Session Type: ePosters
Session Title: ePosters
Authors(s): B.Y. Alhatlani (1), W. Aljabr (2), E. Emmott (3), F. Alhamlan (4), A. Almatroudi (5)
Authors Affiliations(s): (1) Department of Applied Medical Sciences, Unayzah Community College, Qassim University, Saudi Arabia, (2) Research Center, King Fahad Medical City, Saudi Arabia, (3) Institute of Systems, Molecular and Integrative Biology, University of Liverpool, United Kingdom, (4) Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, Saudi Arabia, (5) Department of Clinical Laboratory, College of Applied Medical Sciences, Saudi Arabia
Background:
The Coronaviridae family contains a variety of viruses that cause a wide range of diseases in human and animals. Coronaviruses (CoVs) include two highly pathogenic viruses which are the MERS-CoV responsible for the middle eastern respiratory syndrome and the newly emerged SARS-CoV-2 which is responsible for the COVID-19 pandemic, and both viruses belong to the Betacoronavirus genus. CoVs have the largest genomes among all RNA viruses, and contain enveloped positive-sense, single-stranded RNA molecule (+ve ssRNA). The termini of the CoVs genomes are predicted to fold into highly conserved RNA structures known as cis-acting regulatory elements. In this study, we aimed to identify host factors that interact with the 5’ end of the SARS-CoV-2 and MERS-CoV genomic RNAs.
Methods:- The first 500 nucleotides of the SARS-CoV-2 and MERS-CoV 5’ ends were amplified using PCR with a forward oligo that has the T7 promoter sequence, followed by in vitro transcription.
- RNA transcripts were biotinylated at the 3’ end together with a non-labelled RNA control.
- Cell lysates were prepared from Vero E6 cell lines that were cultured to reach 80% confluence.
- Streptavidin magnetic beads Dynabeads were used for the RNA affinity capture pull down in which the biotinylated viral RNA and biotin labelled control RNA were initially incubated with Dynabeads.
- Biotinylated RNA- streptavidin magnetic beads were then incubated with Vero E6 lysates.
- The RNA/protein/beads complexes were washed with protein washing buffer, eluted in 5X SDS PAGE loading buffer, and the bound proteins were analyzed in 12% SDS-PAGE gels.
- Preliminary results suggested that the 5’ end of the SARS-CoV-2 and MERS-CoV genomes interact with several host proteins.
- These viral/host interactions are identified using mass spectrometry.
- Further work will be required to validate these interactions.
In this study, we used bioinfromatics, molecular, and proteomics techniques in order to identify several host factors that interact with the 5' end of the RNA genome of SARS-CoV-2 and MERS-CoV. The identification of such interactions could facilitate the development of antiviral theraputics and/or vaccines against these important pathogenes.
Keyword(s): Coronaviruses, RNA structures, Viral/host interactionsSession Type: ePosters
Session Title: ePosters
Authors(s): B.Y. Alhatlani (1), W. Aljabr (2), E. Emmott (3), F. Alhamlan (4), A. Almatroudi (5)
Authors Affiliations(s): (1) Department of Applied Medical Sciences, Unayzah Community College, Qassim University, Saudi Arabia, (2) Research Center, King Fahad Medical City, Saudi Arabia, (3) Institute of Systems, Molecular and Integrative Biology, University of Liverpool, United Kingdom, (4) Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, Saudi Arabia, (5) Department of Clinical Laboratory, College of Applied Medical Sciences, Saudi Arabia
Background:
The Coronaviridae family contains a variety of viruses that cause a wide range of diseases in human and animals. Coronaviruses (CoVs) include two highly pathogenic viruses which are the MERS-CoV responsible for the middle eastern respiratory syndrome and the newly emerged SARS-CoV-2 which is responsible for the COVID-19 pandemic, and both viruses belong to the Betacoronavirus genus. CoVs have the largest genomes among all RNA viruses, and contain enveloped positive-sense, single-stranded RNA molecule (+ve ssRNA). The termini of the CoVs genomes are predicted to fold into highly conserved RNA structures known as cis-acting regulatory elements. In this study, we aimed to identify host factors that interact with the 5’ end of the SARS-CoV-2 and MERS-CoV genomic RNAs.
Methods:- The first 500 nucleotides of the SARS-CoV-2 and MERS-CoV 5’ ends were amplified using PCR with a forward oligo that has the T7 promoter sequence, followed by in vitro transcription.
- RNA transcripts were biotinylated at the 3’ end together with a non-labelled RNA control.
- Cell lysates were prepared from Vero E6 cell lines that were cultured to reach 80% confluence.
- Streptavidin magnetic beads Dynabeads were used for the RNA affinity capture pull down in which the biotinylated viral RNA and biotin labelled control RNA were initially incubated with Dynabeads.
- Biotinylated RNA- streptavidin magnetic beads were then incubated with Vero E6 lysates.
- The RNA/protein/beads complexes were washed with protein washing buffer, eluted in 5X SDS PAGE loading buffer, and the bound proteins were analyzed in 12% SDS-PAGE gels.
- Preliminary results suggested that the 5’ end of the SARS-CoV-2 and MERS-CoV genomes interact with several host proteins.
- These viral/host interactions are identified using mass spectrometry.
- Further work will be required to validate these interactions.
In this study, we used bioinfromatics, molecular, and proteomics techniques in order to identify several host factors that interact with the 5' end of the RNA genome of SARS-CoV-2 and MERS-CoV. The identification of such interactions could facilitate the development of antiviral theraputics and/or vaccines against these important pathogenes.
Keyword(s): Coronaviruses, RNA structures, Viral/host interactions
Session Type: ePosters
Session Title: ePosters
Authors(s): B.Y. Alhatlani (1), W. Aljabr (2), E. Emmott (3), F. Alhamlan (4), A. Almatroudi (5)
Authors Affiliations(s): (1) Department of Applied Medical Sciences, Unayzah Community College, Qassim University, Saudi Arabia, (2) Research Center, King Fahad Medical City, Saudi Arabia, (3) Institute of Systems, Molecular and Integrative Biology, University of Liverpool, United Kingdom, (4) Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, Saudi Arabia, (5) Department of Clinical Laboratory, College of Applied Medical Sciences, Saudi Arabia
Background:
The Coronaviridae family contains a variety of viruses that cause a wide range of diseases in human and animals. Coronaviruses (CoVs) include two highly pathogenic viruses which are the MERS-CoV responsible for the middle eastern respiratory syndrome and the newly emerged SARS-CoV-2 which is responsible for the COVID-19 pandemic, and both viruses belong to the Betacoronavirus genus. CoVs have the largest genomes among all RNA viruses, and contain enveloped positive-sense, single-stranded RNA molecule (+ve ssRNA). The termini of the CoVs genomes are predicted to fold into highly conserved RNA structures known as cis-acting regulatory elements. In this study, we aimed to identify host factors that interact with the 5’ end of the SARS-CoV-2 and MERS-CoV genomic RNAs.
Methods:- The first 500 nucleotides of the SARS-CoV-2 and MERS-CoV 5’ ends were amplified using PCR with a forward oligo that has the T7 promoter sequence, followed by in vitro transcription.
- RNA transcripts were biotinylated at the 3’ end together with a non-labelled RNA control.
- Cell lysates were prepared from Vero E6 cell lines that were cultured to reach 80% confluence.
- Streptavidin magnetic beads Dynabeads were used for the RNA affinity capture pull down in which the biotinylated viral RNA and biotin labelled control RNA were initially incubated with Dynabeads.
- Biotinylated RNA- streptavidin magnetic beads were then incubated with Vero E6 lysates.
- The RNA/protein/beads complexes were washed with protein washing buffer, eluted in 5X SDS PAGE loading buffer, and the bound proteins were analyzed in 12% SDS-PAGE gels.
- Preliminary results suggested that the 5’ end of the SARS-CoV-2 and MERS-CoV genomes interact with several host proteins.
- These viral/host interactions are identified using mass spectrometry.
- Further work will be required to validate these interactions.
In this study, we used bioinfromatics, molecular, and proteomics techniques in order to identify several host factors that interact with the 5' end of the RNA genome of SARS-CoV-2 and MERS-CoV. The identification of such interactions could facilitate the development of antiviral theraputics and/or vaccines against these important pathogenes.
Keyword(s): Coronaviruses, RNA structures, Viral/host interactionsSession Type: ePosters
Session Title: ePosters
Authors(s): B.Y. Alhatlani (1), W. Aljabr (2), E. Emmott (3), F. Alhamlan (4), A. Almatroudi (5)
Authors Affiliations(s): (1) Department of Applied Medical Sciences, Unayzah Community College, Qassim University, Saudi Arabia, (2) Research Center, King Fahad Medical City, Saudi Arabia, (3) Institute of Systems, Molecular and Integrative Biology, University of Liverpool, United Kingdom, (4) Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, Saudi Arabia, (5) Department of Clinical Laboratory, College of Applied Medical Sciences, Saudi Arabia
Background:
The Coronaviridae family contains a variety of viruses that cause a wide range of diseases in human and animals. Coronaviruses (CoVs) include two highly pathogenic viruses which are the MERS-CoV responsible for the middle eastern respiratory syndrome and the newly emerged SARS-CoV-2 which is responsible for the COVID-19 pandemic, and both viruses belong to the Betacoronavirus genus. CoVs have the largest genomes among all RNA viruses, and contain enveloped positive-sense, single-stranded RNA molecule (+ve ssRNA). The termini of the CoVs genomes are predicted to fold into highly conserved RNA structures known as cis-acting regulatory elements. In this study, we aimed to identify host factors that interact with the 5’ end of the SARS-CoV-2 and MERS-CoV genomic RNAs.
Methods:- The first 500 nucleotides of the SARS-CoV-2 and MERS-CoV 5’ ends were amplified using PCR with a forward oligo that has the T7 promoter sequence, followed by in vitro transcription.
- RNA transcripts were biotinylated at the 3’ end together with a non-labelled RNA control.
- Cell lysates were prepared from Vero E6 cell lines that were cultured to reach 80% confluence.
- Streptavidin magnetic beads Dynabeads were used for the RNA affinity capture pull down in which the biotinylated viral RNA and biotin labelled control RNA were initially incubated with Dynabeads.
- Biotinylated RNA- streptavidin magnetic beads were then incubated with Vero E6 lysates.
- The RNA/protein/beads complexes were washed with protein washing buffer, eluted in 5X SDS PAGE loading buffer, and the bound proteins were analyzed in 12% SDS-PAGE gels.
- Preliminary results suggested that the 5’ end of the SARS-CoV-2 and MERS-CoV genomes interact with several host proteins.
- These viral/host interactions are identified using mass spectrometry.
- Further work will be required to validate these interactions.
In this study, we used bioinfromatics, molecular, and proteomics techniques in order to identify several host factors that interact with the 5' end of the RNA genome of SARS-CoV-2 and MERS-CoV. The identification of such interactions could facilitate the development of antiviral theraputics and/or vaccines against these important pathogenes.
Keyword(s): Coronaviruses, RNA structures, Viral/host interactions