PC-02 Interaction Between the Viral RNA Leader Sequence and nsp1 in SARS Coronavirus
Start Date
31-3-2023 10:30 AM
End Date
31-3-2023 12:30 PM
Document Type
Poster
Abstract
Introduction and background: Nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus (SARS-CoV), inhibits host translation by blocking the translation initiation site on the 40S ribosome and cleaving host mRNA. Stem-Loop 1 (SL1) of the viral RNA leader sequence has been identified to bind to nsp1, allowing viral RNA to escape translation repression. However, the specific residues on nsp1 and the specific sequences on SL-1 important to binding have not been experimentally verified.
Hypothesis and goals: To investigate this binding, we used gel-shift assay and RNA pull-down to verify the binding between nsp1 and SL1. By mutating SL-1 and nsp1, we seek to map the sequence responsible for this interaction. Interestingly, nsp1 is a small protein with intrinsically unstructured regions at both C- and N- terminal ends of the protein. Based on recent literature we hypothesize that the C-terminal domain binds to the stem structure of SL1 and disrupting the stem region of SL1 will decrease binding between nsp1 and SL-1
Methods and results: To investigate the binding of nsp1 to SL1, we used nsp1 purified from bacterial overexpression using glutathione beads followed by precision protease cleavage of GST-nsp1, and biotinylated RNA. We used chemiluminescence to detect the RNA in a complex with nsp1 using a gel shift assay. Contrary to our hypothesis, we found an increase in nsp1 binding to the RNA carrying stem mutation and a decrease in nsp1 binding to the RNA with the loop mutation. Currently, we are further investigating several mutations in SL1 to identify the actual binding site.
Conclusion: We confirmed the complex formation by nsp1 directly on SL1 using a reconstituted system. Since disrupting the stem-loop region of the RNA decreased binding to nsp1, this region may be critical in allowing viral RNA to escape translation repression.
Acknowledgement: Dr. Nag is supported by the SC INBRE DRP award (2020-23). The nsp1 plasmid was a kind gift from Dr. Makino at UTMB.
PC-02 Interaction Between the Viral RNA Leader Sequence and nsp1 in SARS Coronavirus
Introduction and background: Nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus (SARS-CoV), inhibits host translation by blocking the translation initiation site on the 40S ribosome and cleaving host mRNA. Stem-Loop 1 (SL1) of the viral RNA leader sequence has been identified to bind to nsp1, allowing viral RNA to escape translation repression. However, the specific residues on nsp1 and the specific sequences on SL-1 important to binding have not been experimentally verified.
Hypothesis and goals: To investigate this binding, we used gel-shift assay and RNA pull-down to verify the binding between nsp1 and SL1. By mutating SL-1 and nsp1, we seek to map the sequence responsible for this interaction. Interestingly, nsp1 is a small protein with intrinsically unstructured regions at both C- and N- terminal ends of the protein. Based on recent literature we hypothesize that the C-terminal domain binds to the stem structure of SL1 and disrupting the stem region of SL1 will decrease binding between nsp1 and SL-1
Methods and results: To investigate the binding of nsp1 to SL1, we used nsp1 purified from bacterial overexpression using glutathione beads followed by precision protease cleavage of GST-nsp1, and biotinylated RNA. We used chemiluminescence to detect the RNA in a complex with nsp1 using a gel shift assay. Contrary to our hypothesis, we found an increase in nsp1 binding to the RNA carrying stem mutation and a decrease in nsp1 binding to the RNA with the loop mutation. Currently, we are further investigating several mutations in SL1 to identify the actual binding site.
Conclusion: We confirmed the complex formation by nsp1 directly on SL1 using a reconstituted system. Since disrupting the stem-loop region of the RNA decreased binding to nsp1, this region may be critical in allowing viral RNA to escape translation repression.
Acknowledgement: Dr. Nag is supported by the SC INBRE DRP award (2020-23). The nsp1 plasmid was a kind gift from Dr. Makino at UTMB.