55 percent of their farm produce, implying that farmers have made some transition from subsistence to semi-commercial farming. However, farmers’ priority still remains food self-sufficiency and market participation only takes place after satisfying their home food needs. The results revealed that the significant factors influencing the farmers’ decisions and their extent of participation in output markets were the age, gender, marital status of the household head, primary occupation of household head, size of farm cultivated, government financial support, access to extension services and farmer’s membership of cooperatives. Concerning the impact of output market participation on welfare of smallholders, the Average Treatment on the Treated (ATT) as the measure of change revealed that participation in output markets has a positive impact on welfare of the smallholder farmers through increased incomes. Farmers who participated in output market were at least R838.44 better off than those who did not participate in markets although social grants and remittances made significantly higher contribution to household welfare. The study suggests that despite some improvements in income of market participants, the standards of living of the rural households are still far from what would be considered optimal. Crop farming evidently contributes less than desired, hence the persistence of the widespread poverty. It is urgent to focus interventions on improving agricultural productivity while widening strategies for improving rural livelihoods beyond agriculture to diversify the choices open to rural dwellers. , Thesis (PhD) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-06
- «
- ‹
- 1
- ›
- »