Inhibition of SARS-CoV-2 by Highly Potent Broad-Spectrum Anti-Coronaviral Tylophorine-Based Derivatives

Tylophorine-based compounds and natural cardiotonic steroids (cardenolides and bufadienolides) are two classes of transmissible gastroenteritis coronavirus inhibitors, targeting viral RNA and host cell factors, respectively. We tested both types of compounds against two types of coronaviruses, to compare and contrast their antiviral properties, and with view to their further therapeutic development. Examples of both types of compounds potently inhibited the replication of both feline infectious peritonitis virus and human coronavirus OC43 with EC50 values of up to 8 and 16 nM, respectively. Strikingly, the tylophorine-based compounds tested inhibited viral yields of HCoV-OC43 to a much greater extent (7–8 log magnitudes of p.f.u./ml) than the cardiotonic steroids (about 2–3 log magnitudes of p.f.u./ml), as determined by end point assays. Based on these results, three tylophorine-based compounds were further examined for their anti-viral activities on two other human coronaviruses, HCoV-229E and SARS-CoV-2. These three tylophorine-based compounds inhibited HCoV-229E with EC50 values of up to 6.5 nM, inhibited viral yields of HCoV-229E by 6–7 log magnitudes of p.f.u./ml, and were also found to inhibit SARS-CoV-2 with EC50 values of up to 2.5–14 nM. In conclusion, tylophorine-based compounds are potent, broad-spectrum inhibitors of coronaviruses including SARS-CoV-2, and could be used for the treatment of COVID-19.

Identification of a Gamma Interferon-Activated Inhibitor of Translation-Like RNA Motif at the 3′ End of the Transmissible Gastroenteritis Coronavirus Genome Modulating Innate Immune Response

ABSTRACTA 32-nucleotide (nt) RNA motif located at the 3′ end of the transmissible gastroenteritis coronavirus (TGEV) genome was found to specifically interact with the host proteins glutamyl-prolyl-tRNA synthetase (EPRS) and arginyl-tRNA synthetase (RRS). This RNA motif has high homology in sequence and secondary structure with the gamma interferon-activated inhibitor of translation (GAIT) element, which is located at the 3′ end of several mRNAs encoding proinflammatory proteins. The GAIT element is involved in the translation silencing of these mRNAs through its interaction with the GAIT complex (EPRS, heterogeneous nuclear ribonucleoprotein Q, ribosomal protein L13a, and glyceraldehyde 3-phosphate dehydrogenase) to favor the resolution of inflammation. Interestingly, we showed that the viral RNA motif bound the GAIT complex and inhibited thein vitrotranslation of a chimeric mRNA containing this RNA motif. To our knowledge, this is the first GAIT-like motif described in a positive RNA virus. To test the functional role of the GAIT-like RNA motif during TGEV infection, a recombinant coronavirus harboring mutations in this motif was engineered and characterized. Mutations of the GAIT-like RNA motif did not affect virus growth in cell cultures. However, an exacerbated innate immune response, mediated by the melanoma differentiation-associated gene 5 (MDA5) pathway, was observed in cells infected with the mutant virus compared with the response observed in cells infected with the parental virus. Furthermore, the mutant virus was more sensitive to beta interferon than the parental virus. All together, these data strongly suggested that the viral GAIT-like RNA motif modulates the host innate immune response.IMPORTANCEThe innate immune response is the first line of antiviral defense that culminates with the synthesis of interferon and proinflammatory cytokines to limit virus replication. Coronaviruses encode several proteins that interfere with the innate immune response at different levels, but to date, no viral RNA counteracting antiviral response has been described. In this work, we have characterized a 32-nt RNA motif located at the 3′ end of the TGEV genome that specifically interacted with EPRS and RRS. This RNA motif presented high homology with the GAIT element, involved in the modulation of the inflammatory response. Moreover, the disruption of the viral GAIT-like RNA motif led to an exacerbated innate immune response triggered by MDA5, indicating that the GAIT-like RNA motif counteracts the host innate immune response. These novel findings may be of relevance for other coronaviruses and could serve as the basis for the development of novel antiviral strategies.