Cell-based reporter assay for screening inhibitors of coronavirus RNA-dependent RNA polymerase activity
The present invention relates to a screening composition for a therapeutic agent for coronavirus infection, comprising a CoV RdRp expression vector and a bicistronic reporter vector, a screening kit for a therapeutic agent for coronavirus infection, comprising the composition, and a method for screening a therapeutic agent for coronavirus infection using the composition or kit. When the screening composition for a therapeutic agent for coronavirus infection, provided by the present invention, is used, candidate materials that can have direct influences on the activity of CoV RdRp can be screened more quickly and easily, and thus, the composition can be widely used in the development of therapeutic agents for coronavirus infection.
1 . A screening composition for a therapeutic agent for coronavirus infection, comprising:
(a) a CoV RdRp expression vector comprising a FLAG-labeled CoV (coronavirus) nsp12 gene at the N-terminus; and
(b) a bicistronic reporter vector comprising: a firefly luciferase gene in the sense direction ((+)FLuc) and a NanoLuc luciferase gene in the antisense direction ((−)NLuc); wherein the (−)NLuc forms domains (3′-UTR, NLuc and 5′-UTR) interposed between 3′-UTR and 5′-UTR in the antisense direction derived from CoV; wherein the 3′-UTR, NLuc, and 5′-UTR domains in the antisense direction are in the form in which a ribozyme self-cleaving sequence derived from hepatitis delta virus (HDV) is bound to each of the 5′- and 3′-ends thereof; and a firefly luciferase gene in the sense direction independently of the domain in the antisense direction.
2 . The composition of claim 1 , wherein the CoV nsp12 gene is a MERS-CoV nsp12 gene or a SARS-CoV-2 nsp12 gene.
3 . The composition of claim 2 , wherein the MERS-CoV nsp12 gene comprises a nucleotide sequence of SEQ ID NO: 1.
4 . The composition of claim 2 , wherein the SARS-CoV-2 nsp12 gene comprises a nucleotide sequence of SEQ ID NO: 2.
5 . The composition of claim 1 , wherein the CoV RdRp expression vector is a MERS-CoV RdRp expression vector comprising the MERS-CoV nsp12 gene.
6 . The composition of claim 1 , wherein the CoV RdRp expression vector is a SARS-CoV-2 RdRp expression vector comprising the SARS-CoV-2 nsp12 gene.
7 . The composition of claim 1 , wherein the CoV RdRp expression vector further comprises a gene selected from the group consisting of a CoV nsp7 gene, CoV nsp8 gene, and a combination thereof.
8 . The composition of claim 7 , wherein the CoV nsp7 gene is a MERS-CoV nsp7 gene or a SARS-CoV-2 nsp7 gene.
9 . The composition of claim 8 , wherein the MERS-CoV nsp7 gene comprises a nucleotide sequence of SEQ ID NO: 3.
10 . The composition of claim 8 , wherein the SARS-CoV-2 nsp7 gene comprises a nucleotide sequence of SEQ ID NO: 4.
11 . The composition of claim 7 , wherein the CoV nsp8 gene is a MERS-CoV nsp8 gene or a SARS-CoV-2 nsp8 gene.
12 . The composition of claim 11 , wherein the MERS-CoV nsp8 gene comprises a nucleotide sequence of SEQ ID NO: 5.
13 . The composition of claim 11 , wherein the SARS-CoV-2 nsp8 gene comprises a nucleotide sequence of SEQ ID NO: 6.
14 . The composition of claim 1 , wherein the firefly luciferase gene comprises a nucleotide sequence of SEQ ID NO: 7.
15 . The composition of claim 1 , wherein the NanoLuc luciferase gene comprises a nucleotide sequence of SEQ ID NO: 8.
16 . The composition of claim 1 , wherein the composition is in the form in which each of the CoV RdRp expression vector and the reporter vector are separately included.
17 . The composition of claim 1 , wherein the composition is in the form containing a transfectant, into which the CoV RdRp expression vector and the reporter vector are introduced.
18 . The composition of claim 1 , wherein the coronavirus infection is Middle East respiratory syndrome or Coronavirus disease-19 (COVID-19).
19 . A screening kit for a therapeutic agent for coronavirus, comprising the screening composition for a therapeutic agent for coronavirus of any one of claims 1 and 2 to 18 .
20 . A method for screening a therapeutic agent for coronavirus infection, comprising:
(a) preparing a transfectant into which a CoV RdRp expression vector and a reporter vector included in the screening composition for a therapeutic agent for coronavirus infection according to any one of claims 1 and 2 to 18 are introduced together;
(b) treating the prepared transfectant with a candidate material expected to inhibit the activity of coronavirus-derived RNA-dependent RNA polymerase (CoV RdRp); and
(c) measuring the fluorescence level derived from NanoLuc luciferase after treating the candidate material.
21 . The method of claim 20 , wherein in step (b), the method further comprises pre-measuring the fluorescence level derived from NanoLuc luciferase in the transfectant before treating the candidate material.
22 . The method of claim 20 , wherein in step (c), the method distinguished the candidate as an inhibitor of CoV RdRp activity when the fluorescence level derived from NanoLuc luciferase is reduced.