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:
- 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:
Synthesis, characterisation and evaluation of ferrocene-containing Novobiocin analogues for anticancer and antiplasmodial activity through inhibition of Hsp90
- Authors: Mbaba, Mziyanda
- Date: 2017
- Subjects: Antibiotics Synthesis , Ferrocene , Heat shock proteins , Antimalarials , Cancer Chemotherapy
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65111 , vital:28690
- Description: Novobiocin (Nb) is a coumarin type antibiotic isolated from the bacterium species of Streptomyces and possesses modest anticancer and antimalarial activities. Nb and analogues have been extensively explored as potential anticancer agents through inhibition of the C- terminal domain of heat shock protein 90 (Hsp90), which plays a pivotal role in the proteinfolding machinery of cells. There has been little effort in the exploration of Nb and derivatives for antimalarial activity. Incorporation of organometallic units, such as ferrocene (Fc), into bioactive chemical scaffolds remains an attractive approach for developing new therapeutic agents for treatment of several ailments. The current study sought to investigate the anticancer and antiplasmodial effects of incorporating ferrocene (Fc) into Nb scaffold presumably through inhibition of Hsp90. The ferrocenyl Nb analogues containing simplified structural motifs such as phenyl, benzyl, and piperidine were synthesized in six to nine steps employing conventional synthetic organic protocols adapted from literature, and the compounds were accessed in reasonable yields. For comparison purposes, a selection of organic Nb analogues were also included in the study. The target compounds were characterized by spectroscopic techniques including 1-dimensional nuclear magnetic resonance (1D NMR) and high-resolution mass spectroscopy. The synthesized compounds were evaluated in vitro for potential anticancer and antiplasmodial activities using the breast cancer cell line (HCC38) and chloroquine-sensitive strain (3D7) of the malaria parasite, Plasmodium falciparum. The presence of the Fc unit was found to enhance both anticancer and antiplasmodial activities of the resultant ferrocenyl Nb compounds with IC50 values in the low to mid micromolar range. Hsp90 inhibitory studies of the ferrocenyl Nb analogues possessing superior activities (2.13a and 2.20c) were also conducted using different yeast strains expressing both human and malarial Hsp90 isoforms: hHsp90a/p and PfHsp90, respectively. The results of Hsp90 inhibitory studies suggested no direct correlation between the observed activities of the analogues and Hsp90 inhibition. However, since the conditions of the assay were not optimised due to time constrains of the project, these observed data remained to be confirmed. , Thesis (MSc) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Authors: Mbaba, Mziyanda
- Date: 2017
- Subjects: Antibiotics Synthesis , Ferrocene , Heat shock proteins , Antimalarials , Cancer Chemotherapy
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65111 , vital:28690
- Description: Novobiocin (Nb) is a coumarin type antibiotic isolated from the bacterium species of Streptomyces and possesses modest anticancer and antimalarial activities. Nb and analogues have been extensively explored as potential anticancer agents through inhibition of the C- terminal domain of heat shock protein 90 (Hsp90), which plays a pivotal role in the proteinfolding machinery of cells. There has been little effort in the exploration of Nb and derivatives for antimalarial activity. Incorporation of organometallic units, such as ferrocene (Fc), into bioactive chemical scaffolds remains an attractive approach for developing new therapeutic agents for treatment of several ailments. The current study sought to investigate the anticancer and antiplasmodial effects of incorporating ferrocene (Fc) into Nb scaffold presumably through inhibition of Hsp90. The ferrocenyl Nb analogues containing simplified structural motifs such as phenyl, benzyl, and piperidine were synthesized in six to nine steps employing conventional synthetic organic protocols adapted from literature, and the compounds were accessed in reasonable yields. For comparison purposes, a selection of organic Nb analogues were also included in the study. The target compounds were characterized by spectroscopic techniques including 1-dimensional nuclear magnetic resonance (1D NMR) and high-resolution mass spectroscopy. The synthesized compounds were evaluated in vitro for potential anticancer and antiplasmodial activities using the breast cancer cell line (HCC38) and chloroquine-sensitive strain (3D7) of the malaria parasite, Plasmodium falciparum. The presence of the Fc unit was found to enhance both anticancer and antiplasmodial activities of the resultant ferrocenyl Nb compounds with IC50 values in the low to mid micromolar range. Hsp90 inhibitory studies of the ferrocenyl Nb analogues possessing superior activities (2.13a and 2.20c) were also conducted using different yeast strains expressing both human and malarial Hsp90 isoforms: hHsp90a/p and PfHsp90, respectively. The results of Hsp90 inhibitory studies suggested no direct correlation between the observed activities of the analogues and Hsp90 inhibition. However, since the conditions of the assay were not optimised due to time constrains of the project, these observed data remained to be confirmed. , Thesis (MSc) -- Faculty of Science, Chemistry, 2017
- Full Text:
- «
- ‹
- 1
- ›
- »