The-1 programmed ribosomal frameshifting(-1 PRF)in severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is crucial for keeping the balance between pp1a and pp1ab polyproteins.To date,the host factors influencing...The-1 programmed ribosomal frameshifting(-1 PRF)in severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is crucial for keeping the balance between pp1a and pp1ab polyproteins.To date,the host factors influencing this process remain poorly understood.Using RNA pull-down assays combined with mass spectrometry screening,we discovered five host proteins interacting with-1 PRF RNA,including Stem Loop Binding Protein(SLBP).Our findings revealed that SLBP overexpression enhanced frameshifting and promoted viral replication.Moreover,the interaction between SLBP and-1 PRF RNA was predicted using the PrismNet deep learning tool,which calculated a high binding probability of 0.922.Using Electrophoretic Mobility Shift Assays(EMSAs)and RNA pull down assays,our findings demonstrated SLBP’s direct binding to the SARS-CoV-2 genome,with preferential affinity for the stem loop 3 region of the-1 PRF RNA.Using smFISH assays,we further confirmed their physical colocalization.The role of SLBP in promoting frameshifting was verified using an in vitro translation system.Further investigation showed that SLBP deletions reshaped the host factor pattern around-1 PRF RNA,diminishing interactions with FUBP3 and RPS3A while enhancing RPL10A binding.Together,our findings identify SLBP as a host protein that promotes SARS-CoV-2 frameshifting,highlighting its potential as a druggable target for COVID-19.展开更多
基金supported by the Major Program of the National Natural Science Foundation of China(No.82394462)the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(82221004)+3 种基金State Key Laboratory Special Fund(2060204)the Yunnan Key R&D Project(202103AQ100001)National Key R&D Program of China(2020YFA0707602,2021YFC230170402,2021YFC0864600,2021YFC0863300)Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences(2023-I2M-2-001,2021-I2M-1-038,2021-I2M-1-039).
文摘The-1 programmed ribosomal frameshifting(-1 PRF)in severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is crucial for keeping the balance between pp1a and pp1ab polyproteins.To date,the host factors influencing this process remain poorly understood.Using RNA pull-down assays combined with mass spectrometry screening,we discovered five host proteins interacting with-1 PRF RNA,including Stem Loop Binding Protein(SLBP).Our findings revealed that SLBP overexpression enhanced frameshifting and promoted viral replication.Moreover,the interaction between SLBP and-1 PRF RNA was predicted using the PrismNet deep learning tool,which calculated a high binding probability of 0.922.Using Electrophoretic Mobility Shift Assays(EMSAs)and RNA pull down assays,our findings demonstrated SLBP’s direct binding to the SARS-CoV-2 genome,with preferential affinity for the stem loop 3 region of the-1 PRF RNA.Using smFISH assays,we further confirmed their physical colocalization.The role of SLBP in promoting frameshifting was verified using an in vitro translation system.Further investigation showed that SLBP deletions reshaped the host factor pattern around-1 PRF RNA,diminishing interactions with FUBP3 and RPS3A while enhancing RPL10A binding.Together,our findings identify SLBP as a host protein that promotes SARS-CoV-2 frameshifting,highlighting its potential as a druggable target for COVID-19.
