SAFE CELL REPORTER SYSTEM FOR SEARCHING FOR NUCLEOSIDE AND NON-NUCLEOSIDE INHIBITORS OF RNA-DEPENDENT RNA-POLYMERASE SARS-CoV-2 Russian patent published in 2025 - IPC C12N1/00 

FIELD: biotechnology.

SUBSTANCE: invention relates to molecular biology, medicinal chemistry, virology and microbiology and is intended for screening of potential inhibitors of RNA-dependent RNA-polymerase (RDRP) SARS-CoV-2 in laboratories with the first level of biosaur, not equipped for work with highly pathogenic infections. More specifically, the invention relates to full-size replicons, that is artificially created systems which enable to test potential inhibitors of SARS-CoV-2 RDRP without using high-grade viral particles. As a safe cell system for testing inhibitors of RNA-dependent RNA polymerase SARS-CoV-2, a genetic construct is obtained, which is a cDNA of a virus, which does not contain genes of structural proteins S, E and M, with two blocks of reporter genes, which is cloned into an artificial bacterial chromosome vector. First reporter gene unit, represented by Renilla reniformis luciferase and green fluorescent protein (Rluc-GFP), is under the control of the CMV promoter located in front of 5'UTR SARS-CoV-2. Second reporter gene unit is represented by firefly luciferase Photinus pyralis and red fluorescent protein (Fluc-RFP) and is located after the transcription control sequence (TRS-N). Such arrangement of genes and regulatory sequences (CMV-5'UTR-Rluc-GFP-TRS-N-Fluc-RFP-3'UTR) allows to estimate the level of inhibition of viral RGR, since the second reporter gene unit, located after TRS-N, can be transcribed only by viral polymerase by the intermittent transcription mechanism characteristic of the Coronaviridae family. First block of reporter genes, which is under control of the CMV-promoter, can be transcribed not only by viral RDRP, but also by cellular polymerases. For use as a negative control, a full-length replicon system has been created, in which amino acids (D760A, D761A) are substituted in the active center of the polymerase subunit of RDRP, which lead to loss of catalytic activity of RDRP.

EFFECT: to facilitate the search for nucleoside inhibitors of RDRP without creating phosphorylated depot forms, a cell line Vero E6_TK, which expresses thymidine kinase of herpes simplex virus, which performs the first stage of nucleoside phosphorylation in a cell, is created.

3 cl, 14 dwg, 1 tbl, 8 ex