SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. we screened a 273-compound library using replicon cells and identified three compounds as novel inhibitors of SARS-CoV-2 replication. Altogether, this work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs. IMPORTANCE SARS-CoV-2 replicon systems that have been reported up to date were unsuccessful in deriving stable cell lines harboring non-cytopathic replicons. The transient expression of viral sgmRNA or a reporter gene makes it impractical for industry-scale screening of large compound libraries using these systems. Here, for the first time, we derived stable cell clones harboring the SARS-CoV-2 replicon. These clones may now be conveniently cultured in a standard BSL-2 laboratory for high throughput screen of compound libraries. Additionally, our stable replicon cells represent a new model system to study SARS-CoV-2 replication. transcribed RNA encoding the nucleocapsid protein (NP), which was previously shown to improve launch efficiency (9,C11), into Vero E6, Huh7.5.1, A549, and BHK-21 cells resulted in expression of nanoluciferase to varying extents (Fig. 1B to ?toE).E). However, no viable clones could be recovered after 21?days of selection in G418, suggesting that active replication of the replicon RNA is either unsustainable or cytotoxic. Huh7.5.1 and BHK-21 cells supported higher levels of nanoluciferase expression than Vero E6 and A549, although we cannot rule out that the differences are attributed to different electroporation efficiency of the four cell lines. Because electroporating two different RNA species into the same cell is inefficient, we created a BHK-21 stable cell clone (BHK-21-NPDox-ON) in which NP is expressed in a doxycycline-inducible manner ETP-46464 (Fig. 1F). Electroporation of SARS-CoV-2-Rep-NanoLuc-Neo RNA into BHK-21-NPDox-ON cells resulted in three neomycin-resistant clones out of four million cells. The resulted clones grew very slowly in the presence ETP-46464 of 200?g/mL G418, and no nanoluciferase activity or viral RNA could be detected, indicating the loss of functional replicon RNA. Altogether, persistent replication of SARS-CoV-2-Rep-NanoLuc-Neo could not be achieved in any of the four mammalian cell lines. Open in a separate window FIG 1 Initial design of SARS-CoV-2 replicons. (A) Top, genome organization of SARS-CoV-2. Leader sequence (red), transcriptional regulatory sequence within the leader sequence (TRS-L) and within the body (TRS-B) are highlighted in green. Bottom, the design of SARS-CoV-2-Rep-NanoLuc-Neo. (B, C, D, E) Replication kinetics DPD1 of SARS-CoV-2-Rep-NanoLuc-Neo in Vero E6 (B), A549 (C), Huh7.5.1 (D), and BHK-21 cells (E). Nano luciferase was measured at indicated time points post-electroporation. Notably, nanoluciferase continued to decrease even under G418 selection. (F) Generation of BHK21 stable cells that express NP in a doxycycline-inducible manner. Cells were induced ETP-46464 with 0.5?g/mL doxycycline and lysed at 48 h postinduction for Western blotting with anti-NP and anti-actin antibodies. Numbers on the left refer to the positions of marker proteins that are given in kilodalton ETP-46464 (kDa). An improved SARS-CoV-2 replicon. Coronaviruses have evolved a variety of mechanisms to shut off host transcription and translation (12,C14). Of all SARS-CoV-2 proteins, Nsp1 causes the most severe viability reduction in cells of human lung origin (15). The carboxyl terminus of Nsp1 folds into two helices, which insert into the mRNA entrance channel on the 40S ribosome subunit, preventing both the host mRNA and viral mRNA from gaining access to ribosomes and consequently shutting down translation (16, 17) (Fig. 2A). The first C-terminal helix (residues 153 to 160) makes hydrophobic interactions with the 40S ribosomal protein uS5, and interacts with the 40S ribosomal protein uS3 with salt-bridges (18); the second C-terminal helix (residues 166 to 178) interacts with ribosomal protein eS30 and with the phosphate backbone of h18 of the 18S rRNA via the two conserved arginines R171 and R175 (16). A notably conserved KH dipeptide (K164 and H165) between the two helices forms interactions with h18 through H165 stacking between Uridine 607 (U607) and U630 of 18S rRNA, and through electrostatic interactions between K164 and the rRNA bases G625 and U630 (Fig. 2B). To explore the possibility of disrupting the interaction between Nsp1 and host ribosomes, we performed molecular dynamics simulation followed by ETP-46464 free energy perturbation calculation. The results predicted that mutations of residues K164, R171, R175, H165, and S167 of Nsp1 to alanine will reduce the interaction in the order of impact (Fig. 2C and Fig. S1). We hypothesize that a pair of mutations, such as K164A/H165A that weaken the interaction between C-terminus of Nsp1 and ribosome, will lead to a shorter occupation time of Nsp1 on the ribosome and increase the accessibility of ribosomes to host mRNA. As a result, the Nsp1-mediated.
