1) Researchers studied the internal ribosomal entry site (IRES) in the 5' untranslated region of the canine dicistrovirus (CDV-A) genome.
2) Using computational prediction and SHAPE analysis, they determined the secondary structure of the CDV-A 5'UTR IRES, which resembles the poliovirus IRES structure.
3) In vitro translation assays in rabbit reticulocyte lysate showed that the CDV-A 5'UTR IRES can direct cap-independent translation, and requires the initiation factor eIF4A.
This document discusses research on the mechanism of translation initiation on the genomic RNA of Cadicivirus A, a naturally occurring dicistronic picornavirus. Cadicivirus A has a dicistronic genome containing two open reading frames separated by an intergenic region that functions as an internal ribosomal entry site (IRES). The researchers investigated the structure of the 5'UTR IRES using SHAPE analysis, finding it closely resembled the structure of the poliovirus IRES. They showed the 5'UTR IRES could drive translation in rabbit reticulocyte lysate and required the canonical initiation factor eIF4A. Toeprinting analysis was used to study 48S complex formation on the IRES
1) The study characterized the internal ribosomal entry site (IRES) of Canine dicistronic picornavirus (Cadicivirus A), a naturally occurring dicistronic picornavirus.
2) Predictive modeling and SHAPE analysis were used to predict the secondary structure of the 5'UTR IRES and confirm its predicted folding.
3) Comparisons showed similarities between domains of the Cadicivirus IRES and domains of the poliovirus IRES, but also differences that may influence structure and function.
4) IRESs can be studied in vitro using rabbit reticulocyte lysate to determine their ability to independently initiate translation of a downstream cistron.
The mechani smof t ransl at i on i ni t i at i on on t he genomi c RNA of Cadi ci vi rus A: a nat ural l y occurri ng di ci st roni c pi cornavi rus and t ype member of t he novel genus Di ci pi vi rus. The document discusses the discovery of Canine dicistronic picornavirus (Cadicivirus A, CDV-A) and aims to characterize the mechanism of translation initiation on its genomic RNA through predicting the secondary structure of its 5'UTR using SHAPE analysis and studying its activity in rabbit reticulocyte lysate using various constructs. Toe-printing
This document discusses the potential benefits and risks of using CRISPR/Cas9 gene editing technology. Some key benefits discussed include its relatively low cost compared to other gene editing methods, its ability to efficiently and precisely edit genes, and recent successes using it to correct mutations that cause diseases like Duchenne Muscular Dystrophy in mice models. However, the document also notes potential risks like inducing unintended mutations, difficulties achieving a high enough editing rate, and risks of editing germline cells or human embryos. While promising for treating genetic diseases, the document argues that more research is still needed to minimize risks before clinical use, and that regulations will be important to guide its safe and ethical application.
DNA sequencing determines the order of nucleotides in a DNA molecule. Next-generation sequencing (NGS) methods like pyrosequencing have accelerated research by allowing high-throughput, low-cost sequencing. Pyrosequencing works by detecting pyrophosphate release during DNA synthesis. It has applications in genetics, epigenetics, forensics, medicine, and more. NGS continues to advance sequencing capabilities and make whole genome analysis increasingly accessible.
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
Potential for off-target effects in topically applied dsRNA-based products us...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
Engineering plant immunity using crispr cas9 to generate virus resistanceSheikh Mansoor
Targeted genome editing by use of artificial nucleases has the plausible potential to speed basic research as well as plant breeding by providing the means to modify genomes quickly in a specific and predictable manner but advanced CRISPR-Cas9 based technologies first confirmed in mammalian cell systems are quickly being fitted for use in plants. These new technologies increase CRISPR-Cas9’s utility and effectiveness by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. RNA-guided genome editing using Streptococcus pyogenes CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) has renewed the concept of genome editing in plants. CRISPR-associated surveillance complexes are easily programmable molecular sleds that can target any sequence of choice. These complexes offer new opportunities for implementation in biotechnology. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. The CRISPR/Cas9 platform could also be used for targeted mutagenesis to identify host factors that control plant resistance and susceptibility to viral infection. Thus, CRISPR/Cas9 technology offers a promising approach for under- standing and engineering resistance to single and multiple viral infections in plants.
This document discusses research on the mechanism of translation initiation on the genomic RNA of Cadicivirus A, a naturally occurring dicistronic picornavirus. Cadicivirus A has a dicistronic genome containing two open reading frames separated by an intergenic region that functions as an internal ribosomal entry site (IRES). The researchers investigated the structure of the 5'UTR IRES using SHAPE analysis, finding it closely resembled the structure of the poliovirus IRES. They showed the 5'UTR IRES could drive translation in rabbit reticulocyte lysate and required the canonical initiation factor eIF4A. Toeprinting analysis was used to study 48S complex formation on the IRES
1) The study characterized the internal ribosomal entry site (IRES) of Canine dicistronic picornavirus (Cadicivirus A), a naturally occurring dicistronic picornavirus.
2) Predictive modeling and SHAPE analysis were used to predict the secondary structure of the 5'UTR IRES and confirm its predicted folding.
3) Comparisons showed similarities between domains of the Cadicivirus IRES and domains of the poliovirus IRES, but also differences that may influence structure and function.
4) IRESs can be studied in vitro using rabbit reticulocyte lysate to determine their ability to independently initiate translation of a downstream cistron.
The mechani smof t ransl at i on i ni t i at i on on t he genomi c RNA of Cadi ci vi rus A: a nat ural l y occurri ng di ci st roni c pi cornavi rus and t ype member of t he novel genus Di ci pi vi rus. The document discusses the discovery of Canine dicistronic picornavirus (Cadicivirus A, CDV-A) and aims to characterize the mechanism of translation initiation on its genomic RNA through predicting the secondary structure of its 5'UTR using SHAPE analysis and studying its activity in rabbit reticulocyte lysate using various constructs. Toe-printing
This document discusses the potential benefits and risks of using CRISPR/Cas9 gene editing technology. Some key benefits discussed include its relatively low cost compared to other gene editing methods, its ability to efficiently and precisely edit genes, and recent successes using it to correct mutations that cause diseases like Duchenne Muscular Dystrophy in mice models. However, the document also notes potential risks like inducing unintended mutations, difficulties achieving a high enough editing rate, and risks of editing germline cells or human embryos. While promising for treating genetic diseases, the document argues that more research is still needed to minimize risks before clinical use, and that regulations will be important to guide its safe and ethical application.
DNA sequencing determines the order of nucleotides in a DNA molecule. Next-generation sequencing (NGS) methods like pyrosequencing have accelerated research by allowing high-throughput, low-cost sequencing. Pyrosequencing works by detecting pyrophosphate release during DNA synthesis. It has applications in genetics, epigenetics, forensics, medicine, and more. NGS continues to advance sequencing capabilities and make whole genome analysis increasingly accessible.
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
Potential for off-target effects in topically applied dsRNA-based products us...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
Engineering plant immunity using crispr cas9 to generate virus resistanceSheikh Mansoor
Targeted genome editing by use of artificial nucleases has the plausible potential to speed basic research as well as plant breeding by providing the means to modify genomes quickly in a specific and predictable manner but advanced CRISPR-Cas9 based technologies first confirmed in mammalian cell systems are quickly being fitted for use in plants. These new technologies increase CRISPR-Cas9’s utility and effectiveness by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. RNA-guided genome editing using Streptococcus pyogenes CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) has renewed the concept of genome editing in plants. CRISPR-associated surveillance complexes are easily programmable molecular sleds that can target any sequence of choice. These complexes offer new opportunities for implementation in biotechnology. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. The CRISPR/Cas9 platform could also be used for targeted mutagenesis to identify host factors that control plant resistance and susceptibility to viral infection. Thus, CRISPR/Cas9 technology offers a promising approach for under- standing and engineering resistance to single and multiple viral infections in plants.
Next Generation Sequencing- NGS for COVID19 PPTMesele Tilahun
This document presents a next-generation sequencing (NGS)-based method for diagnosing COVID-19 using a single-step RNA extraction. The method was tested on 336 clinical samples and showed high accuracy compared to RT-PCR, the gold standard method. Key advantages of the NGS approach include higher scalability allowing thousands of samples to be tested simultaneously, and higher sensitivity to detect SARS-CoV-2 RNA compared to RT-PCR. The method provides individual qualitative results for each patient sample along with viral load estimates based on sequencing coverage.
Singapore grouper iridovirus induced parapoptosis-like death in host cells vi...mgray11
Singapore grouper iridovirus (SGIV) induces a type of programmed cell death called parapotosis in host cells. This document summarizes research finding that SGIV activates mitogen-activated protein kinase (MAPK) signaling pathways, including ERK, JNK, and p38 MAPK, to induce parapotosis and facilitate viral replication. Inhibition of the ERK and JNK pathways can delay cytopathic effects and reduce virus production, indicating their role in the viral life cycle. JNK activation in particular promotes viral transcription and protein synthesis. MAPK signaling also modulates the expression of immune genes during infection. Therefore, SGIV utilizes MAPK pathways to induce parapotosis in host cells and
- The mRNA-1273 vaccine induced robust antibody and neutralizing responses against SARS-CoV-2 in all participants after the first dose. A second dose led to even stronger responses, especially in the 100 μg and 250 μg groups. Systemic side effects were mostly mild or moderate. This interim report provides support for mRNA-1273 as a promising COVID-19 vaccine candidate warranting further development and evaluation.
Introduction, History, components, cas9 protein structure and function,gRNA variants, Cas9 nuclease variants, CRISPR in bacteria as the immune system, mechanism, steps of working, Applications, and pros and cons.
This slide will help you understand the basics of CRISPR-Cas9, Mechanism, Application, Advantages, and Disadvantages of CRISPR-Cas9, Future Concerns, Future Possibilities.
The document discusses CRISPR-Cas9 genome editing. It begins by explaining why genome editing is useful for applications like disease modeling, gene therapy, and agriculture. It then provides details on the CRISPR-Cas9 system, describing how it uses the Cas9 enzyme guided by a short RNA to introduce targeted double-stranded breaks in DNA. The document outlines several uses of CRISPR-Cas9 in research, including generating animal models of disease and correcting genetic defects in human cells and stem cells. It also discusses approaches for screening mammalian cells using libraries of guide RNAs to induce mutations.
This document summarizes Jai Kishan's presentation on gene manipulation techniques. It discusses conventional plant breeding, mutation breeding, transgenic plants, and several precise gene editing techniques like ZFN, TALENs, and CRISPR-Cas9. CRISPR-Cas systems provide adaptive immunity in bacteria and archaea by recognizing foreign DNA. The document then reviews the history and components of the CRISPR-Cas system and its applications in cancer therapy, viral diseases, bacterial infections, and genetic disorders. It also discusses challenges like delivery methods, mosaicism, immune responses, and off-target mutations. Key references on the discovery of CRISPR and its use in agriculture and as a genetic manipulation tool are presented.
Join Fight CRC in a webinar about biomarkers. In this session, Dr. Chris Lieu will focus the discussion on the NTRK biomarker, in addition to ctDNA, and Next-Generation Sequencing.
Discovery and Molecular characterization of virus PPT Mesele Tilahun
1) Next-generation sequencing was used to identify novel viruses in wild grass plants. Two putative novel secoviruses, poaceae Liege nepovirus A (PoLNVA) and poaceae Liege virus 1 (PoLV1), were discovered through de novo assembly of sequencing reads.
2) PoLNVA has a bipartite genome consisting of RNA1 (7087 nt) and RNA2 (4056 nt), and shares 77.8% and 60.3% amino acid identity with beet ringspot virus for RNA1 and RNA2, respectively. PoLV1 is a single-stranded RNA virus of 10843 nt that shares 29.8% identity with brassica napus RNA virus
This document discusses a meta-analysis of multiple datasets examining features of viral RNA that correlate with activation of the innate immune system. The analysis found that "A" nucleotide content and minimum folding energy (MFE) were good predictors of immune system activation, more so than other proposed indicators like CpG enrichment. As RNA composition and structure are correlated, further experiments using synthetic sequences are needed to identify the specific viral RNA sensing mechanisms of immune system receptors.
CRISPR/Cas9 is a new genome editing tool that allows geneticists to precisely edit DNA sequences. It uses a bacterial immune system to cut DNA at a specific location so that parts of the genome can be removed, replaced, or added. The system involves a Cas9 enzyme guided by RNA to a targeted DNA site, where it cuts both strands of the DNA. This allows desired modifications to the genome. The summary discusses how CRISPR can be used to create genetic variability, develop disease resistance in crops, and potentially edit human organs for transplantation with less immune rejection risk. It also provides an example of using CRISPR to enhance blast resistance in rice.
This document provides an overview of CRISPR/Cas9 genome editing. It discusses how CRISPR/Cas9 enables precise modification of DNA sequences, outlines the timeline of key discoveries in CRISPR research, and describes the molecular mechanism and potential applications of this technology, including in microbial research, crop improvement, and human gene therapy. It also notes some limitations of the CRISPR/Cas9 system and concludes by emphasizing the opportunities it provides to advance research and address challenges like food security.
mRNA vaccine is a novel vaccine technology, which delivers mRNA that encoding the antigen protein of pathogen to the cell, and expresses the antigen protein, and then stimulates the immune response of the body.
Creative Biolabs has developed non-replicating mRNA vaccine platform, mRNA vaccine platform, mRNA pharmacology optimization platform, and and Self-amplifying mRNA vaccine platform to spport your vaccine researches. If you need more information about mRNA vaccine, please follow us.
Discovering my research interest by eric garson sheffield university 2017Eric Garson
1) The document describes the author's process of discovering his research interest in targeting specific cell types in the brain using viral vectors and manipulating their tropism.
2) It outlines experiments using organotypic slice cultures of rat cerebellum to test the ability of cathepsin K and its inhibitor to shift the tropism of lentiviral vectors toward Bergmann glia or Purkinje neurons, but the results were inconclusive.
3) Going forward, the author is interested in developing more targeted approaches using cell-type specific pseudotyping, promoters, or microRNA sequences to modify viral tropism and enable gene delivery to specific cells involved in diseases
Validation of RNA interference by RNA-Seq: How to see the big picture - Brend...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
Developing Ultra-Sensitive PCR Protocols for HIV Vaccine ResearchKate Barlow
This document summarizes Dr. Catherine N. Kibirige's presentation on developing ultra-sensitive PCR protocols for HIV vaccine research. It discusses (1) the challenges of HIV's genetic diversity and finding conserved sequence regions for assay design, (2) how digital droplet PCR (ddPCR) was useful for quantifying cell line standards but qPCR was better for analyzing archival patient samples due to issues with ddPCR, and (3) two successful studies using the ultra-sensitive qPCR assay - a pilot study found HIV RNA more frequently in virologically suppressed patients with high inflammation, and characterization of the IAVI Viral Inhibition Assay showed HIV RNA detection within 2 days of differences in CD8 inhibition
1) The study investigated how Gram-negative bacterial lipopolysaccharide (LPS), a component of bacteria like Chlamydia and Neisseria that cause STIs, affects expression of HIV receptors in cervical epithelial cells.
2) The results showed that LPS increased expression of the CCR5 HIV co-receptor and other alternative receptors in cervical cells through activation of EGFR, ERK1/2, and COX-2 signaling pathways.
3) This suggests that STIs have the potential to enhance susceptibility to HIV infection in women by regulating expression of HIV receptors in cervical epithelial cells through an inflammatory response.
RETROVIRUS MEDIATED GENE TRANSFER AND EXPRESSION CLONINGSrishtiRoy10
- The retroviral virion is a spherical particle 80-100 nm in diameter composed of a lipid bilayer envelope containing glycoproteins and a capsid containing two copies of the viral RNA genome and enzymes.
- Retroviruses replicate by reverse transcribing their RNA genome into DNA which is then integrated into the host cell genome by an integrase enzyme to become a provirus, allowing transcription of viral genes.
- Retrovirus mediated gene transfer involves the virus producing a DNA copy of its genome using reverse transcriptase, with the DNA then integrating randomly into the host cell genome, allowing investigation of gene function.
This document discusses the molecular biology of hepatitis C virus (HCV). It describes HCV's structure, including its genome and genes that encode structural and non-structural proteins. These proteins and their roles in HCV's lifecycle are explained. The document also discusses HCV genotypes, mutations that contribute to its genetic diversity, and molecular targets for new antiviral drugs against HCV proteins and RNA. Resistance to direct antiviral drugs is a concern, as HCV can rapidly develop mutations due to its high replication rate and lack of a DNA stage. Understanding HCV's molecular biology is important for research, treatment, and developing new antiviral therapies.
Next Generation Sequencing- NGS for COVID19 PPTMesele Tilahun
This document presents a next-generation sequencing (NGS)-based method for diagnosing COVID-19 using a single-step RNA extraction. The method was tested on 336 clinical samples and showed high accuracy compared to RT-PCR, the gold standard method. Key advantages of the NGS approach include higher scalability allowing thousands of samples to be tested simultaneously, and higher sensitivity to detect SARS-CoV-2 RNA compared to RT-PCR. The method provides individual qualitative results for each patient sample along with viral load estimates based on sequencing coverage.
Singapore grouper iridovirus induced parapoptosis-like death in host cells vi...mgray11
Singapore grouper iridovirus (SGIV) induces a type of programmed cell death called parapotosis in host cells. This document summarizes research finding that SGIV activates mitogen-activated protein kinase (MAPK) signaling pathways, including ERK, JNK, and p38 MAPK, to induce parapotosis and facilitate viral replication. Inhibition of the ERK and JNK pathways can delay cytopathic effects and reduce virus production, indicating their role in the viral life cycle. JNK activation in particular promotes viral transcription and protein synthesis. MAPK signaling also modulates the expression of immune genes during infection. Therefore, SGIV utilizes MAPK pathways to induce parapotosis in host cells and
- The mRNA-1273 vaccine induced robust antibody and neutralizing responses against SARS-CoV-2 in all participants after the first dose. A second dose led to even stronger responses, especially in the 100 μg and 250 μg groups. Systemic side effects were mostly mild or moderate. This interim report provides support for mRNA-1273 as a promising COVID-19 vaccine candidate warranting further development and evaluation.
Introduction, History, components, cas9 protein structure and function,gRNA variants, Cas9 nuclease variants, CRISPR in bacteria as the immune system, mechanism, steps of working, Applications, and pros and cons.
This slide will help you understand the basics of CRISPR-Cas9, Mechanism, Application, Advantages, and Disadvantages of CRISPR-Cas9, Future Concerns, Future Possibilities.
The document discusses CRISPR-Cas9 genome editing. It begins by explaining why genome editing is useful for applications like disease modeling, gene therapy, and agriculture. It then provides details on the CRISPR-Cas9 system, describing how it uses the Cas9 enzyme guided by a short RNA to introduce targeted double-stranded breaks in DNA. The document outlines several uses of CRISPR-Cas9 in research, including generating animal models of disease and correcting genetic defects in human cells and stem cells. It also discusses approaches for screening mammalian cells using libraries of guide RNAs to induce mutations.
This document summarizes Jai Kishan's presentation on gene manipulation techniques. It discusses conventional plant breeding, mutation breeding, transgenic plants, and several precise gene editing techniques like ZFN, TALENs, and CRISPR-Cas9. CRISPR-Cas systems provide adaptive immunity in bacteria and archaea by recognizing foreign DNA. The document then reviews the history and components of the CRISPR-Cas system and its applications in cancer therapy, viral diseases, bacterial infections, and genetic disorders. It also discusses challenges like delivery methods, mosaicism, immune responses, and off-target mutations. Key references on the discovery of CRISPR and its use in agriculture and as a genetic manipulation tool are presented.
Join Fight CRC in a webinar about biomarkers. In this session, Dr. Chris Lieu will focus the discussion on the NTRK biomarker, in addition to ctDNA, and Next-Generation Sequencing.
Discovery and Molecular characterization of virus PPT Mesele Tilahun
1) Next-generation sequencing was used to identify novel viruses in wild grass plants. Two putative novel secoviruses, poaceae Liege nepovirus A (PoLNVA) and poaceae Liege virus 1 (PoLV1), were discovered through de novo assembly of sequencing reads.
2) PoLNVA has a bipartite genome consisting of RNA1 (7087 nt) and RNA2 (4056 nt), and shares 77.8% and 60.3% amino acid identity with beet ringspot virus for RNA1 and RNA2, respectively. PoLV1 is a single-stranded RNA virus of 10843 nt that shares 29.8% identity with brassica napus RNA virus
This document discusses a meta-analysis of multiple datasets examining features of viral RNA that correlate with activation of the innate immune system. The analysis found that "A" nucleotide content and minimum folding energy (MFE) were good predictors of immune system activation, more so than other proposed indicators like CpG enrichment. As RNA composition and structure are correlated, further experiments using synthetic sequences are needed to identify the specific viral RNA sensing mechanisms of immune system receptors.
CRISPR/Cas9 is a new genome editing tool that allows geneticists to precisely edit DNA sequences. It uses a bacterial immune system to cut DNA at a specific location so that parts of the genome can be removed, replaced, or added. The system involves a Cas9 enzyme guided by RNA to a targeted DNA site, where it cuts both strands of the DNA. This allows desired modifications to the genome. The summary discusses how CRISPR can be used to create genetic variability, develop disease resistance in crops, and potentially edit human organs for transplantation with less immune rejection risk. It also provides an example of using CRISPR to enhance blast resistance in rice.
This document provides an overview of CRISPR/Cas9 genome editing. It discusses how CRISPR/Cas9 enables precise modification of DNA sequences, outlines the timeline of key discoveries in CRISPR research, and describes the molecular mechanism and potential applications of this technology, including in microbial research, crop improvement, and human gene therapy. It also notes some limitations of the CRISPR/Cas9 system and concludes by emphasizing the opportunities it provides to advance research and address challenges like food security.
mRNA vaccine is a novel vaccine technology, which delivers mRNA that encoding the antigen protein of pathogen to the cell, and expresses the antigen protein, and then stimulates the immune response of the body.
Creative Biolabs has developed non-replicating mRNA vaccine platform, mRNA vaccine platform, mRNA pharmacology optimization platform, and and Self-amplifying mRNA vaccine platform to spport your vaccine researches. If you need more information about mRNA vaccine, please follow us.
Discovering my research interest by eric garson sheffield university 2017Eric Garson
1) The document describes the author's process of discovering his research interest in targeting specific cell types in the brain using viral vectors and manipulating their tropism.
2) It outlines experiments using organotypic slice cultures of rat cerebellum to test the ability of cathepsin K and its inhibitor to shift the tropism of lentiviral vectors toward Bergmann glia or Purkinje neurons, but the results were inconclusive.
3) Going forward, the author is interested in developing more targeted approaches using cell-type specific pseudotyping, promoters, or microRNA sequences to modify viral tropism and enable gene delivery to specific cells involved in diseases
Validation of RNA interference by RNA-Seq: How to see the big picture - Brend...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
Developing Ultra-Sensitive PCR Protocols for HIV Vaccine ResearchKate Barlow
This document summarizes Dr. Catherine N. Kibirige's presentation on developing ultra-sensitive PCR protocols for HIV vaccine research. It discusses (1) the challenges of HIV's genetic diversity and finding conserved sequence regions for assay design, (2) how digital droplet PCR (ddPCR) was useful for quantifying cell line standards but qPCR was better for analyzing archival patient samples due to issues with ddPCR, and (3) two successful studies using the ultra-sensitive qPCR assay - a pilot study found HIV RNA more frequently in virologically suppressed patients with high inflammation, and characterization of the IAVI Viral Inhibition Assay showed HIV RNA detection within 2 days of differences in CD8 inhibition
1) The study investigated how Gram-negative bacterial lipopolysaccharide (LPS), a component of bacteria like Chlamydia and Neisseria that cause STIs, affects expression of HIV receptors in cervical epithelial cells.
2) The results showed that LPS increased expression of the CCR5 HIV co-receptor and other alternative receptors in cervical cells through activation of EGFR, ERK1/2, and COX-2 signaling pathways.
3) This suggests that STIs have the potential to enhance susceptibility to HIV infection in women by regulating expression of HIV receptors in cervical epithelial cells through an inflammatory response.
RETROVIRUS MEDIATED GENE TRANSFER AND EXPRESSION CLONINGSrishtiRoy10
- The retroviral virion is a spherical particle 80-100 nm in diameter composed of a lipid bilayer envelope containing glycoproteins and a capsid containing two copies of the viral RNA genome and enzymes.
- Retroviruses replicate by reverse transcribing their RNA genome into DNA which is then integrated into the host cell genome by an integrase enzyme to become a provirus, allowing transcription of viral genes.
- Retrovirus mediated gene transfer involves the virus producing a DNA copy of its genome using reverse transcriptase, with the DNA then integrating randomly into the host cell genome, allowing investigation of gene function.
This document discusses the molecular biology of hepatitis C virus (HCV). It describes HCV's structure, including its genome and genes that encode structural and non-structural proteins. These proteins and their roles in HCV's lifecycle are explained. The document also discusses HCV genotypes, mutations that contribute to its genetic diversity, and molecular targets for new antiviral drugs against HCV proteins and RNA. Resistance to direct antiviral drugs is a concern, as HCV can rapidly develop mutations due to its high replication rate and lack of a DNA stage. Understanding HCV's molecular biology is important for research, treatment, and developing new antiviral therapies.
African Swine Fever (ASF) virus genomics and diagnosticsILRI
This document summarizes activities related to analyzing the genetics of the African Swine Fever virus (ASFV) in Kenya. It discusses sequencing the whole ASFV genome to analyze diversity and origins of outbreaks. Genotyping using three genetic markers finds that recent Kenyan outbreaks involve genotype IX, the same genotype present in Uganda. While whole genome sequencing and genetic analysis can inform vaccine development and tracing outbreaks, developing low-cost, rapid field diagnostics remains a priority for controlling ASF. Surveillance of pigs in coastal Kenya may also be needed to prevent the spread of genotype IX globally.
The document discusses using genetically encoded fluorescent probes to visualize glutamate neurotransmission in organotypic hippocampal slices. Glutamate is an important neurotransmitter in vertebrates, used in many neural circuits supporting learning and behavior. The probes allow researchers to monitor glutamate release and receptor activation with high spatiotemporal resolution in intact neural circuits. This provides insights into glutamate signaling dynamics during normal and pathological states.
Next Generation Sequencing application in virologyEben Titus
Next Generation Sequencing (NGS) is a promising technique for virus diagnosis that provides several advantages over traditional Sanger sequencing. NGS workflows involve sample preparation, sequencing, and data analysis. NGS has various applications in virology including identifying viral quasispecies, detecting antiviral drug resistance mutations, discovering novel virus genotypes, and performing quality control of live vaccines. While NGS reduces costs and improves throughput over Sanger sequencing, analyzing large NGS datasets requires strong bioinformatics skills. Overall, NGS represents a significant improvement for virus research and diagnosis.
This document discusses reverse genetics techniques used in zebrafish research. It describes several common reverse genetics methods including retrovirus-mediated insertional mutagenesis, the Tol2 transposon system, TILLING, ZFNs, TALENs, and morpholino knockdowns. It provides details on how each technique works and its advantages and limitations. The document also discusses applications of reverse genetics in studying virus biology and potential issues with some reverse genetics experiments.
Dr. Laura Miller - Comparative analysis of signature genes in PRRSV-infected ...John Blue
Comparative analysis of signature genes in PRRSV-infected porcine monocyte-derived dendritic cells at differential activation statuses - Dr. Laura Miller, Virus and Prion Diseases of Livestock Research Unit, National Animal Disease Center, USDA-ARS, from the 2015 North American PRRS Symposium, December 4 - 5, 2015, Chicago, IL, USA.
More presentations at http://www.swinecast.com/2015-north-american-prrs-symposium
Insilico Comprehension of Stop Codon Readthrough in Human Virusesijtsrd
Readthrough is an event in which stop codon is misread, resulting in elongation of polypeptides. Stop codon suppression or termination codon readthrough, is a mechanism of expression of many disorder proteins. Many important cellular functions are carried out by way of the Readthrough process. This could alter the gene function which thereon produces either destructive or constructive effects. Hence, this study aims to diagnose this recoding mechanism in certain selected humans infecting pathogenic viruses through insilico approach. For this target, the 3'UnTranslated Regions of the selected viruses were retrieved from the Genbank database. Each of these 3'UTRs were translated into all their reading frames. Motif search using Interproscan in each of the frames, followed by homology search using BLASTX, and were achieved to identify stop codon readthrough candidates in each of the selected viruses. Finally, the secondary structure of RNA was predicted using RNAFold web server to ensure the stability of the RNA. The 3'UTRs from Aichi Virus 1, Cosa Virus A, Dengue Virus 1, Duvenhage Lyssavirus, Enterovirus A, HepatitisGB Virus B, Human Cosavirus A, Human Pegivirus 2, Langat Virus, Parechovirus A, WestNile Virus and Zika Virus were retrieved. A total of 48 motifs were identified in different reading frames of 3'UTR of the selected viruses. BlastX search recognized 9 homologs in the reading frames of 3'UTR. The secondary structure analysis and search of motifs and homologs resulted in the confirmation of 5 candidates with strong evidence for the readthrough event. These candidates showed homology with proteins of prime importance such as Imidazole glycerol Phosphate synthase protein, 50S ribosomal protein L27, DNA replication, and repair protein, replication origin binding protein, and adenosine deaminase. Hence, we proved that the 3'untranslated regions would undergo translation. This strongly suggests that many such readthrough events are to be determined to exactly unravel the pathogenicity behind Viruses. To design anti viral drugs to impede this viral machinery, it is essential to analyse their 3'UTR regions. Arockiyajainmary M | Balaji S | Sivashankari Selvarajan "Insilico Comprehension of Stop Codon Readthrough in Human Viruses" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 | Issue-4 , June 2020, URL: https://www.ijtsrd.com/papers/ijtsrd31550.pdf Paper Url :https://www.ijtsrd.com/biological-science/biotechnology/31550/insilico-comprehension-of-stop-codon-readthrough-in-human-viruses/arockiyajainmary-m
This document discusses the challenges involved in developing an HIV vaccine. It provides background on HIV, describing it as a retrovirus that targets CD4+ T cells. It reviews past vaccine trials, noting the only modest success of the RV144 trial. It discusses the massive diversity of HIV strains as a major challenge. It outlines various vaccine design strategies that have been pursued, including recombinant proteins, DNA vaccines, viral vectors, and approaches using broadly neutralizing antibodies. Throughout, it emphasizes the need for a vaccine to elicit robust cellular and humoral immune responses against a wide range of HIV subtypes to achieve protective efficacy.
Benjamin P. Steil has over 15 years of experience in virology research. He received his Ph.D. in Microbiology and Immunology from the University of Colorado Denver in 2008, and has since worked as a postdoctoral fellow and staff scientist developing viral vaccine adjuvants. His skills include viral particle production, nucleic acid techniques, mouse models, and immunological assays. Currently he is seeking a position where he can apply his expertise in vaccine design and development.
Gene therapy is a contemporary therapeutic intervention with recent positive results and regulatory approvals either completed or expected in the next several years for various conditions. The evolving view is that gene therapy will ultimately offer hope across a range of otherwise debilitating or difficult to treat conditions. The renaissance in gene therapy has seen major development of both non viral and viral vectors and accelerated preclinical studies and clinical trials. It is therefore timely to address the progress in gene therapy through a special issue presenting reviews on non viral and viral vectors including relevant updates on applications on herpes simplex virus HSV and adeno associated virus AAV vectors. Thus, the purpose of this review is to summarize the general concepts of gene therapy with a specific focus on monogenic rare disease in hematology and central nervous system disorders where burgeoning therapies are currently entering clinical investigations and approaching regulatory approval. Ms. Snehal D. Jadhav | Mr. Jeevan R. Rajguru | Ms. Hina U. Momin | Dr. Mrunal K. Shirsat "Gene Therapy- Challenges & Success" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 | Issue-3 , April 2020, URL: https://www.ijtsrd.com/papers/ijtsrd30612.pdf Paper Url :https://www.ijtsrd.com/medicine/other/30612/gene-therapy-challenges-and-success/ms-snehal-d-jadhav
This study investigated whether the E3L protein from vaccinia virus (VACV) or ectromelia virus (ECTV) could rescue replication of an influenza virus lacking the NS1 protein (ΔNS1 Flu virus). The ECTV E3L protein was cloned and cells were transfected with ECTV E3L, VACV E3L, or wildtype Flu NS1 protein before infecting with ΔNS1 Flu virus or wildtype Flu virus. Flow cytometry was used to measure expression of the influenza hemagglutinin protein as an indicator of viral replication. The results showed that neither VACV E3L nor ECTV E3L rescued replication of the ΔNS1 Flu virus
Novel research aimed at finding a cure for AIDS requires animal models responding to human antiretroviral drugs. However, there have been few antiretrovirals cross-active against the simian viruses. In this study, we expanded the arsenal of drugs active against the simian retrovirus SIVmac251 and showed that this virus is inhibited by the protease inhibitor, darunavir, and the CCR5 blocker, maraviroc. Administration of these two drugs in combination with the reverse transcriptase inhibitors, tenofovir and emtricitabine, and the integrase inhibitor, raltegravir, resulted in prolonged plasma viral loads below assay detection limits, and, surprisingly, restricted the viral reservoir, a marker of which is viral DNA. We then decided to employ this multidrug regimen (termed “highly intensified ART”) in order to increase the potency of a previous strategy based on the gold drug auranofin, which recently proved able to restrict the viral reservoir in vivo. A short course of highly intensified ART following the previous treatment resulted, upon therapy suspension, in a remarkably spontaneous control of the infection, that may pave the way to a persistent suppression of viremia in the absence of ART. These results corroborate the robustness of the macaque AIDS model as a vanguard for potentially future treatments for HIV in humans.
In vitro transcription and transfection of HCV genomic repliconBinodGupta27
Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 μg transcript using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR. Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
In vitro transcription and transfection of HCV genomic repliconBinodGupta27
ABSTRACT:
Introduction: Hepatitis C virus (HCV) is a positive stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Aims & Objectives: The study was conducted to establish the transfection of Huh 7.5 derived cell lines with In-vitro transcript of HCV pF6/JFH-1 for production of infectious virus particles in naïve Huh 7.5 cells, its detection by RT-PCR. Materials and Method: Huh 7.5 cells, a highly permissive cell lines for HCV replication, were grown in Dulbecco’s Modified Eagle’s Medium and pFL-J6/JFH plasmid was linearized with XbaI and subjected to in-vitro transcription using MEGAscript Kit (Ambion, USA) Huh-7.5 cells were transfected with 2.5 μg transcript using Lipofectamine 2000 transfection reagent (Invitrogen, USA) . The culture supernatant was collected after 24, 48 and 72 hr after incubation in fresh media and viral RNAs were isolated from it using Trizol LS reagent (Ambion, USA) and quantified by real-time quantitative RT-PCR. Total RNA was extracted from cells using Trizol reagent (Ambion, USA) and then RNA was subjected to cDNA synthesis using RevertAid reverse transcription (Thermo Fisher Scientific, USA). The PCR products were resolved by electrophoresis in 1.5% (w/v) agarose gels and images were captured by a Chemidoc XRS system (Bio-Rad, USA). Results: We observed Huh7.5 cells were cultured in DMEM. Plasmid FL-J6/JFH1 was linearized with the restriction enzyme XbaI and HCV RNA was obtained by In-vitro transcription and was transfected to grown Huh 7.5 cells shown by band on agarose gel and total RNA isolated after 24 hours of post infection followed by RT-PCR gave distinct band on gel whereas 48 and 72 hr did not. Infection of Huh 7.5 cells with cell culture supernatant from cells transfected with HCV in vitro transcript gave a distinct band. This will help in understanding entire viral life cycle and its non-structural gene products like NS4B and NS5A that enhance the replicative capacity of replicons in Huh 7.5 cell lines for development of drug and vaccines.
DNA fingerprinting involves detecting variations in DNA sequences between individuals to generate unique profiles. There are two main categories of techniques: hybridization-based like Restriction Fragment Length Polymorphism (RFLP) and PCR-based like Randomly Amplified Polymorphic DNA (RAPD). RFLP was the first technique developed and involves digesting DNA with restriction enzymes and comparing fragment lengths on gels. PCR-based techniques do not require blotting/hybridization and can use smaller DNA quantities. DNA fingerprinting has applications in forensics, genetics, and plant/animal breeding.
Dengue Virus: Genomic Insights, Pathogenic Mechanisms, and Therapeutic Approa...SindhBiotech
This lecture is presented by our volunteer Sajid Ali Shah, he is from Islamabad, Pakistan, and he is covering Dengue Virus: Genomic Insights, Pathogenic Mechanisms, and Therapeutic Approaches
for video: https://youtu.be/whrkkKR-NSY
#denguevirus #virology #virologist #genomics #covid19 #virus #pathogen #pathology #immunology
1) The document discusses a study analyzing the impact of gene length on detecting differentially expressed genes using RNA-seq technology.
2) The study will first test the reproducibility of RNA-seq and the effect of normalization. It will then compare different statistical tests for identifying differentially expressed genes.
3) Finally, the study will specifically test how gene length impacts the likelihood of a gene being identified as differentially expressed, as longer genes are easier to map with short reads.
This document discusses biosafety considerations for gene therapy. It provides information on criteria used to categorize biological risk, the history of human gene therapy, and risk management procedures when working with adeno-associated virus vectors. It also discusses criteria used to ensure appropriate containment procedures are followed.
1. Mukta Asnani
Dr. Tatyana Pestova
Dr. Christopher Hellen
Department of Cell Biology, SUNY Downstate Medical Center
2. RNA viruses: Infection and hijacking of cellular translation
apparatus
Viruses depend on the host cell's translation apparatus.
They commonly suppress translation of cellular mRNAs by inhibiting the canonical mechanism
of cap-dependent initiation of translation – to favor viral protein synthesis and to impair host
antiviral responses.
This raises the question:
How does viral translation proceed in these circumstances?
Investigation of this question may reveal unique aspects of viral translation initiation that are
potential targets for therapeutic inhibition.
3. The canonical mechanism of cap-dependent translation initiation and sites of
viral regulation
AUG UAG
AUG UAG
E P A
AUG UAG
E P A
AUG UAG
E P A
AUG
UAG
E P
A
AUG UAG
1. mRNA Activation by
eIF4F cap-binding complex
2. Recruitment of
43S complex
3. 5’ to 3’ Scanning
4. Initiation codon recognition
and
48S complex formation
48S complex
eIF4E
eIF4G
eIF4A
eIF4B
eIF1
eIF1A
eIF2
eIF5
eIF3
43S complex
GTP
GTP
E P A
AUG UAG
5. GTP hydrolysis by eIF2,
release of factors,
60S Subunit joining
6. Hydrolysis of GTP
by eIF5B & release
of eIF5B
80S complex
eIF5B
GTP
GTP
DHX29
GDP
Viral proteases (2A and
3C) synthesized during
infection cleaves host
initiation factors and
hence shuts off the
canonical translation
initiation and allow
selective translation of
viral RNA genome
2A
3C
eIF4F complex
4. The genomes of several families of RNA viruses contain internal ribosomal entry sites
(IRESs), which mediate end-independent initiation, enabling viral mRNAs to bypass
the canonical cap-dependent mechanism
Characteristics of IRES-
1. Long highly structured positioned in 5’-untranslated region of mRNA, which serves the function of interacting with many
canonical initiation factors and other cellular factors.
2. Reduced requirement of initiation factors particularly cap-binding eIF4F complex.
3. Recruits 40S directly onto the mRNA in the vicinity of initiation codon.
4. Requires certain cellular factors called ITAFs (IRES-trans acting factors) which is generally not required during canonical
cap-dependent translation. In addition to modulating IRES activity, these ITAFs also plays an important role in various
cellular functions.
This alternative mechanism of translation initiation was first observed to be used by poliovirus RNA
genome in infected cells in late 1980s.
Poliovirus genome
Poliovirus IRES (~450 nt)
eIF4Gm
PCBP2
PCBP2 – ITAF
eIF4Gm – cleaved eIF4G
Sweeney et. al. (EMBO, 2014)
5. Classification of Viral IRESs
Family Genus Example IRES
class
Key
interaction
ITAFs (IRES Trans acting
factors)
Picornaviridae Aphthovirus Foot-and-mouth disease virus (FMDV)
Type 2 eIF4G
PTB, ITAF45
Cardiovirus Encephalomayocarditis virus (EMCV) PTB
Enterovirus Polio virus
Type 1
eIF4G
PTB
Rhinovirus Human rhinovirus (HRV) PTB, PCBP2, La, hnRNP A1, unr?
Flaviviridae Hepatitis C virus (HCV))
Type 3
40S subunit
Cripaviridae Cricket paralysis virus (CrPV) Type 4 40S subunit
IRESs are classified into different types depending on their secondary structure and initiation factors
requirements.
Non-canonical interactions of IRESs with
canonical components of the translational
apparatus
Poliovirus
Encephalomyocarditis
(EMCV)
Hepatitis C virus
(HCV)
Cricket paralysis
virus (CrPV)
IRES/eIF4G
IRES/eIF4G IRES/40S IRES/40S
6. Internal Ribosomal Entry Site (IRES) links to past of the translation
initiation mechanism ??
Canonical initiation-
In 1988 first IRES was found in
Poliovirus and EMCV
In 1991 first cellular IRES was found in
IgG heavy chain binding protein (BiP)
Quick response under stress
condition such as hypoxia, DNA
damage by UV, nutrient deprivation
etc.
Highly regulated process
(Cap-dependent)
Relic of the past and
evolved in matured
eukaryotes ??
Evolved in eukaryotes to
regulate gene expression
under stress ??
IRES study will shed light on past
of the translation initiation
mechanism
Cap-Independent
7. Viral Zoonoses – Cause of Human Infectious Diseases
Animals like bats and migratory water birds are always found to be reservoir host of zoonotic pathogens.
Cross species transmission has given rise to 70% zoonotic diseases in humans by host switching and adaption
leading to outbreaks in new hosts.
Thus zoonotic viruses always pose a threat to human health.
Understanding of these viruses might prevent the dreadful epidemic.
Bean et. al. (Nature, 2013)
8. Why is it important to study IRES - dependent Translation?
To understand not only the translation mechanism used by different viruses but also the
processes and regulation of cellular mRNA translation.
To understand how does cells and viruses impart specific translation of mRNAs in sea of
competent transcripts.
The understanding of IRES mediated translation and role of various initiation factors in
stimulating their activity can be extended to the cellular translation as well.
Understanding of the viral IRESs can also help to understand the translation of various
cellular IRESs present in the transcript encoding proteins expressed under compromised
conditions such as apoptosis, differentiation, hypoxia and nutrient deprivation when cap-
dependent translation is inhibited.
To study various antiviral and signaling pathways activated during viral infection.
The study of one virus IRES can be extrapolated to understand the mechanism of translation
used by novel or already known IRESs.
Thus there is always a constant hunt for the new viruses from different species.
9. Dicistroviridae
Before genome
sequencing era
(2 families were unrelated)
Picornaviridae
?
After genome
sequencing era
(both are related)
Picornavirus –like
superfamily
Multiple steps of translocation and
IRES deletion/duplication
Found in arthropods such as shrimps,
honey bee and insect pests of
agricultural and medical importance (eg-
triatoma virus cause chagas’ disease,
infected many Latin Americans)
Found in humans and wide variety of
animals in which they can cause
respiratory, cardiac, hepatic,
neurological diseases.
Hosts different
but contain same
gene contents
Different genome organization
Search of new viruses – To understand evolutionary past
Woo et. al. (J Virol, 2012)
10. Discovery of Canine dicistronic picornavirus (Cadicivirus A, CDV-A)
•In order to study picornavirus family and distantly related members, current screening efforts have
identified growing numbers of picornaviruses with 5'UTRs that diverge from known IRES types, and that
may therefore contain novel IRESs or variants of known IRESs.
• We became interested in Canine dicistronic picornavirus (Cadicivirus A or CDV) which was recently
characterized in the course of efforts to identify novel viruses in dogs. This was undertaken because
viruses occasionally gain the ability to spread within new hosts, leading to the emergence of new
epidemic diseases. An understanding of mechanisms underlying viral emergence is necessary for the
rational design of antiviral control strategies, and cross-species transmission of viruses from dogs is
possible because of their long history of cohabitation with humans.
•Cadicivirus A has a dicistronic genome with a 982nt-long 5'UTR and a 588nt-long intergenic region
(IGR).These noncoding regions have both been shown to function as IRESs.
• 982 bases
• 42% G-C rich
• 3’ end shows strong
sequence similarity to stem
loop V of the poliovirus IRES
5’UTR IRES
844 amino acids 1406 amino acids
IGR IRES
• 588 bases
• 3’ end shows strong sequence
similarity to stem loop V of the
poliovirus IRES
My Topic of Interest
11. Prediction of 5’UTR IRES Structure of CDV-A and analyzation using
SHAPE (Selective 2’-hydroxyl acylation analyzed by primer extension)
Binding sites for primers used for probing modifications across the RNA
Reverse
transcriptase
Primer-extension analysis
of modified RNA using
radiolabeled primer
A
B
C
D
F
G
H
I
J
K
L
M
N
AUG
983
NMIA
(N-methylnitroisatoic
anhydride)
Sequence of DNA
- + NMIA
Full length
RNA
Modified
nucleotides
C T A G
Predicted Structure using sequence co-variation
analysis and MFold software
Mechanism of Action
Different primers used to probe the modification along
the IRES
1
2
3
4
5
6
7
8
9
Jennifer et. al. (JACS, 2012)
12. Correlation of SHAPE analysis with the predicted structure
SHAPE data almost perfectly fit the predicted structure of the IRES
and hence confirmed the predicted structure.
B
C
D
F
G
H
I
J
K
L
M
N
Representative gel using primer 2
13. II
III
IV
V
VI
VII
py
AUG
Comparison between the structures of Cadicivirus-A 5’UTR
and poliovirus IRESs
A
B
C
D
F
G
H
I
J
K
L
M
N
UUG
AUG
983
py
GNRA
Tetraloop
Poliovirus IRES
GNRA
Tetraloop
Highlights-
1. CDV-A domain M resembles domain V of the poliovirus IRES.
2. CDV-A domain N (∆G = -4.2 kcal/mol) containing UUG-951 is much less stable
than poliovirus domain VI (∆G = -17.1 kcal/mol).
3. The GNRA tetraloop in CDV-A Domain K is rotated 90 degree clockwise compared
to that in domain IV of poliovirus.
4. Domain L (∆G = -5.9 kcal/mol) separates domain K and M by a greater distance
than that between domains IV and V. This greater distance may confer flexibility to
domain K so that the GNRA tetraloop can be oriented in a proper conformation.
28 nts 22 nts
CDV-A 5’-UTR IRES
14. How are IRESs studied in in-vitro?
• IRES-mediated translation of Cistron 2 occurs independently of translation of the upstream Cistron 1
• It is unaffected when Cistron 1 translation is abrogated by inserting a hairpin at a cap-proximal position that prevents
ribosomal attachment.
RRL (Rabbit
Reticulocyte Lysate) RNA construct
+ S35-Methionine (radioactive
amino acid)
@37C, 60’
Protein expressed is exposed to
film after running on gel
Expected protein size
Marker
Cistron 1 Cistron 2
Expression Expression
+
+
+
+
+
_
_
_
RNA construct
IRES
Cistron 1 Cistron 2
Inter-cistronic
region
IRES
IRES
5’
5’
5’
5’
3’
3’
3’
3’
IRES
5’ 3’ +_
Dicistronic
construct
Dicistronic
construct (ΔIRES)
Dicistronic
construct (stem)
Monocistronic
construct (stem)
Different RNA constructs with IRES inserted in the intergenic region are in-vitro translated in mammalian system
such as rabbit reticulocyte lysate (RRL) and protein expressed determines the IRES activity.
In-vitro Translation in RRL
15. Translational activity of 5’UTR CDV IRES in
Rabbit Reticulocyte lysate (RRL)
Conclusions –
• The CDV-A 5’UTR IRES can promote translation in
RRL and requires eIF4A for its activity.
Next Step -
• The activity of these IRESs in RRL justifies the use
of (a) our mammalian in vitro reconstituted system
and (b) Toe-printing analysis of 48S complex
formation in RRL to investigate their mechanisms of
action.
- + 4AR362QMono-cistronic
(stem)
Di-cistronic
Di-cistronic
(stem)
5’UTRCDV RNA
constructs
IRES dependent 2nd
cistron
5’-cap dependent 1st
cistron
25
35
40
55
70
15
Inhibition of translation of an mRNA by a
dominant-negative form of eIF4A indicates
that initiation on the mRNA occurs by an
eIF4G/eIF4A-dependent mechanism.
- + -
Mechanism of Action of
eIF4AR362Q mutant
16. Toe-printing technique RNA
48S/80S
complexes
sequence
Full-length cDNA
48S/80S complex
(15-17 nts from the P-site codon (AUG))
P-site codon
(AUG)
Analysis of 48S/80S complex formed in RRL and in in-vitro
reconstitution system using Toeprinting approach
2) In vitro reconstituted system1) Arresting 48S/80S in RRL
All the initiation factors and ITAFs required for the
activity of the CDV-A 5’UTR IRES are present in RRL.
80S complexes formed in RRL are then arrested using
cycloheximide.
Using
Cycloheximide
(CHX), a protein
translation inhibitor
- Arrest translation after the first
cycle of elongation
5’
E P A
48S complex
E P A
5’
80S
RTRT
AUG AUG
Initiation factors:
2, 3, 4A, 4B, 4F, 1, 1A, 5, 5B
40S and 60S
subunits
Met-tRNAi
Met
mRNA
E P A
5’
80S
AUG
E P A
48S complex
AUG5’
DHX29
5’
E P A
48S complex
R
AUG
Initiation factors:
2, 3, 4A, 4B, 4F, 1, 1A, 5, 5B
40S and 60S
subunits
Met-tRNAi
Met
mRNA
E P A
48S complex
AUG5’
DHX29
Reverse Transcription
The required initiation factors and ITAFs are either
purified from RRL or expressed recombinantly in E.coli
and then added to the reaction in-vitro separately to
assemble 48S complex on the desired messenger RNA.
17. Toe-printing analysis of 48S complex formation on 5’UTR IRES of CDV in
RRL and in-vitro reconstitution system
C T A G
-
+Recomb.itRNA
+EcoliitRNA
+NativeitRNA
+PCBP1
+PCBP2
+40S/1/1A/2/3/
4A/4B/4G
Native
itRNA
AUG 983
UUG 951
UUG 974
48Son AUG
48Son UUG 974
RRL
80S on AUG 983
40S/eIF1/1A/2/3/4A/4B/4Gm
Ecoli itRNA
RRL
Cycloheximide (20ug)
- - - -
- - - -
- + + +
- - + +
Conclusions –
1. 48S complexes form on the authentic AUG both in
the in vitro reconstituted mammalian system and in
RRL.
2. In the absence of ITAFs, 48S complexes formed
on the authentic CDV-A initiation codon (AUG-983)
and upstream near cognate UUG 974 with E.coli
and in vitro transcribed mammalian Met-tRNAMet
i,
but not with native crude mammalian Met-tRNAMet
i,
in which case 48S complex formation additionally
required PCBP2.
3. The contaminants present in native tRNAMet
i (total)
compete with the IRES for RNA binding proteins
such as eIF4G or eIF4A and thus do not allow 48S
complexes to assemble on this IRES. PCBP2
enables the IRES to win this competition either by
increasing the binding of initiation factors or by
changing the conformation of IRES to facilitate
attachment of 43S complexes.
Next Step –
1. To test which canonical initiation factors are
necessary for assembly of 48S complexes on the
5’UTR IRES.
18. Conclusion -
•eIF2, 3, 4A and 4G are essential for 48S
assembly, while eIF4B stimulated the
activity of this IRES.
• In the absence of eIF1 or 1A, near-
cognate codons such as UUG951 and
UUG974 upstream of the authentic
AUG983 were selected. Selection of the
authentic initiation codon is thus
determined by eIF1/1A.
• The 43S pre-initiation complex attaches
to the IRES upstream of domain N and
scans downstream towards the authentic
codon AUG983.
•The eIF4G-eIF3 interaction is not
obligatory for ribosome loading onto the
CDV-A IRES (in contrast to poliovirus).
•Next Step-
• Since the upstream UUG951 and
UUG974 were selected, IRES mutants will
be designed to determine the earliest
point from which incoming 43S
complexes can begin inspection of the
mRNA.
Initiation Factor requirements for 48S complex formation on the
CDV-A 5’UTR IRES
C T A G
-40S
-eIF1
-eIF1A
-eIF2
-eIF3
-eIF4A
-eIF4B
-eIF4Gm
40S/Native itRNA + PCBP2 +
initiation factors except
AUG 983
UUG 951
UUG 974
48S AUG 983
48S UUG 974
48S UUG 951
C T A G
-40S
-eIF1
-eIF1A
-eIF2
-eIF3
-eIF4A
-eIF4B
-eIF4Gm
40S/Native itRNA + PCBP2 +
initiation factors except
AUG 983
UUG 951
UUG 974
48S AUG 983
48S UUG 974
48S UUG 951
5’UTR MC RNA
40S/ Native itRNA/eIF1/1A/2/3/4A/4B/PCBP2
eIF4F
eIF4Gm 736-1115
eIF4G 736-1008
eIF4G 736-988
+ + + + + +
+ + + + +
+
+
+
+
48S AUG 983
5’UTR MC RNA
40S/ Native itRNA/eIF1/1A/2/3/4A/4B/PCBP2
eIF4F
eIF4Gm 736-1115
eIF4G 736-1008
eIF4G 736-988
+ + + + + +
+ + + + +
+
+
+
+
48S AUG 983
N
N
PABP eIF4E eIF4A eIF4A Mnk1eIF3
eIF4G1
2Apro
1 1599
746
992
1015
1104
eIF4G736-1115 (eIF4Gm)
eIF4G736-1008
eIF4G736-988
eIF3eIF4A
eIF4A
eIF4A
951
974 983
AUG 983 is the authentic initiation codon
19. UUG 951 – good AUG 950
Introduced AUG950 is in-frame with the authentic AUG983
Conclusion -
•An optimized AUG triplet introduced at nt. 950 (upstream of the
authentic AUG983) is active in the in vitro reconstitution system and
functions independently of PCBP2.
Mutational Analysis of the CDV-A 5’UTR IRES to locate the point from which
ribosomal inspection of the mRNA begins
28
38
49
62
-
Lucf.
Wt.IRESmRNA
GoodAUG950mRNA
Product from
AUG 950
Product from
AUG 983
28
38
49
62
-
Lucf.
Wt.IRESmRNA
GoodAUG950mRNA
Product from
AUG 950
Product from
AUG 983
N
951
974 983
In-vitro Translation
20. Fe(III) 1- (p-Bromoacetamidobenzyl)
ethylene diamine tetraacetic acid
Iron –EDTA (chelating agent)
Mechanism of Action of HRC Assay
Locating binding site and Orienting Initiation factors and PCBP2
on 5’UTR CDV IRES
Cleavages generated are
then analyzed using
Reverse transcription.
Fe-BABE
The sulfhydryl group of endogenous
cysteine or single cysteine mutants of the
protein are reacted with bromoacetyl
moiety of FeBABE. (Site specific iron
chelates)
The hydroxyl radicals are generated
using ascorbic acid and H2O2. The target
nucleic acid if known to be bound by the
chelated protein, the radicals will cleave
the nucleic acid in the vicinity of binding
site.
Reacted with target
RNA
+ Ascorbic acid,
H2O2, @37C, 10’
Fenton
Reaction
Fenton
Reaction
A480C
mutant of
protein
In order to locate the binding site of initiation factors such as eIF4G and eIF4A and ITAF (PCBP2)
which are known to bind the poliovirus IRES, I used Hydroxyl Radical cleavage assay (HRC)
Schematic
diagram
21. 1) Locating binding site of Initiation factors eIF4G and eIF4A
-
wt.
C-less
D928DC
C830
Wt.
C-less
D928DC
C830
+ eIF4A + eIF4G
C T A G
S33C
S42C
Cys-less
S33C
S42C
Cys-less
Cysteine
mutants
a) FeBABE-eIF4G wt/mutants b) FeBABE-eIF4A mutants
Conclusions –
• eIF4G interacts with domain M.
• The interaction is enhanced by eIF4A
•eIF4A does not bind directly but is
recruited by eIF4G in the
vicinity of domain M.
G905 - U911
A804 - C812
C897- C903
G819 - C829
C872 – U877
A882- U885
A835
C T A G
Cysteine
mutants
22. Comparison of eIF4G and eIF4A binding site on PV1 and CDV-A IRESs
Cys929
Wt/ Cys819/821/847/919/934/936
Cys829
Cys33
Cys42
eIF4GI736–1115
eIF4A
Poliovirus
(domain V)
Cadicivirus A
(domain M)
23. KH3
domain
KH1
domain
KH2
domain
E34C
S141C
A308C
S330C
C54
C118
119aa linker
GXXG
motif
GXXG
motif
GXXG
motif
• Common ITAF necessary for Type1 IRESs.
• It binds to Type 1 IRESs via cooperative interactions
at distinct sites.
•Three hnRNP K-homology domains – KH1 and KH2 are
arranged back to back while KH3 is mobile, being
separated by 119 amino acid from KH1-KH2 domains.
•Each KH domain accommodates 4 nucleic bases in the
binding cleft formed by
α1, α2 and invariant GXXG connecting motif on
one side
Β2 and a variable loop on the other
•Forms hetero-multimers with PCBP1
Locating PCBP2’s binding site on the CDV-A IRES using directed
hydroxyl radical cleavage
KH1 KH2 KH3
13 81 97 169 288 356
Protein
(cys-less)
Surface exposed Single cysteine
mutants
PCBP2 E34C, S54C, S118C, S141C, A308C,
S330C
24. Locating PCBP2’s binding site on the CDV-A IRES using directed
hydroxyl radical cleavage
-
C-less
C54
C34
C118
C141
C308
C330
C T A G
A748-U753
G734-U737
G734-U737
A718-U724
A718-U724
U710-A717
U700-U705
A703-U707
C666-A674
U673-G679
C664-U678
C657-G663
U650-U655
A610-C613
A600-A603
G881-C599
U573-C578
A450-G452
C436-C441
U418-G420
Conclusion –
•As seen for Poliovirus, KH2 and KH3 domains are close to each
other when bound to IRES. KH1 gave strong cleavages near GNRA
loop of 5’UTR CDV IRES.
• Being flexible, Domain 3 can also bind to a distant stem of domain
H/I.
Domain K of
5’UTR CDV IRES Domain IV of
Poliovirus IRES
GNRA loop
KH 1
KH 2
KH 3
GNRA loop
25. Conclusions
Similarities and Differences between the mechanisms of 48S complex formation on the CDV-A IRES and
on the Type 1 (poliovirus) IRES
SIMILARITIES
1. Initiation depends on specific binding of eIF4G’s central domain to homologous, conserved domains of these IRESs.
2. Initiation requires eIF4A, which is recruited by eIF4G to the same site on both IRESs.
3. Both IRESs depend on the same ITAF, PCBP2, that binds to structurally similar sites on both.
4. Following attachment to the IRES, the 43S complex reaches the initiation codon by scanning.
DIFFERENCES
1. The PCBP2 binding site is differently arranged in CDV-A and Type 1 IRESs.
eIF4G
eIF4A
PCBP2
eIF4G
eIF4A
PCBP2
2. Domain VI is unwound
‘Poor context’ AUG is not inspected
eIF1 is not required.
3. eIF3 – eIF4G is required
2. Domain N is unwound
‘Near-cognate’ UUG is inspected
eIF1/1A is required.
3. eIF3 – eIF4G is not required
5’UTR CDV-A IRESPoliovirus IRES
26. 5’UTR CDV IRES –
1. To locate the exact ribosomal loading site
2. To map the 5’-terminal border of the IRES
Future Plans
∆nt. 341 – 982 (domain H – N)
∆nt. 518 – 982 (domain K – N)
∆nt. 553 – 982 (domain ΔK-N)
A
B
C
D
F
G
H
I
J
K
L
M
N
UUG
AUG
983
Placing good AUG upstream
of Domain N at 944 in the wild
type construct
a) By replacing domain N of CDV-A with
Domian VI of poliovirus IRES
b) By placing good context AUG at 944
upstream of Domain N of wild type
construct.
a) By truncating the IRES from 5’ end
of the IRES and testing its activity in
RRL.
27. Acknowledgements
• Mentor –
Dr. Tatyana Pestova
Dr. Christopher Hellen
• SUNY Downstate Medical Center
• And SigmaXi for hosting this showcase.