Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clinical use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (M^pro) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 M^pro bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of M^pro, map the critical recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed M^pro inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.

Influenza viruses are enveloped, negative-sense single-stranded RNA viruses covered in a dense layer of glycoproteins. Hemagglutinin (HA) accounts for 80 to 90% of influenza glycoprotein and plays a role in host cell binding and membrane fusion. While previous studies have characterized structures of purified receptor-free and receptor-bound HA, the effect of receptor binding on HA organization and structure on virions remains unknown. Here, we used cryoelectron tomography to visualize influenza virions bound to a sialic acid receptor mimic. Overall, receptor binding did not result in significant changes in viral morphology; however, we observed rearrangements of HA trimer organization and orientation. Compared to the even interglycoprotein spacing of unliganded HA trimers, receptor binding promotes HA trimer clustering and the formation of a triplet of trimers. Subtomogram averaging and refinement yielded 8 to 10 Å reconstructions that allowed us to visualize specific contacts between HAs from neighboring trimers and identify molecular features that mediate clustering. Taken together, we present structural evidence that receptor binding triggers clustering of HA trimers, revealing an additional layer of HA dynamics and plasticity.

With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (M^pro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 M^pro, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 M^pro. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors.

Drugs that target the main protease (M ^pro ) of SARS-CoV-2 are effective therapeutics that have entered clinical use. Wide-scale use of these drugs will apply selection pressure for the evolution of resistance mutations. To understand resistance potential in M ^pro , we performed comprehensive surveys of amino acid changes that can cause resistance in a yeast screen to nirmatrelvir (contained in the drug Paxlovid), and ensitrelvir (Xocova) that is currently in phase III trials. The most impactful resistance mutation (E166V) recently reported in multiple viral passaging studies with nirmatrelvir showed the strongest drug resistance score for nirmatrelvir, while P168R had the strongest resistance score for ensitrelvir. Using a systematic approach to assess potential drug resistance, we identified 142 resistance mutations for nirmatrelvir and 177 for ensitrelvir. Among these mutations, 99 caused apparent resistance to both inhibitors, suggesting a strong likelihood for the evolution of cross-resistance. Many mutations that exhibited inhibitor-specific resistance were consistent with distinct ways that each inhibitor protrudes beyond the substrate envelope. In addition, mutations with strong drug resistance scores tended to have reduced function. Our results indicate that strong pressure from nirmatrelvir or ensitrelvir will select for multiple distinct resistant lineages that will include both primary resistance mutations that weaken interactions with drug while decreasing enzyme function and secondary mutations that increase enzyme activity. The comprehensive identification of resistance mutations enables the design of inhibitors with reduced potential of developing resistance and aids in the surveillance of drug resistance in circulating viral populations.

SUMMARY: Influenza viruses are enveloped, negative sense single-stranded RNA viruses covered in a dense layer of glycoproteins. Hemagglutinin (HA) accounts for 80-90% of influenza glycoprotein and plays a role in host cell binding and membrane fusion. While previous studies have characterized structures of receptor-free and receptor-bound HA in vitro, the effect of receptor binding on HA organization and structure on virions remains unknown. Here, we used cryo-electron tomography (cryoET) to visualize influenza virions bound to a sialic acid receptor mimic. Overall, receptor binding did not result in significant changes in viral morphology; however, we observed rearrangements of HA trimer organization and orientation. Compared to the even inter-glycoprotein spacing of unliganded HA trimers, receptor binding promotes HA trimer clustering and formation of a triplet of trimers. Subtomogram averaging and refinement yielded 8-10 Å reconstructions that allowed us to visualize specific contacts between HAs from neighboring trimers and identify molecular features that mediate clustering. Taken together, we present new structural evidence that receptor binding triggers clustering of HA trimers, revealing an additional layer of HA dynamics and plasticity.