A Cell-Based Reporter Assay for Screening Inhibitors of MERS Coronavirus RNA-Dependent RNA Polymerase Activity

Abstract

Severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19) are emerging zoonotic diseases caused by coronavirus (CoV) infections. The viral RNA-dependent RNA polymerase (RdRp) has been suggested as a valuable target for antiviral therapeutics because the sequence homology of CoV RdRp is highly conserved. We established a cell-based reporter assay for MERS-CoV RdRp activity to test viral polymerase inhibitors. The cell-based reporter system was composed of the bicistronic reporter construct and the MERS-CoV nsp12 plasmid construct. Among the tested nine viral polymerase inhibitors, ribavirin, sofosbuvir, favipiravir, lamivudine, zidovudine, valacyclovir, vidarabine, dasabuvir, and remdesivir, only remdesivir exhibited a dose-dependent inhibition. Meanwhile, the Z-factor and Z′-factor of this assay for screening inhibitors of MERS-CoV RdRp activity were 0.778 and 0.782, respectively. Ribavirin and favipiravir did not inhibit the MERS-CoV RdRp activity, and non-nucleoside HCV RdRp inhibitor, dasabuvir, partially inhibited MERS-CoV RdRp activity. Taken together, the cell-based reporter assay for MERS-CoV RdRp activity confirmed remdesivir as a direct inhibitor of MERS-CoV RdRp in cells. A cell-based MERS-CoV RdRp activity reporter assay is reliable and accurate for screening MERS-CoV RdRp-specific inhibitors. It may provide a valuable platform for developing antiviral drugs for emerging CoV infections.

Lycorine, a non-nucleoside RNA dependent RNA polymerase inhibitor, as potential treatment for emerging coronavirus infections

“…The IC 50 of remdesivir was 6.335 ± 0.731 μM ( Fig. 2 D), consistent with our previous results which showed inhibition of MERS-CoV RdRp activity in this assay ( Min et al, 2020 ). To exclude the possibility that decreased expression of MERS-CoV RdRp by lycorine caused the decreased activity of MERS-CoV RdRp, we confirmed the consistent expression level of MERS-CoV RdRp by western blot analysis (Supplement Fig.…”

“…Previously, we established a cell-based MERS-CoV RdRp activity reporter assay ( Min et al, 2020 ) by modifying the cell-based HCV RdRp activity assay ( Lee et al, 2010 ). The cell-based reporter system consisted of the MERS-CoV nsp12 plasmid and the bicistronic reporter plasmid, p(+)FLuc-(−)UTR-NLuc which contains the sense orientation firefly luciferase gene (+)FLuc and the antisense orientation (−)NLuc, which is flanked by the antisense orientation 3′- and 5′-UTR of MERS-CoV and the hepatitis delta virus (HDV) ribozyme self-cleavage sequence.…”

“…To determine MERS-CoV RdRp activity in cells, we used a previously described method ( Min et al, 2020 ). Briefly, HEK293T were transfected with MERS-CoV RdRp-expressing plasmid (pN-termFlag-nsp12) and reporter plasmids [p(+)FLuc-(−)UTR-NLuc].…”

Corilagin inhibits SARS-CoV-2 replication by targeting viral RNA-dependent RNA polymerase

“…We established a cell-based SARS-CoV-2 RdRp reporter assay system by modifying the previously developed system 40 , 41 . The plasmids nsp12, nsp7, nsp8, nsp10, and nsp14 were used to express codon-optimized Flag-nsp12, Flag-nsp7, Flag-nsp8, Flag-nsp10, and Flag-nsp14, respectively, all of which contain a Flag tag at the C-terminus.…”

Nucleoside Analogs and Nucleoside Precursors as Drugs in the Fight against SARS-CoV-2 and Other Coronaviruses

“…Amongst the coronavirus targets that were studied, or are currently being investigated, in the fight against the three most pathogenic HCoVs, particular relevance is given to the spike (S) protein [ 17 , 18 , 19 , 20 , 21 ], RNA-dependent RNA-polymerase (RdRp) [ 83 , 84 , 85 , 86 , 87 ], papain-like protease (PL pro ) [ 88 , 89 ] and main protease (M pro , 3CL pro ) [ 90 , 91 ]. In particular, this latter, which proteolytically cleaves the polyproteins to functional proteins essential for viral replication, occupies a special place in pharmaceutical research [ 92 ].…”