| The COVID-19 pandemic is still spreading all over the world.Human life and health are seriously threatened,and people’s daily life and social and economic development are hindered.A complete understanding of the life cycle and the pathogenic mechanism of the SARS-CoV-2will help us to develop vaccines and therapeutic drugs.The molecular mechanism of replication and transcription of SARS-CoV-2 in infected cells remains unknown.It has been confirmed that host proteins play an essential role in the expression and regulation of the viral genome,so identification and analysis of their functions will help to understand the pathogenic mechanism of the virus.After SARS-CoV-2 virus infects cells,the full-length positive-strand genomic RNA acts as a template for replication and transcription to generate full-length negative-strand RNA for genome replication and negative-strand subgenomic RNA replication.The subgenomic RNA shares the same 5’leader sequence and 3’UTR as the genomic RNA.The UTRs of coronaviruses play an important role in viral replication,translation and evasion of cellular antiviral processes.Subgenomic RNA and genomic RNA share the same 5’leader sequence and 3’UTR.The UTR of negative-strand RNA and negative-strand subgenomic RNA is also an important region where the replication-transcription complex bind to initiate the synthesis of positive-strand RNA.However,the host proteins that bind to UTR are little known.Therefore,we will screen the host proteins that interact with the UTR of the positive and negative strands to gain a deeper understanding of the pathogenic mechanism of SARS-CoV-2,and design,synthesize and screen DNazymes that cleave viral UTRs as antiviral drugs.Through this study,we hope to gain a deeper understanding of the pathogenic mechanism of coronavirus and provide a theoretical basis for the development of anti-coronavirus drugs.In this paper,the pGM-T vector was used to construct the cloning vector of SARS-CoV-2UTR.After enzymatic digestion of four PGM-T-UTR vectors,RNAs were synthesized by in vitro transcription,and the 3’ end of RNA was connected with biotinylated molecules,and the host proteins interacting with the UTRs were obtained by RNA pulldown.And protein molecules were identified and analyzed by mass spectroscopy.The SARS-CoV-2 positivestrand 5’UTR and 3’UTR respectively identified 5 and 36 potentially binding host proteins,and the negative-strand 3’UTR identified 62 potentially binding host proteins.These proteins are involved in biological processes such as translation regulation,actin filament assembly,and cytoskeleton regulation,etc.The 103 proteins mentioned above were analyzed using the pharmacogenetic interaction database,and 14 proteins of them already have inhibitors.At the same time,the 5’UTR and 3’UTR secondary structures of the positive and negative strands of SARS-CoV-2 were predicted.AU bases in the four RNA sequences were selected as the cleavage sites,and 61 specific DNazymes were designed and synthesized.The effects of DNazymes in vitro and in cells were evaluated by the cleavage assay and the dual luciferase reporter gene assay.22 DNAzymes with significant cleavage activity were obtained by comprehensive evaluation.These results provide an experimental basis for further elucidation of the interaction between host proteins and SARS-CoV-2 RNA,elucidating the pathogenic mechanism of SARS-CoV-2,and provide a basis for the development of SARS-CoV-2 drugs. |
