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Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral non-structural proteins: nsp7, nsp8 and nsp12. Most of our understanding of coronavirus molecular biology comes from the betacoronaviruses like SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of COVID-19. In contrast, members of the alphacoronavirus genus are relatively understudied despite their importance in human and animal health. Here we have used cryoelectron microscopy to determine the structure of the alphacoronavirus porcine epidemic diarrhea virus (PEDV) core polymerase complex bound to RNA. Our structure shows an unexpected nsp8 stoichiometry in comparison to other published coronavirus polymerase structures. Biochemical analysis shows that the N-terminal extension of one nsp8 is not required forin vitroRNA synthesis for alpha and betacoronaviruses as previously hypothesized. Our work shows the importance of studying diverse coronaviruses to reveal aspects of coronavirus replication while also identifying areas of conservation to be targeted by antiviral drugs.Significance StatementCoronaviruses are important human and animal pathogens with a history of crossing over from animal reservoirs into humans leading to epidemics or pandemics. Betacoronaviruses, such as SARS-CoV and SARS-CoV-2, have been the focus of research efforts in the field of coronaviruses, leaving other genera (alpha, gamma, and delta) understudied. To broaden our understanding, we studied an alphacoronavirus polymerase complex. We solved the first structure of a non-betacoronavirus replication complex, and in doing so identified previously unknown, and conserved, aspects of polymerase cofactor interactions. Our work displays the importance of studying coronaviruses from all genera and provides important insight into coronavirus replication that can be used for antiviral drug development.

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3′-to-5′ proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3′-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3′-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.

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.

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.

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design., Description, CryoEM and next-generation sequencing were used to identify monoclonal antibodies elicited by HIV Env vaccine candidates.

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.

The solution structure of SARS-CoV-2 nonstructural protein 7 (nsp7) at pH 7.0 has been determined by NMR spectroscopy. nsp7 is conserved in the coronavirinae subfamily and is an essential co-factor of the viral RNA-dependent RNA polymerase for active and processive replication. Similar to the previously deposited structures of SARS-CoV-1 nsp7 at acidic and basic conditions, SARS-CoV-2 nsp7 has a helical bundle folding at neutral pH. Remarkably, the α4 helix shows gradual dislocation from the core α2-α3 structure as pH increases from 6.5 to 7.5. The protonation state of residue H36 contributes to the change of nsp7’s intramolecular interactions, and thus, to the structural variation near-neutral pH. Spin-relaxation results revealed that all three loop regions in nsp7 possess dynamic properties associated with this structural variation.

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

Ebola virus is an emerging virus that is capable of causing a deadly disease in humans. Replication, transcription and packaging of the viral genome are carried out by the viral nucleocapsid. The nucleocapsid is a complex of the viral nucleoprotein, RNA and several other viral proteins. The nucleoprotein forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins. The 3.1 Å resolution single-particle cryo-electron microscopy structure of the nucleoprotein–RNA helical filament presented here resembles previous structures determined at lower resolution, while providing improved molecular details of protein–protein and protein–RNA interactions. The higher resolution of the structure presented here will facilitate the design and characterization of novel and specific Ebola virus therapeutics targeting the nucleocapsid.

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.

SummaryCoronaviruses, like SARS-CoV-2, encode a nucleotidyl transferase in the N-terminal NiRAN domain of thenon-structuralprotein (nsp) 12 protein within theRNA dependentRNApolymerase (RdRP)1-3. Though the substrate targets of the viral nucleotidyl transferase are unknown, NiRAN active sites are highly conserved and essential for viral replication3. We show, for the first time, the detection and sequence location of GMP-modified amino acids in nidovirus RdRP-associated proteins using heavy isotope-assisted MS and MS/MS peptide sequencing. We identified lys-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of nucleotidylation in vitro that uses a phosphoramide bond to covalently attach with GMP. In SARS-CoV-2 replicase proteins, we demonstrate a unique O-linked GMP attachment on nsp7 ser-1, whose formation required the presence of nsp12. It is clear that additional nucleotidylation sites remain undiscovered, which includes the possibility that nsp12 itself may form a transient GMP adduct in the NiRAN active site that has eluted detection in these initial studies due to instability of the covalent attachment. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.

The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, 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 nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.TeaserWe revise Remdesivir’s mechanism of action and reveal SARS-CoV-2 ability to evade interferon-induced antiviral ddhCTP

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 additional 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 with regard to their ability to be incorporated by the RdRps in the polymerase reaction and then prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (Carbovir triphosphate, Ganciclovir triphosphate, Stavudine triphosphate, Entecavir triphosphate, 3’-O-methyl UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2’-O-methyl UTP), and 3 did not terminate the polymerase reaction (2’-fluoro-dUTP, 2’-amino-dUTP and Desthiobiotin-16-UTP). The coronavirus genomes encode an exonuclease that apparently requires a 2’ -OH group to excise mismatched bases at the 3’-terminus. In this study, all of the nucleoside triphosphate analogues we evaluated form Watson-Cricklike base pairs. All the nucleotide analogues which demonstrated termination either lack a 2’-OH, have a blocked 2’-OH, or show delayed termination. These nucleotides may thus have the potential to resist exonuclease activity, a property that we will investigate in the future. Furthermore, prodrugs of five of these nucleotide analogues (Brincidofovir/Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA approved for other viral infections, and their safety profile is well known. Thus, they can be evaluated rapidly as potential therapies for COVID-19.

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.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also determined high-resolution structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines.

Summary: Rational immunogen design aims to focus antibody responses to vulnerable sites on primary antigens. Given the size of these antigens, there is, however, potential for eliciting unwanted, off-target responses. Here, we use our electron microscopy polyclonal epitope mapping approach to describe the antibody specificities elicited by immunization of non-human primates with soluble HIV envelope trimers and subsequent repeated viral challenge. An increased diversity of epitopes recognized and the approach angle by which these antibodies bind constitute a hallmark of the humoral response in most protected animals. We also show that fusion peptide-specific antibodies are likely responsible for some neutralization breadth. Moreover, cryoelectron microscopy (cryo-EM) analysis of a fully protected animal reveals a high degree of clonality within a subset of putatively neutralizing antibodies, enabling a detailed molecular description of the antibody paratope. Our results provide important insights into the immune response against a vaccine candidate that entered into clinical trials in 2019. : Nogal et al. use electron microscopy polyclonal epitope mapping of BG505 Env-immunized and matched SHIVBG505-challenged non-human primates to identify hallmarks of protection. Additionally, cryo-EM polyclonal analysis of a fully protected animal reveals a high degree of clonality, allowing detailed characterization of a putative neutralizing paratope.

Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus responsible for significant morbidity and mortality in pigs. A key determinant of viral tropism and entry, the PEDV spike protein is a key target for the host antibody response and a good candidate for a protein-based vaccine immunogen. We used electron microscopy to evaluate the PEDV spike structure, as well as pig polyclonal antibody responses to viral infection. The structure of the PEDV spike reveals a configuration similar to that of HuCoV-NL63. Several PEDV protein-protein interfaces are mediated by non-protein components, including a glycan at Asn264 and two bound palmitoleic acid molecules. The polyclonal antibody response to PEDV infection shows a dominance of epitopes in the S1 region. This structural and immune characterization provides insights into coronavirus spike stability determinants and explores the immune landscape of viral spike proteins., Graphical Abstract, Kirchdoerfer et al. use cryoelectron microscopy of the porcine epidemic diarrhea virus spike ectodomain to identify glycans and fatty acids in protein-protein interfaces and delineate epitopes targeted by the pig immune response to infection.

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.

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measure transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 2013–2016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP’s roles in protein structure, virion budding, and transcription and replication.

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as a highly transmissible pathogenic human betacoronavirus. The viral spike glycoprotein (S) utilizes angiotensin-converting enzyme 2 (ACE2) as a host protein receptor and mediates fusion of the viral and host membranes, making S essential to viral entry into host cells and host species tropism. As SARS-CoV enters host cells, the viral S is believed to undergo a number of conformational transitions as it is cleaved by host proteases and binds to host receptors. We recently developed stabilizing mutations for coronavirus spikes that prevent the transition from the pre-fusion to post-fusion states. Here, we present cryo-EM analyses of a stabilized trimeric SARS-CoV S, as well as the trypsin-cleaved, stabilized S, and its interactions with ACE2. Neither binding to ACE2 nor cleavage by trypsin at the S1/S2 cleavage site impart large conformational changes within stabilized SARS-CoV S or expose the secondary cleavage site, S2′.

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.

The crystal structure of the Reston ebolavirus VP30 C-terminal domain shows a rotated interface in comparison to the previous structure of the Zaire ebolavirus VP30 C-terminal domain., The ebolaviruses can cause severe hemorrhagic fever. Essential to the ebolavirus life cycle is the protein VP30, which serves as a transcriptional cofactor. Here, the crystal structure of the C-terminal, NP-binding domain of VP30 from Reston ebolavirus is presented. Reston VP30 and Ebola VP30 both form homodimers, but the dimeric interfaces are rotated relative to each other, suggesting subtle inherent differences or flexibility in the dimeric interface.

Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus , respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV. IMPORTANCE Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics.

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.

In the postantibiotic era, available treatment options for severe bacterial infections caused by methicillin-resistant Staphylococcus aureus have become limited. Therefore, new and innovative approaches are needed to combat such life-threatening infections. Virulence factor expression in S. aureus is regulated in a cell density-dependent manner using "quorum sensing," which involves generation and secretion of autoinducing peptides (AIPs) into the surrounding environment to activate a bacterial sensor kinase at a particular threshold concentration. Mouse monoclonal antibody AP4-24H11 was shown previously to blunt quorum sensing-mediated changes in gene expression in vitro and protect mice from a lethal dose of S. aureus by sequestering the AIP signal. We have elucidated the crystal structure of the AP4-24H11 Fab in complex with AIP-4 at 2.5 Å resolution to determine its mechanism of ligand recognition. A key Glu(H95) provides much of the binding specificity through formation of hydrogen bonds with each of the four amide nitrogens in the AIP-4 macrocyclic ring. Importantly, these structural data give clues as to the interactions between the cognate staphylococcal AIP receptors AgrC and the AIPs, as AP4-24H11·AIP-4 binding recapitulates features that have been proposed for AgrC-AIP recognition. Additionally, these structural insights may enable the engineering of AIP cross-reactive antibodies or quorum quenching vaccines for use in active or passive immunotherapy for prevention or treatment of S. aureus infections.

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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.

In jawless vertebrates, variable lymphocyte receptors (VLRs) play a crucial role in the recognition of antigens as part of the adaptive immune system. Leucine-rich repeat (LRR) modules and the highly variable insert (HVI) of VLRs contribute to the specificity and diversity of antigen recognition. VLR2913, the antigen of which is not known, contains the same HVI amino-acid sequence as that of VLR RBC36, which recognizes the H-trisaccharide from human blood type O erythrocytes. Since the HVI sequence is rarely identical among all known VLRs, identification of the antigen for VLR2913 and the main contributing factors for antigen recognition based on a comparison of VLR2913 and VLR RBC36 has been attempted. To initiate and facilitate this structural approach, the ectodomain of VLR2913 was fused with the N-terminal domain of internalin B (InlB-VLR2913-ECD). Three amino-acid residues on the concave surface of the LRR modules of InlB-VLR2913-ECD were mutated, considering important residues for hydrogen bonds in the recognition of H-trisaccharide by VLR RBC36. InlB-VLR2913-ECD was overexpressed in Escherichia coli and was crystallized at 295 K using the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 2.04 Å resolution and could be indexed in the tetragonal space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = 91.12, b = 91.12, c = 62.87 Å. Assuming that one monomer molecule was present in the crystallographic asymmetric unit, the calculated Matthews coefficient (V(M)) was 2.75 Å(3) Da(-1) and the solvent content was 55.2%. Structural determination of InlB-VLR2913-ECD by molecular replacement is in progress.

SummaryVariable lymphocyte receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin fold-based T and B cell receptors, lymphocyte-like cells of jawless fish express VLRs (VLRA, VLRB, or VLRC) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA and VLRC) in function. Here, we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis, and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores.

Very little is known about the conformation of polypeptides emerging from the ribosome during protein biosynthesis. Here, we explore the dynamics of ribosome-bound nascent polypeptides and proteins in Escherichia coli by dynamic fluorescence depolarization and assess the population of cotranslationally active chaperones trigger factor (TF) and DnaK. E. coli cell-free technology and fluorophore-linked E. coli Met-tRNA f Met enable selective site-specific labeling of nascent proteins at the N-terminal methionine. For the first time, direct spectroscopic evidence captures the generation of independent nascent chain motions for a single-domain protein emerging from the ribosome (apparent rotational correlation time approximately 5 ns), during the intermediate and late stages of polypeptide elongation. Such motions are detected only for a sequence encoding a globular protein and not for a natively unfolded control, suggesting that the independent nascent chain dynamics may be a signature of folding-competent sequences. In summary, we observe multicomponent, severely rotationally restricted, and strongly chain length/sequence-dependent nascent chain dynamics.

A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely-related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short and narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow for entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody combining site with a closed binding pocket, suggestive of an induced fit mechanism for ligand binding. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will now aid in further development of an antibody-based therapy against bacterial pathogens by interference with quorum sensing.

Proper characterization of aminoacyl- and peptidyl-tRNA, key components of protein biosynthesis, is of crucial importance in the study of the multifaceted aspects of translation. Analysis of ribosome-associated aminoacyl- and peptidyl-tRNAs has often been approached via sucrose gradients followed by scintillation counting [1]. This method is suitable to gain information about the entire ribosomal complex but it fails to provide direct knowledge on the molecular size of aminoacyl- and peptidyl-tRNA. Previous attempts to perform direct analysis on peptidyl-tRNA were based on native gel electrophoresis [2]. On the preparative scale, aminoacyl- and peptidyl-tRNAs have been isolated by ion exchange [3,4], reverse phase, size exclusion or affinity chromatography. However, the latter approaches are not as convenient as gel electrophoresis and may require larger amount of samples.