Nonstructural Proteins Of SARS-CoV-2 In Mediating ER Stress Studies

The novel coronavirus pneumonia(Corona Virus Disease 2019,COVID-19)has greatly affected human health and social development.The RNA synthesis of its pathogen,the novel coronavirus(SARS-CoV-2),relies on 16 non-structural proteins(Non-structural Proteins,NSPs)produced by hydrolysis of the ORF1 gene of the 5’ genomic sequence after expression.Similar to other coronaviruses,the NSPs of SARS-CoV-2 forms a cell-like organelle suitable for viral RNA synthesis by modifying the host cell endoplasmic reticulum(ER)and other cell membrane systems.On one hand,we explore the key pathways of host cell ER changes caused through overexpressing of 16 NSPs of SARS-CoV-2,respectively;on the other hand,we construct a SARS-CoV-2-deficient Interfering RNA(DI RNA,replicon),which mimics viral replication and provides support for analyzing the effect of NSPs on viral RNA synthesis in routine laboratories.The main results of this study are as follows:(1)Effects of different NSPs on the signaling pathway of the ER stress responseIn this study,NSP1-NSP16 overexpression plasmids are constructed.Except for NSP6,the rest of the plasmids are well expressed normally in 293 T cells.We observed that NSP1,NSP2,NSP3,NSP4,NSP6,NSP9,NSP10,and NSP16 can induce significant upregulation of HSPA5 protein.NSP4 significantly upregulates HSPA5 m RNA expression at both 24 h,48h,and 72 h,indicating that NSP4 plays a key role in inducing host cell ER stress response.The q PCR results showed that the overexpression of NSP2,N-NSP3,C-NSP3,and NSP16 at 24 h and 72 h do not cause significant upregulation of HSPA5 m RNA,but the experimental data at 48 h showed that NSP2,N-NSP3,C-NSP3,NSP4,and NSP16 significantly upregulated HSPA5 m RNA,indicating most of NSPs of SARS-CoV-2 can induce ER stress response.Then,We analyzed the function of NSPs and found that N-NSP3,NSP4,and NSP16 significantly causes XBP1 cleavage in the IRE1α pathway.Overexpression of NSP2 and N-NSP3 significantly induce cleavage of ATF6,indicating that N-NSP3,NSP4 and NSP16 act on the IRE1α pathway in the unfolded protein reaction(UPR),and NSP2 and NSP3-N mainly act on the ATF6 pathway.(2)Construction and preliminary verification of SARS-CoV-2 defective interfering RNATo make the study that analyzes the effects of NSPs on viral RNA synthesis in a routine laboratory possibility,we firstly constructed the replicon containing hammerhead ribozyme,5’UTR sequence of SARS-CoV-2,120 bp ORF1ab sequence,m Cherry reporter gene,ORF10,3’UTR,and Hepatitis delta virus ribozyme named it as Rep-s.Then the N gene was inserted between the m Cherry reporter gene and ORF10 of Rep-s to obtain the recombinant plasmid N-Rep-s.To study the effect of ORF1 ab on the replicon,we extends the 5’UTR-ORF1 ab fragment to 502 bp,751bp,and 1501 bp,respectively for constructing recombinant plasmids Rep-502-s,Rep-751-s,Rep-1501-s and N-Rep-1501-s.We also constructs the negative-strand replicon named as Rep-as,Rep-502-as,Rep-751-as,N-Rep-as,and N-Rep-1501-as.After that,these plasmids were transfected into He La-h ACE2 cells,we observed that the positive strand replicon can be transcribed normally,and the ORF1-m Cherry fusion protein also showed a red fluorescent signal,indicating that the protein translation was correct,but the fluorescence intensity mildly decreased at the 5’UTR-ORF1 ab extended to 1501 bp.However,the negative-strand replicon cannot normally translate red fluorescent protein.To verify whether the replicon can mimic virus replication in He La-h ACE2 cells with infecting SARS-CoV-2,we testify the probable RNA synthesis of the replicon in the condition of a helper virus which provide the expressions of NSPs required by SARS-CoV-2 RNA synthesis.We found that the positive-strand DI RNA can be replicated and expressed,but its fluorescence intensity is not significantly affected by helper virus.Most of the negative-strand replicon dose not express and does not show fluorescence by helper virus,while the negative-strand replicon containing the N gene shows weak fluorescence by helper virus.It is preliminarily believed that the length of ORF1 ab and the N gene have key effects on SARS-CoV-2 RNA synthesis.Due to the insensitivity of the m Cherry reporter gene,we believe that the currently constructed replicon cannot reflect the virus replication efficiently,and further exploration of the replicon structure is needed.Based on the above research,the function of NSP was analyzed and the replication subsystem was established,which is of great significance for further understanding of SARS-CoV-2.