BMB-9 Interaction Between the Viral RNA Leader Sequence and NSP1 in SARS-Coronavirus-1 and 2
Abstract
Nonstructural protein 1 (nsp1) of recently identified human coronaviruses (SARS-CoV-1, SARS-CoV-2) acts as the host shutoff protein that inhibits host mRNA translation by blocking the translation initiation site on ribosomal proteins and initiating cleavage and decay of host mRNA, thus suppressing host gene expression. However, viral RNA selectively evades this host shutoff mechanism and continues to translate viral proteins due to a specific stem-loop structure in the viral leader sequence (SL1). Previous work done in our lab with SARS-CoV-1 SL1 and nsp1 showed that nsp1 binds to SL1 of the viral RNA forming multiple complexes, and mutations in this region impacted nsp1 binding and changed the nature of complex formation. To further investigate this binding, we used gel-shift assay and RNA pull-down to verify binding between nsp1 and SL1 in SARS-CoV-1, SARS-CoV-2, and crossed between the two viruses due to the literature indicating a markedly similar structure for nsp1 and SL1 in both. By mutating SL1 and attempting cross binding between the two related viruses, we seek to identify specific nucleotides of SL1 that bind to nsp1.
To investigate the binding of nsp1 to SL1, we used nsp1 purified from GST-nsp1 from bacterial lysate using glutathione beads followed by precision protease cleavage, and biotinylated RNA. LightShift Chemiluminescence RNA EMSA Kit (Promega) was used to detect the RNA in complex with nsp1 using a gel shift assay. In our cross binding experiments, we found that SL1 from SARS-CoV-1 and SARS-CoV-2 can bind with nsp1 from either virus, including with mutant RNA altering the stem and/or loop of SL1. However, the gel shift assay indicates the formation of complexes of varying molecular weights based on which variation of nsp1 and SL1 is used. We hypothesize that this is likely due to a difference in binding sites, with heavier complexes possibly even having more than one nsp1 bound to the viral RNA. This hypothesis will be investigated further along with continued experiments with mutant RNA to identify specific nucleotides of SL1 that bind to nsp1 for each virus.
Keywords
SARS-Coronavirus-1, SARS-Coronavirus-2, RNA, NSP1
BMB-9 Interaction Between the Viral RNA Leader Sequence and NSP1 in SARS-Coronavirus-1 and 2
University Readiness Center Greatroom
Nonstructural protein 1 (nsp1) of recently identified human coronaviruses (SARS-CoV-1, SARS-CoV-2) acts as the host shutoff protein that inhibits host mRNA translation by blocking the translation initiation site on ribosomal proteins and initiating cleavage and decay of host mRNA, thus suppressing host gene expression. However, viral RNA selectively evades this host shutoff mechanism and continues to translate viral proteins due to a specific stem-loop structure in the viral leader sequence (SL1). Previous work done in our lab with SARS-CoV-1 SL1 and nsp1 showed that nsp1 binds to SL1 of the viral RNA forming multiple complexes, and mutations in this region impacted nsp1 binding and changed the nature of complex formation. To further investigate this binding, we used gel-shift assay and RNA pull-down to verify binding between nsp1 and SL1 in SARS-CoV-1, SARS-CoV-2, and crossed between the two viruses due to the literature indicating a markedly similar structure for nsp1 and SL1 in both. By mutating SL1 and attempting cross binding between the two related viruses, we seek to identify specific nucleotides of SL1 that bind to nsp1.
To investigate the binding of nsp1 to SL1, we used nsp1 purified from GST-nsp1 from bacterial lysate using glutathione beads followed by precision protease cleavage, and biotinylated RNA. LightShift Chemiluminescence RNA EMSA Kit (Promega) was used to detect the RNA in complex with nsp1 using a gel shift assay. In our cross binding experiments, we found that SL1 from SARS-CoV-1 and SARS-CoV-2 can bind with nsp1 from either virus, including with mutant RNA altering the stem and/or loop of SL1. However, the gel shift assay indicates the formation of complexes of varying molecular weights based on which variation of nsp1 and SL1 is used. We hypothesize that this is likely due to a difference in binding sites, with heavier complexes possibly even having more than one nsp1 bound to the viral RNA. This hypothesis will be investigated further along with continued experiments with mutant RNA to identify specific nucleotides of SL1 that bind to nsp1 for each virus.