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The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity., Competing Interests: Declaration of interests I.F., N.S., E.B., P.L., J.B., O.S., and F.A.R. have a provisional patent on anti-TMPRSS2 nanobodies. F.A.R. is a founder of Meletios Therapeutics and a member of its scientific advisory board., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Coronaviruses (CoVs) have complex genomes that encode a fixed array of structural and nonstructural components, as well as a variety of accessory proteins that differ even among closely related viruses. Accessory proteins often play a role in the suppression of immune responses and may represent virulence factors. Despite their relevance for CoV phenotypic variability, information on accessory proteins is fragmentary. We applied a systematic approach based on homology detection to create a comprehensive catalogue of accessory proteins encoded by CoVs. Our analyses grouped accessory proteins into 379 orthogroups and 12 super-groups. No orthogroup was shared by the four CoV genera and very few were present in all or most viruses in the same genus, reflecting the dynamic evolution of CoV genomes. We observed differences in the distribution of accessory proteins in CoV genera. Alphacoronaviruses harboured the largest diversity of accessory open reading frames (ORFs), deltacoronaviruses the smallest. However, the average number of accessory proteins per genome was highest in betacoronaviruses. Analysis of the evolutionary history of some orthogroups indicated that the different CoV genera adopted similar evolutionary strategies. Thus, alphacoronaviruses and betacoronaviruses acquired phosphodiesterases and spike-like accessory proteins independently, whereas horizontal gene transfer from reoviruses endowed betacoronaviruses and deltacoronaviruses with fusion-associated small transmembrane (FAST) proteins. Finally, analysis of accessory ORFs in annotated CoV genomes indicated ambiguity in their naming. This complicates cross-communication among researchers and hinders automated searches of large data sets (e.g., PubMed, GenBank). We suggest that orthogroup membership is used together with a naming system to provide information on protein function., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Seven types of Coronaviruses (CoVs) have been identified that can cause infection in humans, including HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV, HCoV-MERS, and SARS-CoV-2. In this study, we investigated the genetic structure, the homology of the structural protein sequences, as well as the investigation of the active site of structural proteins. The active site of structural proteins was determined based on the previous studies, and the homology of their amino acid sequences and structure was compared. Multiple sequence alignment of Spike protein of HCoVs showed that the receptor-binding domain of SARS-CoV-2, SARS-CoV, and MERS-CoV was located at a similar site to the S1 subunit. The binding motif of PDZ (postsynaptic density-95/disks large/zona occludens-1) of the envelope protein, was conserved in SARS-CoV and SARS-CoV-2 according to multiple sequence alignment but showed different changes in the other HCoVs. Overall, spike protein showed the most variation in its active sites, but the other structural proteins were highly conserved. In this study, for the first time, the active site of all structural proteins of HCoVs as a drug target was investigated. The binding site of these proteins can be suitable targets for drugs or vaccines among HCoVs., (© 2021 John Wiley & Sons A/S.)

We introduce multiple interface string alignment (MISA), a visualization tool to display coherently various sequence and structure based statistics at protein-protein interfaces (SSE elements, buried surface area, Δ ASA , B factor values, etc). The amino acids supporting these annotations are obtained from Voronoi interface models. The benefit of MISA is to collate annotated sequences of (homologous) chains found in different biological contexts, that is, bound with different partners or unbound. The aggregated views MISA/SSE, MISA/BSA, MISA/ΔASA, and so forth, make it trivial to identify commonalities and differences between chains, to infer key interface residues, and to understand where conformational changes occur upon binding. As such, they should prove of key relevance for knowledge-based annotations of protein databases such as the Protein Data Bank. Illustrations are provided on the receptor binding domain of coronaviruses, in complex with their cognate partner or (neutralizing) antibodies. MISA computed with a minimal number of structures complement and enrich findings previously reported. The corresponding package is available from the Structural Bioinformatics Library (http://sbl.inria.frand https://sbl.inria.fr/doc/Multiple_interface_string_alignment-user-manual.html)., (© 2021 Wiley Periodicals LLC.)

Understanding the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) is critical to overcome the current coronavirus disease (COVID-19) pandemic. Efforts are being made to understand the potential cross-protective immunity of memory T cells, induced by prior encounters with seasonal coronaviruses, in providing protection against severe COVID-19. In this study we assessed T-cell responses directed against highly conserved regions of SARS-CoV-2. Epitope mapping revealed 16 CD8 + T-cell epitopes across the nucleocapsid (N), spike (S), and open reading frame (ORF)3a proteins of SARS-CoV-2 and five CD8 + T-cell epitopes encoded within the highly conserved regions of the ORF1ab polyprotein of SARS-CoV-2. Comparative sequence analysis showed high conservation of SARS-CoV-2 ORF1ab T-cell epitopes in seasonal coronaviruses. Paradoxically, the immune responses directed against the conserved ORF1ab epitopes were infrequent and subdominant in both convalescent and unexposed participants. This subdominant immune response was consistent with a low abundance of ORF1ab encoded proteins in SARS-CoV-2 infected cells. Overall, these observations suggest that while cross-reactive CD8 + T cells likely exist in unexposed individuals, they are not common and therefore are unlikely to play a significant role in providing broad preexisting immunity in the community. IMPORTANCE T cells play a critical role in protection against SARS-CoV-2. Despite being highly topical, the protective role of preexisting memory CD8 + T cells, induced by prior exposure to circulating common coronavirus strains, remains less clear. In this study, we established a robust approach to specifically assess T cell responses to highly conserved regions within SARS-CoV-2. Consistent with recent observations we demonstrate that recognition of these highly conserved regions is associated with an increased likelihood of milder disease. However, extending these observations we observed that recognition of these conserved regions is rare in both exposed and unexposed volunteers, which we believe is associated with the low abundance of these proteins in SARS-CoV-2 infected cells. These observations have important implications for the likely role preexisting immunity plays in controlling severe disease, further emphasizing the importance of vaccination to generate the immunodominant T cells required for immune protection.

Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here, we report the crystal structure of papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with the Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in a moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that 2-residue-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alphacoronavirus PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for the substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating (DUB) and proteolytic functions of PEDV PLP2 from a structural perspective. IMPORTANCE Current challenges in coronaviruses (CoVs) include a comprehensive understanding of the mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structural basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed that (i) three Ub core-interacting residues are essential for DUB function, (ii) 2-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only structural insights into the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.

The Complex Portal (www.ebi.ac.uk/complexportal) is a manually curated, encyclopaedic database of macromolecular complexes with known function from a range of model organisms. It summarizes complex composition, topology and function along with links to a large range of domain-specific resources (i.e. wwPDB, EMDB and Reactome). Since the last update in 2019, we have produced a first draft complexome for Escherichia coli, maintained and updated that of Saccharomyces cerevisiae, added over 40 coronavirus complexes and increased the human complexome to over 1100 complexes that include approximately 200 complexes that act as targets for viral proteins or are part of the immune system. The display of protein features in ComplexViewer has been improved and the participant table is now colour-coordinated with the nodes in ComplexViewer. Community collaboration has expanded, for example by contributing to an analysis of putative transcription cofactors and providing data accessible to semantic web tools through Wikidata which is now populated with manually curated Complex Portal content through a new bot. Our data license is now CC0 to encourage data reuse. Users are encouraged to get in touch, provide us with feedback and send curation requests through the 'Support' link., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Identifying the epitope of an antibody is a key step in understanding its function and its potential as a therapeutic. Sequence-based clonal clustering can identify antibodies with similar epitope complementarity, however, antibodies from markedly different lineages but with similar structures can engage the same epitope. We describe a novel computational method for epitope profiling based on structural modelling and clustering. Using the method, we demonstrate that sequence dissimilar but functionally similar antibodies can be found across the Coronavirus Antibody Database, with high accuracy (92% of antibodies in multiple-occupancy structural clusters bind to consistent domains). Our approach functionally links antibodies with distinct genetic lineages, species origins, and coronavirus specificities. This indicates greater convergence exists in the immune responses to coronaviruses than is suggested by sequence-based approaches. Our results show that applying structural analytics to large class-specific antibody databases will enable high confidence structure-function relationships to be drawn, yielding new opportunities to identify functional convergence hitherto missed by sequence-only analysis., Competing Interests: The authors have declared that no competing interests exist.

The COVID-19 pandemic has highlighted the urgent need for the identification of new antiviral drug therapies for a variety of diseases. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2, while other related human coronaviruses cause diseases ranging from severe respiratory infections to the common cold. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of a range of human coronavirus diseases. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cell-based experimental assessment reveals several clinically-relevant drug repurposing candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies., (© 2021. The Author(s).)

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.

Coronaviruses are a large family of important pathogens that cause human and animal diseases. At the end of 2019, a pneumonia epidemic caused by a novel coronavirus brought attention to coronaviruses. Exploring the interaction between the virus and its receptor will be helpful in developing preventive vaccines and therapeutic drugs. The coronavirus spike protein (S) plays an important role in both binding to receptors on host cells and fusion of the viral membrane with the host cell membrane. This review introduces the structure and function of the S protein and its receptor, focusing on the binding mode and binding region of both.

Coronaviruses are a large group of RNA viruses that infect a wide range of animal species. The replication strategy of coronaviruses involves recombination and mutation events that lead to the possibility of cross-species transmission. The high plasticity of the viral receptor due to a continuous modification of the host species habitat may be the cause of cross-species transmission that can turn into a threat to other species including the human population. The successive emergence of highly pathogenic coronaviruses such as the Severe Acute Respiratory Syndrome (SARS) in 2003, the Middle East Respiratory Syndrome Coronavirus in 2012, and the recent SARS-CoV-2 has incentivized a number of studies on the molecular basis of the coronavirus and its pathogenesis. The high degree of interrelatedness between humans and wild and domestic animals and the modification of animal habitats by human urbanization, has favored new viral spreads. Hence, knowledge on the main clinical signs of coronavirus infection in the different hosts and the distinctive molecular characteristics of each coronavirus is essential to prevent the emergence of new coronavirus diseases. The coronavirus infections routinely studied in veterinary medicine must be properly recognized and diagnosed not only to prevent animal disease but also to promote public health., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Currently, the whole world is facing the coronavirus disease-19 pandemic. As of now, approximately 0.15 million people around the globe are infected with the novel coronavirus. In the last decade, two strains of the coronavirus family, severe acute respiratory syndrome-related coronavirus and Middle East respiratory syndrome coronavirus, also resulted in epidemics in south Asian and the Middle Eastern countries with high mortality rate. This scenario demands the development of a putative vaccine which may provide immunity against all current and new evolving coronavirus strains. In this study, we designed an epitope-based vaccine using an immunoinformatic approach. This vaccine may protect against all coronavirus strains. The vaccine is developed by considering the geographical distribution of coronavirus strains and host genetics (Chinese population). Nine experimentally validated epitopes sequences from coronavirus strains were used to derive the variants considering the conservancy in all strains. Further, the binding affinities of all derived variants were checked with most abundant human leukocyte antigen alleles in the Chinese population. Three major histocompatibility complex (MHC) Class I epitopes from spike glycoprotein and nucleoprotein showed sufficient binding while one MHC Class II epitope from spike glycoprotein was found to be an effective binder. A cocktail of these epitopes gave more than 95% population coverage in the Chinese population. Moreover, molecular dynamics simulation supported the aforementioned predictions. Further, in vivo studies are needed to confirm the immunogenic potential of these vaccines., (© 2021 The Societies and John Wiley & Sons Australia, Ltd.)

In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the "down" conformation, indicating they are more prone to adopt the receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. We further identified critical residues in the RBD underlying different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes. These results collectively indicate that tight RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.

This paper presents a computer simulation of a virtual robot that behaves as a peptide chain of the Hemagglutinin-Esterase protein (HEs) from human coronavirus. The robot can learn efficient protein folding policies by itself and then use them to solve HEs folding episodes. The proposed robotic unfolded structure inhabits a dynamic environment and is driven by a self-taught neural agent. The neural agent can read sensors and control the angles and interactions between individual amino acids. During the training phase, the agent uses reinforcement learning to explore new folding forms that conduce toward more significant rewards. The memory of the agent is implemented with neural networks. These neural networks are noise-balanced trained to satisfy the look for future conditions required by the Bellman equation. In the operating phase, the components merge into a wise up protein folding robot with look-ahead capacities, which consistently solves a section of the HEs protein., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Coronavirus Disease 2019 named as COVID-19 imposing a huge burden on public health as well as global economies, is caused by a new strain of betacoronavirus named as SARS-CoV-2. The high transmission rate of the virus has resulted in current havoc which highlights the need for a fast and effective approach either to prevent or treat the deadly infection. Development of vaccines can be the most prominent approach to prevent the virus to cause COVID-19 and hence will play a vital role in controlling the spread of the virus and reducing mortality. The virus uses its spike proteins for entering into the host by interacting with a specific receptor called angiotensin converting enzyme-2 (ACE2) present on the surface of alveolar cells in the lungs. Researchers all over the world are targeting the spike protein for the development of potential vaccines. Here, we discuss the immunopathological basis of vaccine designing that can be approached for vaccine development against SARS-CoV-2 infection and different platforms that are being used for vaccine development. We believe this review will increase our understanding of the vaccine designing against SARS-CoV-2 and subsequently contribute to the control of SARS-CoV-2 infections. Also, it gives an insight into the current status of vaccine development and associated outcomes reported at different phases of trial., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Viruses deploy genetically encoded strategies to coopt host machinery and support viral replicative cycles. Here, we use protein structure similarity to scan for molecular mimicry, manifested by structural similarity between viral and endogenous host proteins, across thousands of cataloged viruses and hosts spanning broad ecological niches and taxonomic range, including bacteria, plants and fungi, invertebrates, and vertebrates. This survey identified over 6,000,000 instances of structural mimicry; more than 70% of viral mimics cannot be discerned through protein sequence alone. We demonstrate that the manner and degree to which viruses exploit molecular mimicry varies by genome size and nucleic acid type and identify 158 human proteins that are mimicked by coronaviruses, providing clues about cellular processes driving pathogenesis. Our observations point to molecular mimicry as a pervasive strategy employed by viruses and indicate that the protein structure space used by a given virus is dictated by the host proteome. A record of this paper's transparent peer review process is included in the Supplemental Information., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem macrodomains within nonstructural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated antiviral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 domains exhibit similar structural folds, and all 3 proteins bound to ADP-ribose with affinities in the low micromolar range. Importantly, using ADP-ribose-detecting binding reagents in both a gel-based assay and novel enzyme-linked immunosorbent assays (ELISAs), we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate than the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused more than 1.2 million deaths worldwide. With no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic posttranslational process that is increasingly being recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose and describe its ADP-ribose binding and hydrolysis activities in direct comparison to those of SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain., (Copyright © 2021 Alhammad et al.)

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp8 2 -nsp12-nsp13 2 -RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Coronaviruses (CoVs) are causing a number of human and animal diseases because of their zoonotic nature such as Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19). These viruses can infect respiratory, gastrointestinal, hepatic and central nervous systems of human, livestock, birds, bat, mouse, and many wild animals. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerging respiratory virus and is causing CoVID-19 with high morbidity and considerable mortality. All CoVs belong to the order Nidovirales, family Coronaviridae, are enveloped positive-sense RNA viruses, characterised by club-like spikes on their surfaces and large RNA genome with a distinctive replication strategy. Coronavirus have the largest RNA genomes (~26-32 kilobases) and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral RNA polymerases. Non-structural proteins (nsp) 7-16 are cleaved from two large replicase polyproteins and guide the replication and processing of coronavirus RNA. Coronavirus replicase has more or less universal activities, such as RNA polymerase (nsp 12) and helicase (nsp 13), as well as a variety of unusual or even special mRNA capping (nsp 14, nsp 16) and fidelity regulation (nsp 14) domains. Besides that, several smaller subunits (nsp 7- nsp 10) serve as essential cofactors for these enzymes and contribute to the emerging "nsp interactome." In spite of the significant progress in studying coronaviruses structural and functional properties, there is an urgent need to understand the coronaviruses evolutionary success that will be helpful to develop enhanced control strategies. Therefore, it is crucial to understand the structure, function, and interactions of coronaviruses RNA synthesizing machinery and their replication strategies., (Copyright © 2020. Published by Elsevier Ltd.)

Our evolutionary and structural analyses revealed that the severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) spike gene is a complex mosaic resulting from several recombination events. Additionally, the fixation of variants has mainly been driven by purifying selection, suggesting the presence of conserved structural features. Our dynamic simulations identified two main long-range covariant dynamic movements of the novel glycoprotein, and showed that, as a result of the evolutionary duality, they are preserved. The first movement involves the receptor binding domain with the N -terminal domain and the C -terminal domain 2 and is maintained across human, bat and pangolin coronaviruses. The second is a complex network of long-range dynamics specific to SARS-CoV-2 involving the novel PRRA and the conserved KR*SF cleavage sites, as well as conserved segments in C -terminal domain 3. These movements, essential for host cell binding, are maintained by hinges conserved across human, bat, and pangolin coronaviruses glycoproteins. The hinges, located around Threonine 333 and Proline 527 within the N -terminal domain and C -terminal domain 2, represent candidate targets for the future development of novel pan-coronavirus inhibitors. In summary, we show that while recombination created a new configuration that increased the covariant dynamic movements of the SARS-CoV-2 glycoprotein, negative selection preserved its inter-domain structure throughout evolution in different hosts and inter-species transmissions.

Coronaviruses (CoVs) is showing obvious interspecies transmission, such as the SARS-CoV, MERS-CoV and SARS-CoV-2. Here, the emerging porcine deltacoronavirus (PDCoV) strain, isolated from Shanghai, China, broadly infects porcine, human and chicken cells in vitro. Previously studies by our group and others have confirmed that PDCoV nucleocapsid (N) protein performs an important role in antagonizing retinoic acid-induced gene I-like receptor (RLR) activation. However, the mechanism of PDCoV N protein suppressing porcine type I IFN production remains unclear, especially the downstream of porcine RLR signaling pathway. In the present study, porcine IRF7 (poIRF7) was identified as the interaction protein of PDCoV N protein through LC-MS/MS. The poIRF7 (268-487aa) was the key region of binding PDCoV N protein. Although IRF7 is a conserved functional protein in species, the PDCoV N protein has been confirmed to interact with only poIRF7 and significantly decrease poIRF7-induced type I IFN production, but not human or chicken IRF7. Furthermore, PDCoV N protein can promote poIRF7 degradation via the ubiquitin-proteasome pathway, which directly increased the K6, K11, and K29-linked polyubiquitination of poIRF7. Lysine 359 of poIRF7 was a key site in PDCoV N protein inducing poIRF7 degradation. Taken together, our results reveal a novel mechanism that PDCoV N protein could species-specifically interact with poIRF7 and then promote its degradation to suppress porcine type I IFN production. The novel findings provide a new insight into PDCoV and other zoonotic coronavirus evading the innate immune response of different species., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Methods previously developed by the author are applied to uncover several sites of interest in the spike glycoproteins of all known human coronaviruses (hCoVs), including SARS-CoV-2 that causes COVID-19. The sites comprise three-dimensional neighborhoods of peptides characterized by four key properties: (1) they pinpoint regions of high free energy in the backbone whose obstruction might interrupt function; (2) by their very definition, they occur rarely in the universe of all gene-encoded proteins that could obviate host response to compounds designed for their interference; (3) they are common to all known hCoV spikes, possibly retaining activity in light of inevitable viral mutation; and (4) they are exposed in the molecular surface of the glycoprotein. These peptides in SARS-CoV-2 are given by the triples of residues (131, 117, 134), (203, 227, 228), and (1058, 730, 731) in its spike.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a single-stranded RNA genome that encodes 14 open reading frames (ORFs), eight of which encode accessory proteins that allow the virus to infect the host and promote virulence. The genome expresses around 29 structural and nonstructural protein products. The accessory proteins of SARS-CoV-2 are not essential for virus replication but do affect viral release, stability, and pathogenesis and finally contribute to virulence. This paper has attempted the structure prediction and functional analysis of two such accessory proteins, 9b and ORF14, in the absence of experimental structures. Sequence analysis, structure prediction, functional characterization, and evolutionary analysis based on the UniProtKB reviewed the amino acid sequences of SARS-CoV-2 9b (P0DTD2) and ORF14 (P0DTD3) proteins. Modeling has been presented with the introduction of hybrid comparative and ab initio modeling. QMEANDisCo 4.0.0 and ProQ3 for global and local (per residue) quality estimates verified the structures as high quality, which may be attributed to structure-based drug design targets. Tunnel analysis revealed the presence of 1-2 highly active tunneling sites, perhaps which will able to provide certain inputs for advanced structure-based drug design or to formulate potential vaccines in the absence of a complete experimental structure. The evolutionary analysis of both proteins of human SARS-CoV-2 indicates close relatedness to the bat coronavirus. The whole-genome phylogeny indicates that only the new bat coronavirus followed by pangolin coronaviruses has a close evolutionary relationship with the novel SARS-CoV-2., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2020 Chittaranjan Baruah et al.)

As the number of people infected with the newly identified 2019 novel coronavirus (SARS-CoV2) is continuously increasing every day, development of potential therapeutic platforms is vital. Based on the comparatively high similarity of receptor-binding domain (RBD) in SARS-CoV2 and SARS-CoV, it seems crucial to assay the cross-reactivity of anti-SARS-CoV monoclonal antibodies (mAbs) with SARS-CoV2 spike (S)-protein. Indeed, developing mAbs targeting SARS-CoV2 S-protein RBD could show novel applications for rapid and sensitive development of potential epitope-specific vaccines (ESV). Herein, we present an overview on the discovery of new CoV followed by some explanation on the SARS-CoV2 S-protein RBD site. Furthermore, we surveyed the novel therapeutic mAbs for targeting S-protein RBD such as S230, 80R, F26G18, F26G19, CR3014, CR3022, M396, and S230.15. Afterwards, the mechanism of interaction of RBD and different mAbs were explained and it was suggested that one of the SARS-CoV-specific human mAbs, namely CR3022, could show the highest binding affinity with SARS-CoV2 S-protein RBD. Finally, some ongoing challenges and future prospects for rapid and sensitive advancement of therapeutic mAbs targeting S-protein RBD were discussed. In conclusion, it may be proposed that this review may pave the way for recognition of RBD and different mAbs to develop potential therapeutic ESV., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The pandemic due to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has emerged as a serious global public health issue. Since the start of the outbreak, the importance of hand-hygiene and respiratory protection to prevent the spread of the virus has been the prime focus for infection control. Health regulatory organisations have produced guidelines for the formulation of hand sanitisers to the manufacturing industries. This review summarises the studies on alcohol-based hand sanitisers and their disinfectant activity against SARS-CoV-2 and related viruses. The literature shows that the type and concentration of alcohol, formulation and nature of product, presence of excipients, applied volume, contact time and viral contamination load are critical factors that determine the effectiveness of hand sanitisers.

In less than 20 years, three deadly coronaviruses, SARS-CoV, MERS-CoV and SARS-CoV-2, have emerged in human population causing hundreds to hundreds of thousands of deaths. Other coronaviruses are causing epizootic representing a significant threat for both domestic and wild animals. Members of this viral family have the longest genome of all RNA viruses, and express up to 29 proteins establishing complex interactions with the host proteome. Deciphering these interactions is essential to identify cellular pathways hijacked by these viruses to replicate and escape innate immunity. Virus-host interactions also provide key information to select targets for antiviral drug development. Here, we have manually curated the literature to assemble a unique dataset of 1311 coronavirus-host protein-protein interactions. Functional enrichment and network-based analyses showed coronavirus connections to RNA processing and translation, DNA damage and pathogen sensing, interferon production, and metabolic pathways. In particular, this global analysis pinpointed overlooked interactions with translation modulators (GIGYF2-EIF4E2), components of the nuclear pore, proteins involved in mitochondria homeostasis (PHB, PHB2, STOML2), and methylation pathways (MAT2A/B). Finally, interactome data provided a rational for the antiviral activity of some drugs inhibiting coronaviruses replication. Altogether, this work describing the current landscape of coronavirus-host interactions provides valuable hints for understanding the pathophysiology of coronavirus infections and developing effective antiviral therapies.

The virus that causes COVID-19, SARS-CoV-2, has a large RNA genome that encodes numerous proteins that might be targets for antiviral drugs. Some of these proteins, such as the RNA-dependent RNA polymerase, helicase, and main protease, are well conserved between SARS-CoV-2 and the original SARS virus, but several others are not. This study examines one of the proteins encoded by SARS-CoV-2 that is most different, a macrodomain of nonstructural protein 3 (nsp3). Although 26% of the amino acids in this SARS-CoV-2 macrodomain differ from those observed in other coronaviruses, biochemical and structural data reveal that the protein retains the ability to bind ADP-ribose, which is an important characteristic of beta coronaviruses and a potential therapeutic target.

Global emergencies caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 significantly endanger human health. The spike (S) glycoprotein is the key antigen and its conserved S2 subunit contributes to viral entry by mediating host-viral membrane fusion. However, structural information of the post-fusion S2 from these highly pathogenic human-infecting coronaviruses is still lacking. We used single-particle cryo-electron microscopy to show that the post-fusion SARS-CoV S2 forms a further rotated HR1-HR2 six-helix bundle and a tightly bound linker region upstream of the HR2 motif. The structures of pre- and post-fusion SARS-CoV S glycoprotein dramatically differ, resembling that of the Mouse hepatitis virus (MHV) and other class I viral fusion proteins. This structure suggests potential targets for the development of vaccines and therapies against a wide range of SARS-like coronaviruses.

This paper continues a recent study of the spike protein sequence of the COVID-19 virus (SARS-CoV-2). It is also in part an introductory review to relevant computational techniques for tackling viral threats, using COVID-19 as an example. Q-UEL tools for facilitating access to knowledge and bioinformatics tools were again used for efficiency, but the focus in this paper is even more on the virus. Subsequence KRSFIEDLLFNKV of the S2' spike glycoprotein proteolytic cleavage site continues to appear important. Here it is shown to be recognizable in the common cold coronaviruses, avian coronaviruses and possibly as traces in the nidoviruses of reptiles and fish. Its function or functions thus seem important to the coronaviruses. It might represent SARS-CoV-2 Achilles' heel, less likely to acquire resistance by mutation, as has happened in some early SARS vaccine studies discussed in the previous paper. Preliminary conformational analysis of the receptor (ACE2) binding site of the spike protein is carried out suggesting that while it is somewhat conserved, it appears to be more variable than KRSFIEDLLFNKV. However compounds like emodin that inhibit SARS entry, apparently by binding ACE2, might also have functions at several different human protein binding sites. The enzyme 11β-hydroxysteroid dehydrogenase type 1 is again argued to be a convenient model pharmacophore perhaps representing an ensemble of targets, and it is noted that it occurs both in lung and alimentary tract. Perhaps it benefits the virus to block an inflammatory response by inhibiting the dehydrogenase, but a fairly complex web involves several possible targets., Competing Interests: Declaration of competing interest This paper is provided to the community to promote the more general applications of the thinking of Professor Paul A. M. Dirac in human and animal medicine in accordance with the charter of The Dirac Foundation, to emphasize the advantages and simplicity of the basic form of the Hyperbolic Dirac Net, to encourage its use, and to propose at least some of the principles of the associated Q-UEL, a universal exchange language for medicine, as a basis for a standard for interoperability. These mathematical and engineering principles are used, amongst many others in an integrated way, in the algorithms and internal architectural features of the BioIngine.com, a distributed system developed by Ingine Inc. Cleveland, Ohio, for the mining of, and inference from, Very Big Data for commercial purposes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was recently identified as the causative agent for the coronavirus disease 2019 (COVID-19) outbreak that has generated a global health crisis. We use a combination of genomic analysis and sensitive profile-based sequence and structure analysis to understand the potential pathogenesis determinants of this virus. As a result, we identify several fast-evolving genomic regions that might be at the interface of virus-host interactions, corresponding to the receptor binding domain of the Spike protein, the three tandem Macro fold domains in ORF1a, and the uncharacterized protein ORF8. Further, we show that ORF8 and several other proteins from alpha- and beta-CoVs belong to novel families of immunoglobulin (Ig) proteins. Among them, ORF8 is distinguished by being rapidly evolving, possessing a unique insert, and having a hypervariable position among SARS-CoV-2 genomes in its predicted ligand-binding groove. We also uncover numerous Ig domain proteins from several unrelated metazoan viruses, which are distinct in sequence and structure but share comparable architectures to those of the CoV Ig domain proteins. Hence, we propose that SARS-CoV-2 ORF8 and other previously unidentified CoV Ig domain proteins fall under the umbrella of a widespread strategy of deployment of Ig domain proteins in animal viruses as pathogenicity factors that modulate host immunity. The rapid evolution of the ORF8 Ig domain proteins points to a potential evolutionary arms race between viruses and hosts, likely arising from immune pressure, and suggests a role in transmission between distinct host species. IMPORTANCE The ongoing COVID-19 pandemic strongly emphasizes the need for a more complete understanding of the biology and pathogenesis of its causative agent SARS-CoV-2. Despite intense scrutiny, several proteins encoded by the genomes of SARS-CoV-2 and other SARS-like coronaviruses remain enigmatic. Moreover, the high infectivity and severity of SARS-CoV-2 in certain individuals make wet-lab studies currently challenging. In this study, we used a series of computational strategies to identify several fast-evolving regions of SARS-CoV-2 proteins which are potentially under host immune pressure. Most notably, the hitherto-uncharacterized protein encoded by ORF8 is one of them. Using sensitive sequence and structural analysis methods, we show that ORF8 and several other proteins from alpha- and beta-coronavirus comprise novel families of immunoglobulin domain proteins, which might function as potential immune modulators to delay or attenuate the host immune response against the viruses., (Copyright © 2020 Tan et al.)

A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans, causing COVID-19 1,2 . 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 humans 3,4 . 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.

The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This paper concerns study of the genome of the Wuhan Seafood Market isolate believed to represent the causative agent of the disease COVID-19. This is to find a short section or sections of viral protein sequence suitable for preliminary design proposal for a peptide synthetic vaccine and a peptidomimetic therapeutic, and to explore some design possibilities. The project was originally directed towards a use case for the Q-UEL language and its implementation in a knowledge management and automated inference system for medicine called the BioIngine, but focus here remains mostly on the virus itself. However, using Q-UEL systems to access relevant and emerging literature, and to interact with standard publically available bioinformatics tools on the Internet, did help quickly identify sequences of amino acids that are well conserved across many coronaviruses including 2019-nCoV. KRSFIEDLLFNKV was found to be particularly well conserved in this study and corresponds to the region around one of the known cleavage sites of the SARS virus that are believed to be required for virus activation for cell entry. This sequence motif and surrounding variations formed the basis for proposing a specific synthetic vaccine epitope and peptidomimetic agent. The work can, nonetheless, be described in traditional bioinformatics terms, and readily reproduced by others, albeit with the caveat that new data and research into 2019-nCoV is emerging and evolving at an explosive pace. Preliminary studies using molecular modeling and docking, and in that context the potential value of certain known herbal extracts, are also described., Competing Interests: Declaration of competing interest This paper is provided to the community to promote the more general applications of the thinking of Professor Paul A. M. Dirac in human and animal medicine in accordance with the charter of The Dirac Foundation, to emphasize the advantages and simplicity of the basic form of the Hyperbolic Dirac Net, to encourage its use, and to propose at least some of the principles of the associated Q-UEL, a universal exchange language for medicine, as a basis for a standard for interoperability. These mathematical and engineering principles are used, amongst many others in an integrated way, in the algorithms and internal architectural features of the BioIngine.com, a distributed system developed by Ingine Inc. Cleveland, Ohio, for the mining of, and inference from, Very Big Data for commercial purposes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Coronaviruses (CoVs) primarily cause enzootic infections in birds and mammals but, in the last few decades, have shown to be capable of infecting humans as well. The outbreak of severe acute respiratory syndrome (SARS) in 2003 and, more recently, Middle-East respiratory syndrome (MERS) has demonstrated the lethality of CoVs when they cross the species barrier and infect humans. A renewed interest in coronaviral research has led to the discovery of several novel human CoVs and since then much progress has been made in understanding the CoV life cycle. The CoV envelope (E) protein is a small, integral membrane protein involved in several aspects of the virus' life cycle, such as assembly, budding, envelope formation, and pathogenesis. Recent studies have expanded on its structural motifs and topology, its functions as an ion-channelling viroporin, and its interactions with both other CoV proteins and host cell proteins., Main Body: This review aims to establish the current knowledge on CoV E by highlighting the recent progress that has been made and comparing it to previous knowledge. It also compares E to other viral proteins of a similar nature to speculate the relevance of these new findings. Good progress has been made but much still remains unknown and this review has identified some gaps in the current knowledge and made suggestions for consideration in future research., Conclusions: The most progress has been made on SARS-CoV E, highlighting specific structural requirements for its functions in the CoV life cycle as well as mechanisms behind its pathogenesis. Data shows that E is involved in critical aspects of the viral life cycle and that CoVs lacking E make promising vaccine candidates. The high mortality rate of certain CoVs, along with their ease of transmission, underpins the need for more research into CoV molecular biology which can aid in the production of effective anti-coronaviral agents for both human CoVs and enzootic CoVs.

Nidovirus endoribonucleases (NendoUs) include nonstructural protein 15 (nsp15) from coronaviruses and nsp11 from arteriviruses, both of which have been reported to participate in the viral replication process and in the evasion of the host immune system. Results from a previous study of coronaviruses SARS-CoV, HCoV-229E, and MHV nsp15 indicate that it mainly forms a functional hexamer, whereas nsp11 from the arterivirus PRRSV is a dimer. Here, we found that porcine Deltacoronavirus (PDCoV) nsp15 primarily exists as dimers and monomers in vitro Biological experiments reveal that a PDCoV nsp15 mutant lacking the first 27 amino acids of the N-terminal domain (Asn-1-Asn-27) forms more monomers and displays decreased enzymatic activity, indicating that this region is important for its dimerization. Moreover, multiple sequence alignments and three-dimensional structural analysis indicated that the C-terminal region (His-251-Val-261) of PDCoV nsp15 is 10 amino acids shorter and forms a shorter loop than that formed by the equivalent sequence (Gln-259-Phe-279) of SARS-CoV nsp15. This result may explain why PDCoV nsp15 failed to form hexamers. We speculate that NendoUs may have originated from XendoU endoribonucleases (XendoUs) forming monomers in eukaryotic cells, that NendoU from arterivirus gained the ability to form dimers, and that the coronavirus variants then evolved the capacity to assemble into hexamers. We further propose that PDCoV nsp15 may be an intermediate in this evolutionary process. Our findings provide a theoretical basis for improving our understanding of NendoU evolution and offer useful clues for designing drugs and vaccines against nidoviruses., (© 2018 Zheng et al.)

The coronavirus nonstructural protein 3 (nsp3) is a multifunctional protein that comprises multiple structural domains. This protein assists viral polyprotein cleavage, host immune interference, and may play other roles in genome replication or transcription. Here, we report the solution NMR structure of a protein from the "SARS-unique region" of the bat coronavirus HKU9. The protein contains a frataxin fold or double-wing motif, which is an α + β fold that is associated with protein/protein interactions, DNA binding, and metal ion binding. High structural similarity to the human severe acute respiratory syndrome (SARS) coronavirus nsp3 is present. A possible functional site that is conserved among some betacoronaviruses has been identified using bioinformatics and biochemical analyses. This structure provides strong experimental support for the recent proposal advanced by us and others that the "SARS-unique" region is not unique to the human SARS virus, but is conserved among several different phylogenetic groups of coronaviruses and provides essential functions., (© 2017 The Protein Society.)

Accumulating evidence indicates that MERS-CoV originated from bat coronaviruses (BatCoVs). Previously, we demonstrated that both MERS-CoV and BatCoV HKU4 use CD26 as a receptor, but how the BatCoVs evolved to bind CD26 is an intriguing question. Here, we solved the crystal structure of the S1 subunit C-terminal domain of HKU5 (HKU5-CTD), another BatCoV that is phylogenetically related to MERS-CoV but cannot bind to CD26. We observed that the conserved core subdomain and those of other betacoronaviruses (betaCoVs) have a similar topology of the external subdomain, indicating the same ancestor of lineage C betaCoVs. However, two deletions in two respective loops located in HKU5-CTD result in conformational variations in CD26-binding interface and are responsible for the non-binding of HKU5-CTD to CD26. Combined with sequence variation in the HKU5-CTD receptor binding interface, we propose the necessity for surveilling the mutation in BatCoV HKU5 spike protein in case of bat-to-human interspecies transmission., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. The first outbreak of PDCoV was announced from the United States in 2014, followed by reports in Asia. The nonstructural protein nsp5 is a 3C-like protease of coronavirus, and our previous study showed that PDCoV nsp5 inhibits type I interferon (IFN) production. In this study, we found that PDCoV nsp5 significantly inhibited IFN-stimulated response element (ISRE) promoter activity and transcription of IFN-stimulated genes (ISGs), suggesting that PDCoV nsp5 also suppresses IFN signaling. Detailed analysis showed that nsp5 cleaved signal transducer and activator of transcription 2 (STAT2) but not Janus kinase 1 (JAK1), tyrosine kinase 2 (TYK2), STAT1, and interferon regulatory factor 9 (IRF9), key molecules of the JAK-STAT pathway. STAT2 cleavage was dependent on the protease activity of nsp5. Interestingly, nsp5 cleaved STAT2 at two sites, glutamine 685 (Q685) and Q758, and similar cleavage was observed in PDCoV-infected cells. As expected, cleaved STAT2 impaired the ability to induce ISGs, demonstrating that STAT2 cleavage is an important mechanism utilized by PDCoV nsp5 to antagonize IFN signaling. We also discussed the substrate selection and binding mode of PDCoV nsp5 by homologous modeling of PDCoV nsp5 with the two cleaved peptide substrates. The results of our study demonstrate that PDCoV nsp5 antagonizes type I IFN signaling by cleaving STAT2 and provides structural insights for comprehending the cleavage mechanism of PDCoV nsp5, revealing a potential new function for PDCoV nsp5 in type I IFN signaling. IMPORTANCE The 3C-like protease encoded by nsp5 is a major protease of coronaviruses; thus, it is an attractive target for development of anticoronavirus drugs. Previous studies have revealed that the 3C-like protease of coronaviruses, including PDCoV and porcine epidemic diarrhea virus (PEDV), antagonizes type I IFN production by targeting the NF-κB essential modulator (NEMO). Here, for the first time, we demonstrate that overexpression of PDCoV nsp5 also antagonizes IFN signaling by cleaving STAT2, an essential component of transcription factor complex ISGF3, and that PDCoV infection reduces the levels of STAT2, which may affect the innate immune response., (Copyright © 2017 American Society for Microbiology.)

ADP-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. To regulate this activity, macrodomain proteins enzymatically remove covalently attached ADP-ribose from protein targets. All members of the Coronavirinae, a subfamily of positive-sense RNA viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3). However, its function or targets during infection remain unknown. We identified several macrodomain mutations that greatly reduced nsp3's de-ADP-ribosylation activity in vitro Next, we created recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) strains with these mutations. These mutations led to virus attenuation and a modest reduction of viral loads in infected mice, despite normal replication in cell culture. Further, macrodomain mutant virus elicited an early, enhanced interferon (IFN), interferon-stimulated gene (ISG), and proinflammatory cytokine response in mice and in a human bronchial epithelial cell line. Using a coinfection assay, we found that inclusion of mutant virus in the inoculum protected mice from an otherwise lethal SARS-CoV infection without reducing virus loads, indicating that the changes in innate immune response were physiologically significant. In conclusion, we have established a novel function for the SARS-CoV macrodomain that implicates ADP-ribose in the regulation of the innate immune response and helps to demonstrate why this domain is conserved in CoVs., Importance: The macrodomain is a ubiquitous structural domain that removes ADP-ribose from proteins, reversing the activity of ADP-ribosyltransferases. All coronaviruses contain a macrodomain, suggesting that ADP-ribosylation impacts coronavirus infection. However, its function during infection remains unknown. Here, we found that the macrodomain is an important virulence factor for a highly pathogenic human CoV, SARS-CoV. Viruses with macrodomain mutations that abrogate its ability to remove ADP-ribose from protein were unable to cause lethal disease in mice. Importantly, the SARS-CoV macrodomain suppressed the innate immune response during infection. Our data suggest that an early innate immune response can protect mice from lethal disease. Understanding the mechanism used by this enzyme to promote disease will open up novel avenues for coronavirus therapies and give further insight into the role of macrodomains in viral pathogenesis., (Copyright © 2016 Fehr et al.)

The suggested bat origin for Middle East respiratory syndrome coronavirus (MERS-CoV) has revitalized the studies of other bat-derived coronaviruses with respect to interspecies transmission potential. Bat coronavirus (BatCoV) HKU9 is an important betacoronavirus (betaCoV) that is phylogenetically affiliated with the same genus as MERS-CoV. The bat surveillance data indicated that BatCoV HKU9 has been widely spreading and circulating in bats. This highlights the necessity of characterizing the virus for its potential to cross species barriers. The receptor binding domain (RBD) of the coronavirus spike (S) protein recognizes host receptors to mediate virus entry and is therefore a key factor determining the viral tropism and transmission capacity. In this study, the putative S RBD of BatCoV HKU9 (HKU9-RBD), which is homologous to other betaCoV RBDs that have been structurally and functionally defined, was characterized via a series of biophysical and crystallographic methods. By using surface plasmon resonance, we demonstrated that HKU9-RBD binds to neither SARS-CoV receptor ACE2 nor MERS-CoV receptor CD26. We further determined the atomic structure of HKU9-RBD, which as expected is composed of a core and an external subdomain. The core subdomain fold resembles those of other betaCoV RBDs, whereas the external subdomain is structurally unique with a single helix, explaining the inability of HKU9-RBD to react with either ACE2 or CD26. Via comparison of the available RBD structures, we further proposed a homologous intersubdomain binding mode in betaCoV RBDs that anchors the external subdomain to the core subdomain. The revealed RBD features would shed light on the evolution route of betaCoV.

Middle East respiratory syndrome (MERS) is an emerging infectious disease of growing global importance, which has caused severe acute respiratory disease in more than 1,700 people, resulting in almost 600 deaths. MERS is caused by a novel betacoronavirus (MERS-CoV). All cases of MERS have been linked through travel to or residence in countries in or near the Arabian Peninsula. Dromedary camels are considered natu- ral reservoirs for MERS-CoV. MERS-CoV is mainly transmitted from infected dromedary camels to human beings, and it is transmitted among human beings by droplets, contact, and perhaps airborne spread. Both community-acquired and hospital-acquired cases have been reported with little human-to-human transmis- sion reported in the community. The largest known outbreak of MERS outside the Arabian Peninsula oc- curred in the Republic of Korea in 2015, with 186 cases. The outbreak was associated with a traveler re- turning from the Arabian Peninsula. Clinical features of MERS range from asymptomatic or mild disease to acute respiratory distress syndrome and multi-organ failure resulting in death, especially in individuals with underlying comorbidities. MERS is suspected in the presence of febrile acute respiratory illness and close contact with MERS-CoV, and can be confirmed by the detection of viral nucleic acid through RT-PCR or se- rology. No specific drug treatment exists for MERS; however, the neutralizing antibodies, ribavirin and interferon have been shown to be potentially useful anti-MERS-CoV drugs. Rigorous infection prevention and control measures with droplet and contact precautions are crucial to prevent the spread in health-care facilities. [Review].

HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease, and is related to the zoonotic SARS and MERS betacoronaviruses, which have high fatality rates and pandemic potential. Cell tropism and host range is determined in part by the coronavirus spike (S) protein, 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 Å 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.

Coronavirus particles serve three fundamentally important functions in infection. The virion provides the means to deliver the viral genome across the plasma membrane of a host cell. The virion is also a means of escape for newly synthesized genomes. Lastly, the virion is a durable vessel that protects the genome on its journey between cells. This review summarizes the available X-ray crystallography, NMR, and cryoelectron microscopy structural data for coronavirus structural proteins, and looks at the role of each of the major structural proteins in virus entry and assembly. The potential wider conservation of the nucleoprotein fold identified in the Arteriviridae and Coronaviridae families and a speculative model for the evolution of corona-like virus architecture are discussed., (© 2016 Elsevier Inc. All rights reserved.)

The 3'-terminal domain of the most conserved ORF1b in three of the four families of the order Nidovirales (except for the family Arteriviridae) encodes a (putative) 2'-O-methyltransferase (2'-O-MTase), known as non structural protein (nsp) 16 in the family Coronaviridae and implicated in methylation of the 5' cap structure of nidoviral mRNAs. As with coronavirus transcripts, arterivirus mRNAs are assumed to possess a 5' cap although no candidate MTases have been identified thus far. To address this knowledge gap, we analysed the uncharacterized nsp12 of arteriviruses, which occupies the ORF1b position equivalent to that of the nidovirus 2'-O-MTase (coronavirus nsp16). In our in-depth bioinformatics analysis of nsp12, the protein was confirmed to be family specific whilst having diverged much further than other nidovirus ORF1b-encoded proteins, including those of the family Coronaviridae. Only one invariant and several partially conserved, predominantly aromatic residues were identified in nsp12, which may adopt a structure with alternating α-helices and β-strands, an organization also found in known MTases. However, no statistically significant similarity was found between nsp12 and the twofold larger coronavirus nsp16, nor could we detect MTase activity in biochemical assays using recombinant equine arteritis virus (EAV) nsp12. Our further analysis established that this subunit is essential for replication of this prototypic arterivirus. Using reverse genetics, we assessed the impact of 25 substitutions at 14 positions, yielding virus phenotypes ranging from WT-like to non-viable. Notably, replacement of the invariant phenylalanine 109 with tyrosine was lethal. We concluded that nsp12 plays an essential role during EAV replication, possibly by acting as a co-factor for another enzyme.

Objective: The spike (S) protein of porcine hemagglutinating encephalomyelitis virus (PHEV) may mediate infection by binding to a cellular neural cell adhesion molecule (NCAM). This study aimed to identify the crucial domain of the S1 subunit of the S protein that interacts with NCAM., Methods: Three truncated segments (S(1-291), S(277-794) and S(548-868)) of the S gene of PHEV and the NCAM gene were cloned individually into the Escherichia coli expression vectors and yeast two-hybrid expression vectors. The interaction between S(1-291), S(277-794), S(548-868) and NCAM were detected by a GST pull-down experiment and yeast two-hybrid assay., Results: Three fusion proteins (S(1-291), S(277-794) and S(548-868)) were screened for their interactions with NCAM by protein-protein interaction assays. The results of these assays clarified that S(277-794) interacted with NCAM, while S(1-291) and S(548-868) did not., Conclusions: A small fragment (258-amino-acid fragment, residues 291-548) on the PHEV S protein was posited to be the minimum number of amino acids necessary to interact with NCAM. This fragment may be the receptor-binding domain that mediates PHEV binding to NCAM., (© 2015 S. Karger AG, Basel.)

Coronavirus genome replication is mediated by a multi-subunit protein complex that is comprised of more than a dozen virally encoded and several cellular proteins. Interactions of the viral replicase complex with cis-acting RNA elements located in the 5' and 3'-terminal genome regions ensure the specific replication of viral RNA. Over the past years, boundaries and structures of cis-acting RNA elements required for coronavirus genome replication have been extensively characterized in betacoronaviruses and, to a lesser extent, other coronavirus genera. Here, we review our current understanding of coronavirus cis-acting elements located in the terminal genome regions and use a combination of bioinformatic and RNA structure probing studies to identify and characterize putative cis-acting RNA elements in alphacoronaviruses. The study suggests significant RNA structure conservation among members of the genus Alphacoronavirus but also across genus boundaries. Overall, the conservation pattern identified for 5' and 3'-terminal RNA structural elements in the genomes of alpha- and betacoronaviruses is in agreement with the widely used replicase polyprotein-based classification of the Coronavirinae, suggesting co-evolution of the coronavirus replication machinery with cognate cis-acting RNA elements., (Copyright © 2014 Elsevier B.V. All rights reserved.)