A proofreader in SARS-CoV-2: Although vaccines provide protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there remains a need for antivirals to treat COVID-19. Nucleotide analog drugs such as remdesivir, which target the viral RNA polymerase, have potential but are compromised by exoribonuclease (ExoN) activity that removes incorrect nucleotides from newly synthesized RNA. Liu et al . determined the structure of the complex that harbors the ExoN activity (nsp10–nsp-14) bound to a mimic of RNA that has incorporated an incorrect nucleotide. The structure shows how the RNA is recognized and suggests how ExoN specifically removes mismatched nucleotides. It also provides clues for designing nucleotide analogs that may evade excision. —VV

Coronaviruses recognize a variety of receptors using different domains of their envelope-anchored spike protein. How these diverse receptor recognition patterns affect viral entry is unknown. Mouse hepatitis coronavirus (MHV) is the only known coronavirus that uses the N-terminal domain (NTD) of its spike to recognize a protein receptor, CEACAM1a. Here we determined the cryo-EM structure of MHV spike complexed with mouse CEACAM1a. The trimeric spike contains three receptor-binding S1 heads sitting on top of a trimeric membrane-fusion S2 stalk. Three receptor molecules bind to the sides of the spike trimer, where three NTDs are located. Receptor binding induces structural changes in the spike, weakening the interactions between S1 and S2. Using protease sensitivity and negative-stain EM analyses, we further showed that after protease treatment of the spike, receptor binding facilitated the dissociation of S1 from S2, allowing S2 to transition from pre-fusion to post-fusion conformation. Together these results reveal a new role of receptor binding in MHV entry: in addition to its well-characterized role in viral attachment to host cells, receptor binding also induces the conformational change of the spike and hence the fusion of viral and host membranes. Our study provides new mechanistic insight into coronavirus entry and highlights the diverse entry mechanisms used by different viruses. Coronaviruses recognize many receptors using their envelope-anchored spike protein. The role of receptor binding in coronavirus entry into host cells is a fundamental question in virology. Mouse hepatitis coronavirus (MHV) is unique among all coronaviruses in that it uses the N-terminal domain (NTD) of its spike protein to bind a protein receptor CEACAM1a. While extensive research has been performed on the cell entry mechanisms of coronaviruses that use a different domain of their spike protein for receptor binding, the cell entry mechanism for MHV is still elusive. Here we determined the cryo-EM structure of MHV spike protein complexed with CEACAM1a. The structure reveals unique features of receptor binding by MHV spike that facilitate the structural changes of MHV spike and promote cell entry of MHV. We further confirmed the structural results with biochemical and negative-stain EM analyses. These results suggest that receptor binding plays dual roles in MHV entry: it promotes both viral attachment to host cells and the fusion of host and viral membranes. Our study provides insight into the molecular mechanism of MHV entry, demonstrating how cell entry of MHV has been adapted to its unique way of receptor binding.

Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved –35 and –10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19–20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.

The copper efflux regulator (CueR), a representative member of mercury resistance regulator (MerR) family metalloregulators, controls expression of copper homeostasis-regulating genes in bacteria. The mechanism of transcription activation by CueR and other MerR family regulators is bending the spacer domain of promoter DNA. Here, we report the cryo-EM structures of the intact CueR-dependent transcription activation complexes. The structures show that CueR dimer bends the 19-bp promoter spacer to realign the −35 and −10 elements for recognition by σ70-RNA polymerase holoenzyme and reveal a previously unreported interaction between the DNA-binding domain (DBD) from one CueR subunit and the σ70 nonconserved region (σNCR). Functional studies have shown that the CueR-σNCR interaction plays an auxiliary role in CueR-dependent transcription, assisting the activation mechanism of bending promoter DNA by CueR dimer. Because DBDs are highly conserved in sequence and structure, this transcription-activating mechanism could be generally used by MerR family regulators.

SARS-CoV-2’s remarkable resistance to nucleotide analog antivirals such as remdesivir, which thwarts RNA synthesis by inhibiting viral polymerase (RdRp), challenges available therapies. We reveal that remdesivir incorporation destabilizes RdRp–RNA complex while enhancing RNA binding to the proofreading exoribonuclease (ExoN), facilitating remdesivir excision. Conserved ExoN determinants for remdesivir recognition and excision underpin ExoN-mediated resistance across all coronaviruses. These findings inform the design of next-generation antivirals and combination therapies capable of overcoming ExoN-mediated resistance.