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The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date1–7. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes3,8. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor-binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

The absence of ‘shovel-ready’ anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases., eLife digest To multiply and spread from cell to cell, the virus responsible for COVID-19 (also known as SARS-CoV-2) must first replicate its genetic information. This process involves a ‘polymerase’ protein complex making a faithful copy by assembling a precise sequence of building blocks, or nucleotides. The only drug approved against SARS-CoV-2 by the US Food and Drug Administration (FDA), remdesivir, consists of a nucleotide analog, a molecule whose structure is similar to the actual building blocks needed for replication. If the polymerase recognizes and integrates these analogs into the growing genetic sequence, the replication mechanism is disrupted, and the virus cannot multiply. Most approaches to study this process seem to indicate that remdesivir works by stopping the polymerase and terminating replication altogether. Yet, exactly how remdesivir and other analogs impair the synthesis of new copies of the virus remains uncertain. To explore this question, Seifert, Bera et al. employed an approach called magnetic tweezers which uses a magnetic field to manipulate micro-particles with great precision. Unlike other methods, this technique allows analogs to be integrated under conditions similar to those found in cells, and to be examined at the level of a single molecule. The results show that contrary to previous assumptions, remdesivir does not terminate replication; instead, it causes the polymerase to pause and backtrack (which may appear as termination in other techniques). The same approach was then applied to other nucleotide analogs, some of which were also found to target the SARS-CoV-2 polymerase. However, these analogs are incorporated differently to remdesivir and with less efficiency. They also obstruct the polymerase in distinct ways. Taken together, the results by Seifert, Bera et al. suggest that magnetic tweezers can be a powerful approach to reveal how analogs interfere with replication. This information could be used to improve currently available analogs as well as develop new antiviral drugs that are more effective against SARS-CoV-2. This knowledge will be key at a time when treatments against COVID-19 are still lacking, and may be needed to protect against new variants and future outbreaks.

Summary: Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into the human population in 2012 and has caused substantial morbidity and mortality. Potently neutralizing antibodies targeting the receptor-binding domain (RBD) on MERS-CoV spike (S) protein have been characterized, but much less is known about antibodies targeting non-RBD epitopes. Here, we report the structural and functional characterization of G2, a neutralizing antibody targeting the MERS-CoV S1 N-terminal domain (S1-NTD). Structures of G2 alone and in complex with the MERS-CoV S1-NTD define a site of vulnerability comprising two loops, each of which contain a residue mutated in G2-escape variants. Cell-surface binding studies and in vitro competition experiments demonstrate that G2 strongly disrupts the attachment of MERS-CoV S to its receptor, dipeptidyl peptidase-4 (DPP4), with the inhibition requiring the native trimeric S conformation. These results advance our understanding of antibody-mediated neutralization of coronaviruses and should facilitate the development of immunotherapeutics and vaccines against MERS-CoV. : Wang et al. report the structural and functional characterization of the Middle East respiratory syndrome coronavirus (MERS-CoV)-neutralizing antibody G2. G2 recognizes a conserved epitope on the MERS-CoV S1 N-terminal domain (S1-NTD) and neutralizes MERS-CoV by interfering with binding to host receptor dipeptidyl peptidase-4 (DPP4). The findings are relevant for understanding the viral attachment mechanism and for the development of S1-NTD-based vaccines. Keywords: MERS-CoV, coronavirus, crystal structure, electron microscopy, DPP4, receptor-binding, membrane fusion

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We use a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide evidence that the RNA-dependent RNA polymerase (RdRp) uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enable the direct observation of coronavirus polymerase deep and long-lived backtracking that is strongly stimulated by secondary structures in the template. The framework we present here elucidates one of the most important structure-dynamics-function relationships in human health today and will form the grounds for understanding the regulation of this complex., Graphical abstract, Bera et al. reveal the complete nucleotide addition cycle of the SARS-CoV-2 polymerase and show that the SARS-CoV-2 polymerase is a processive RNA polymerase that backtracks when elongating through RNA template containing secondary structures.

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 pandemic. We previously demonstrated that four nucleotide analogues (specifically, the active triphosphate forms of Sofosbuvir, Alovudine, AZT and Tenofovir alafenamide) inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Tenofovir and emtricitabine are the two components in DESCOVY and TRUVADA, the two FDA-approved medications for use as pre-exposure prophylaxis (PrEP) to prevent HIV infection. This is a preventative method in which individuals who are HIV negative (but at high-risk of contracting the virus) take the combination drug daily to reduce the chance of becoming infected with HIV. PrEP can stop HIV from replicating and spreading throughout the body. We report here that the triphosphates of tenofovir and emtricitabine, the two components in DESCOVY and TRUVADA, act as terminators for the SARS-CoV-2 RdRp catalyzed reaction. These results provide a molecular basis to evaluate the potential of DESCOVY and TRUVADA as PrEP for COVID-19.

SARS‐CoV‐2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA‐approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS‐CoV‐2. Here, using model polymerase extension experiments, we demonstrate that the active triphosphate form of Sofosbuvir is incorporated by low‐fidelity polymerases and SARS‐CoV RNA‐dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the active triphosphate form of Sofosbuvir is not incorporated by a host‐like high‐fidelity DNA polymerase. Using the same molecular insight, we selected 3’‐fluoro‐3’‐deoxythymidine triphosphate and 3’‐azido‐3’‐deoxythymidine triphosphate, which are the active forms of two other anti‐viral agents, Alovudine and AZT (an FDA‐approved HIV/AIDS drug) for evaluation as inhibitors of SARS‐CoV RdRp. We demonstrate the ability of two of these HIV reverse transcriptase inhibitors to be incorporated by SARS‐CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS‐CoV and SARS‐CoV‐2 RdRps, we expect these nucleotide analogues would also inhibit the SARS‐CoV‐2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad‐spectrum anti‐coronavirus agents., The triphosphate form of Sofosbuvir is incorporated by SARS‐CoV polymerase to terminate further primer extension, potentially preventing replication of the virus.

SARS-CoV-2 is responsible for the current COVID-19 pandemic. On the basis of our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues (the triphosphates of Sofosbuvir, Alovudine, and AZT) inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). We also demonstrated that a library of additional nucleotide analogues terminate RNA synthesis catalyzed by the SARS-CoV-2 RdRp, a well-established drug target for COVID-19. Here, we used polymerase extension experiments to demonstrate that the active triphosphate form of Sofosbuvir (an FDA-approved hepatitis C drug) is incorporated by SARS-CoV-2 RdRp and blocks further incorporation. Using the molecular insight gained from the previous studies, we selected the active triphosphate forms of six other antiviral agents, Alovudine, Tenofovir alafenamide, AZT, Abacavir, Lamivudine, and Emtricitabine, for evaluation as inhibitors of the SARS-CoV-2 RdRp and demonstrated the ability of these viral polymerase inhibitors to be incorporated by SARS-CoV-2 RdRp, where they terminate further polymerase extension with varying efficiency. These results provide a molecular basis for inhibition of the SARS-CoV-2 RdRp by these nucleotide analogues. If sufficient efficacy of some of these FDA-approved drugs in inhibiting viral replication in cell culture is established, they may be explored as potential COVID-19 therapeutics.

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2′ or 3′ modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3′-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2′-OMe-UTP), and 3 did not terminate the polymerase reaction (2′-F-dUTP, 2′–NH2–dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2′-OH at the 3′-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2′-OH, have a blocked 2′-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19., Highlights • Cidofovir triphosphate is a delayed terminator for SARS-CoV-2 RNA polymerase. • Abacavir, Ganciclovir, and Stavudine triphosphates inhibit SARS-CoV-2 polymerase. • 2′-O-methylated UTP substantially terminates the SARS-CoV-2 polymerase reaction.

Summary Ebolavirus NP oligomerizes into helical filaments found at the core of the virion, encapsidates the viral RNA genome, and serves as a scaffold for additional viral proteins within the viral nucleocapsid. We identified a portion of the phosphoprotein homolog VP35 that binds with high affinity to nascent NP and regulates NP assembly and viral genome binding. Removal of the VP35 peptide leads to NP self-assembly via its N-terminal oligomerization arm. NP oligomerization likely causes a conformational change between the NP N- and C-terminal domains, facilitating RNA binding. These functional data are complemented by crystal structures of the NP°-VP35 complex at 2.4 A resolution. The interactions between NP and VP35 illuminated by these structures are conserved among filoviruses and provide key targets for therapeutic intervention.

Since their first identification 50 years ago, marburgviruses have emerged several times, with 83%–90% lethality in the largest outbreaks. Although no vaccines or therapeutics are available for human use, the human antibody MR191 provides complete protection in non-human primates when delivered several days after inoculation of a lethal marburgvirus dose. The detailed neutralization mechanism of MR191 remains outstanding. Here we present a 3.2 A crystal structure of MR191 complexed with a trimeric marburgvirus surface glycoprotein (GP). MR191 neutralizes by occupying the conserved receptor-binding site and competing with the host receptor Niemann-Pick C1. The structure illuminates previously disordered regions of GP including the stalk, fusion loop, CX_6CC switch, and an N-terminal region of GP2 that wraps about the outside of GP1 to anchor a marburgvirus-specific “wing” antibody epitope. Virus escape mutations mapped far outside the MR191 receptor-binding site footprint suggest a role for these other regions in the GP quaternary structure.

Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using in vitro biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures of the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis., Author Summary Filoviruses use a system of proteins and RNA to regulate viral RNA genome transcription and replication. Here, we have determined crystal structures and the biological functions of the protein complex formed by the filovirus transcriptional activator, VP30, and the core component of the nucleocapsid machinery, NP. The complex of these two essential players represses Ebola virus RNA synthesis and may have played a role in the evolution of filoviruses to tune viral RNA synthesis activity to a level ideal for infection. This interaction is conserved across the filoviruses and may provide an opportunity for therapeutic development.

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A 4.0 A resolution cryo-electron microscopy structure of the pre-fusion form of the trimeric spike from the human coronavirus HKU1 provides insight into how the spike protein mediates host-cell attachment and membrane fusion. Coronaviruses are responsible for respiratory infections worldwide, many of them mild, but also including severe pneumonia and the recent SARS and MERS outbreaks. The entry of coronaviruses into cells is mediated by the virus glycoprotein spike trimer, which contains the receptor-binding domain, as well as membrane fusion domains. Two papers published in this issue of Nature provide high-resolution (4A) cryo-electron microscopy structures of pre-fusion coronavirus spike trimers. David Veesler and colleagues studied the trimer from murine hepatitis virus; Andrew Ward and colleagues used the human betacoronavirus HKU1, a cause of mild respiratory disease. The structures reveal mechanistic insights into the viral fusion process and architectural similarities to paramyxovirus F proteins, suggesting that these fusion proteins may have evolved from a distant common ancestor. HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease1, and is related to the zoonotic SARS2 and MERS3 betacoronaviruses, which have high fatality rates and pandemic potential. Cell tropism and host range is determined in part by the coronavirus spike (S) protein4, which binds cellular receptors and mediates membrane fusion. As the largest known class I fusion protein, its size and extensive glycosylation have hindered structural studies of the full ectodomain, thus preventing a molecular understanding of its function and limiting development of effective interventions. Here we present the 4.0 A resolution structure of the trimeric HKU1 S protein determined using single-particle cryo-electron microscopy. In the pre-fusion conformation, the receptor-binding subunits, S1, rest above the fusion-mediating subunits, S2, preventing their conformational rearrangement. Surprisingly, the S1 C-terminal domains are interdigitated and form extensive quaternary interactions that occlude surfaces known in other coronaviruses to bind protein receptors. These features, along with the location of the two protease sites known to be important for coronavirus entry, provide a structural basis to support a model of membrane fusion mediated by progressive S protein destabilization through receptor binding and proteolytic cleavage. These studies should also serve as a foundation for the structure-based design of betacoronavirus vaccine immunogens.

Influenza Revealed Influenza virus, a single-stranded RNA virus, is responsible for substantial morbidity and mortality worldwide. The influenza ribonucleoprotein (RNP) complex, which carries out viral replication and transcription, is central to the virus life-cycle and to viral host adaptation (see the Perspective by Tao and Zheng ). Structural characterization of the viral RNP has been challenging, but Moeller et al. (p. 1631 , published online 22 November) and Arranz et al. (p. 1634 , published online 22 November) now report the structure and assembly of this complex, using cryo-electron microscopy and negative-stain electron microscopy. The structures reveal how the viral polymerase, RNA genome, and nucleoprotein interact in the RNP providing insight into mechanisms for influenza genome replication and transcription.