Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors.

Vaccines and drugs have helped reduce disease severity and blunt the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, ongoing virus transmission, continuous evolution, and increasing selective pressures have the potential to yield viral variants capable of resisting these interventions. Here, we investigate the susceptibility of natural variants of the main protease [Mpro; 3C-like protease (3CLpro)] of SARS-CoV-2 to protease inhibitors. Multiple single amino acid changes in Mpro confer resistance to nirmatrelvir (the active component of Paxlovid). An additional clinical-stage inhibitor, ensitrelvir (Xocova), shows a different resistance mutation profile. Importantly, phylogenetic analyses indicate that several of these resistant variants have pre-existed the introduction of these drugs into the human population and are capable of spreading. These results encourage the monitoring of resistance variants and the development of additional protease inhibitors and other antiviral drugs with different mechanisms of action and resistance profiles for combinatorial therapy.

Structural basis for mouse receptor recognition by SARS-CoV-2 omicron variant.

The sudden emergence and rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant has raised questions about its animal reservoir. Here, we investigated receptor recognition of the omicron's receptor-binding domain (RBD), focusing on four of its mutations (Q493R, Q498R, N501Y, and Y505H) surrounding two mutational hotspots. These mutations have variable effects on the RBD's affinity for human angiotensin-converting enzyme 2 (ACE2), but they all enhance the RBD's affinity for mouse ACE2. We further determined the crystal structure of omicron RBD complexed with mouse ACE2. The structure showed that all four mutations are viral adaptations to mouse ACE2: three of them (Q493R, Q498R, and Y505H) are uniquely adapted to mouse ACE2, whereas the other one (N501Y) is adapted to both human ACE2 and mouse ACE2. These data reveal that the omicron RBD was well adapted to mouse ACE2 before omicron started to infect humans, providing insight into the potential evolutionary origin of the omicron variant.

SARS-CoV-2 nsp14 Exoribonuclease Removes the Natural Antiviral 3'-Deoxy-3',4'-didehydro-cytidine Nucleotide from RNA.

The on-going global pandemic of COVID-19 is caused by SARS-CoV-2, which features a proofreading mechanism to facilitate the replication of its large RNA genome. The 3'-to-5' exoribonuclease (ExoN) activity of SARS-CoV-2 non-structural protein 14 (nsp14) removes nucleotides misincorporated during RNA synthesis by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and thereby compromises the efficacy of antiviral nucleoside/nucleotide analogues. Here we show biochemically that SARS-CoV-2 nsp14 can excise the natural antiviral chain-terminating nucleotide, 3'-deoxy-3',4'-didehydro-cytidine 5'-monophosphate (ddhCMP), incorporated by RdRp at the 3' end of an RNA strand. Nsp14 ExoN processes an RNA strand terminated with ddhCMP more efficiently than that with a non-physiological chain terminator 3'-deoxy-cytidine monophosphate (3'-dCMP), whereas RdRp is more susceptible to chain termination by 3'-dCTP than ddhCTP. These results suggest that nsp14 ExoN could play a role in protecting SARS-CoV-2 from ddhCTP, which is produced as part of the innate immune response against viral infections, and that the SARS-CoV-2 enzymes may have adapted to minimize the antiviral effect of ddhCTP.

Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 Mpro/3CLpro in Living Cells.

The main protease, Mpro, of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here, we report quantitative reporters for Mpro function in living cells in which protease inhibition by genetic or chemical methods results in robust signal readouts by fluorescence (enhanced green fluorescent protein [eGFP]) or bioluminescence (firefly luciferase). These gain-of-signal systems are scalable to high-throughput platforms for quantitative discrimination between Mpro mutants and/or inhibitor potencies as evidenced by validation of several reported inhibitors. Additional utility is shown by single Mpro amino acid variants and structural information combining to demonstrate that both inhibitor conformational dynamics and amino acid differences are able to influence inhibitor potency. We further show that a recent variant of concern (Omicron) has an unchanged response to a clinically approved drug, nirmatrelvir, whereas proteases from divergent coronavirus species show differential susceptibility. Together, we demonstrate that these gain-of-signal systems serve as robust, facile, and scalable assays for live cell quantification of Mpro inhibition, which will help expedite the development of next-generation antivirals and enable the rapid testing of emerging variants. IMPORTANCE The main protease, Mpro, of SARS-CoV-2 is an essential viral protein required for the earliest steps of infection. It is therefore an attractive target for antiviral drug development. Here, we report the development and implementation of two complementary cell-based systems for quantification of Mpro inhibition by genetic or chemical approaches. The first is fluorescence based (eGFP), and the second is luminescence based (firefly luciferase). Importantly, both systems rely upon gain-of-signal readouts such that stronger inhibitors yield higher fluorescent or luminescent signal. The high versatility and utility of these systems are demonstrated by characterizing Mpro mutants and natural variants, including Omicron, as well as a panel of existing inhibitors. These systems rapidly, safely, and sensitively identify Mpro variants with altered susceptibilities to inhibition, triage-nonspecific, or off-target molecules and validate bona fide inhibitors, with the most potent thus far being the first-in-class drug nirmatrelvir.

Structural Basis for Human Receptor Recognition by SARS-CoV-2 Omicron Variant BA.1.

The highly contagious and fast-spreading omicron variant of SARS-CoV-2 infects the respiratory tracts efficiently. The receptor-binding domain (RBD) of the omicron spike protein recognizes human angiotensin-converting enzyme 2 (ACE2) as its receptor and plays a critical role in the tissue tropism of SARS-CoV-2. Here, we showed that the omicron RBD (strain BA.1) binds to ACE2 more strongly than does the prototypic RBD from the original Wuhan strain. We also measured how individual omicron mutations affect ACE2 binding. We further determined the crystal structure of the omicron RBD (engineered to facilitate crystallization) complexed with ACE2 at 2.6 Å. The structure shows that omicron mutations caused significant structural rearrangements of two mutational hot spots at the RBD/ACE2 interface, elucidating how each omicron mutation affects ACE2 binding. The enhanced ACE2 binding by the omicron RBD may facilitate the omicron variant's infection of the respiratory tracts where ACE2 expression level is low. Our study provides insights into the receptor recognition and tissue tropism of the omicron variant. IMPORTANCE Despite the scarcity of the SARS-CoV-2 receptor-human angiotensin-converting enzyme 2 (ACE2)-in the respiratory tract, the omicron variant efficiently infects the respiratory tract, causing rapid and widespread infections of COVID-19. The omicron variant contains extensive mutations in the receptor-binding domain (RBD) of its spike protein that recognizes human ACE2. Here, using a combination of biochemical and X-ray crystallographic approaches, we showed that the omicron RBD binds to ACE2 with enhanced affinity and also elucidated the role of each of the omicron mutations in ACE2 binding. The enhanced ACE2 binding by the omicron RBD may contribute to the omicron variant's new viral tropism in the respiratory tract despite the low level of ACE2 expression in the tissue. These findings help us to understand tissue tropism of the omicron variant and shed light on the molecular evolution of SARS-CoV-2.

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN.

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.

Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography.

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

The development of Nanosota-1 as anti-SARS-CoV-2 nanobody drug candidates.

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking viral receptor angiotensin-converting enzyme 2 (ACE2). The lead drug candidate possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD ~3000 times more tightly than ACE2 did and inhibited SARS-CoV-2 pseudovirus ~160 times more efficiently than ACE2 did. Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy against live SARS-CoV-2 infection in both hamster and mouse models. Unlike conventional antibodies, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented an excellent in vivo stability and a high tissue bioavailability. As effective and inexpensive drug candidates, Nanosota-1 may contribute to the battle against COVID-19.

Structural basis of receptor recognition by SARS-CoV-2.

A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans, causing COVID-191,2. A key to tackling this pandemic is to understand the receptor recognition mechanism of the virus, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor-angiotensin-converting enzyme 2 (ACE2)-in humans3,4. Here we determined the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 (engineered to facilitate crystallization) in complex with ACE2. In comparison with the SARS-CoV RBD, an ACE2-binding ridge in SARS-CoV-2 RBD has a more compact conformation; moreover, several residue changes in the SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD-ACE2 interface. These structural features of SARS-CoV-2 RBD increase its ACE2-binding affinity. Additionally, we show that RaTG13, a bat coronavirus that is closely related to SARS-CoV-2, also uses human ACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in ACE2 recognition shed light on the potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies that target receptor recognition by SARS-CoV-2.