The SARS-CoV-2 main protease (M^pro) is the drug target of Pfizer's oral drug nirmatrelvir. The emergence of SARS-CoV-2 variants with mutations in M^pro raised the alarm of potential drug resistance. To identify potential clinically relevant drug-resistant mutants, we systematically characterized 102 naturally occurring M^pro mutants located at 12 residues at the nirmatrelvir-binding site, among which 22 mutations in 5 residues, including S144M/F/A/G/Y, M165T, E166 V/G/A, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (k_cat/K_m < 10-fold change) while being resistant to nirmatrelvir (K_i > 10-fold increase). X-ray crystal structures were determined for six representative mutants with and/or without GC-376/nirmatrelvir. Using recombinant SARS-CoV-2 viruses generated from reverse genetics, we confirmed the drug resistance in the antiviral assay and showed that M^pro mutants with reduced enzymatic activity had attenuated viral replication. Overall, our study identified several drug-resistant hotspots in M^pro that warrant close monitoring for possible clinical evidence of nirmatrelvir resistance, some of which have already emerged in independent viral passage assays conducted by others.

Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5′-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type−infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.

The emergence of SARS-CoV-2 variants and drug-resistant mutants calls for additional oral antivirals. The SARS-CoV-2 papain-like protease (PL ^pro ) is a promising but challenging drug target. We designed and synthesized 85 noncovalent PL ^pro inhibitors that bind to a recently discovered ubiquitin binding site and the known BL2 groove pocket near the S4 subsite. Leads inhibited PL ^pro with the inhibitory constant K _i values from 13.2 to 88.2 nanomolar. The co-crystal structures of PL ^pro with eight leads revealed their interaction modes. The in vivo lead Jun12682 inhibited SARS-CoV-2 and its variants, including nirmatrelvir-resistant strains with EC ₅₀ from 0.44 to 2.02 micromolar. Oral treatment with Jun12682 improved survival and reduced lung viral loads and lesions in a SARS-CoV-2 infection mouse model, suggesting that PL ^pro inhibitors are promising oral SARS-CoV-2 antiviral candidates.

The ever-evolving SARS-CoV-2 variants necessitate the development of additional oral antivirals. This study presents the systematic design of quinoline-containing SARS-CoV-2 papain-like protease (PL^pro) inhibitors as potential oral antiviral drug candidates. By leveraging the recently discovered Val70^Ub binding site in PL^pro, we designed a series of quinoline analogs demonstrating potent PL^pro inhibition and antiviral activity. Notably, the X-ray crystal structures of 6 lead compounds reveal that the 2-aryl substitution can occupy either the Val70^Ub site as expected or the BL2 groove in a flipped orientation. The in vivo lead Jun13296 exhibits favorable pharmacokinetic properties and potent inhibition against SARS-CoV-2 variants and nirmatrelvir-resistant mutants. In a mouse model of SARS-CoV-2 infection, oral treatment with Jun13296 significantly improves survival, reduces body weight loss and lung viral titers, and prevents lung tissue damage. These results underscore the potential of quinoline PL^pro inhibitors as promising oral SARS-CoV-2 antiviral candidates, instilling hope for the future of SARS-CoV-2 treatment.

The tumor suppressor p53 as an innate antiviral regulator contributes to restricting Japanese encephalitis virus (JEV) replication, but the mechanism is still unclear. The interferon-induced transmembrane protein 3 (IFITM3) is an intrinsic barrier to a range of virus infection, whether IFITM3 is responsible for the p53-mediated anti-JEV response remains elusive. Here, we found that IFITM3 significantly inhibited JEV replication in a protein-palmitoylation-dependent manner and incorporated into JEV virions to diminish the infectivity of progeny viruses. Palmitoylation was also indispensible for keeping IFITM3 from lysosomal degradation to maintain its protein stability. p53 up-regulated IFITM3 expression at the protein level via enhancing IFITM3 palmitoylation. Screening of palmitoyltransferases revealed that zinc finger DHHC domain-containing protein 1 (ZDHHC1) was transcriptionally up-regulated by p53, and consequently ZDHHC1 interacted with IFITM3 to promote its palmitoylation and stability. Knockdown of IFITM3 significantly impaired the inhibitory role of ZDHHC1 on JEV replication. Meanwhile, knockdown of either ZDHHC1 or IFITM3 expression also compromised the p53-mediated anti-JEV effect. Interestingly, JEV reduced p53 expression to impair ZDHHC1 mediated IFITM3 palmitoylation for viral evasion. Our data suggest the existence of a previously unrecognized p53-ZDHHC1-IFITM3 regulatory pathway with an essential role in restricting JEV infection and provide a novel insight into JEV-host interaction.