TMPRSS2 and glycan receptors synergistically facilitate coronavirus entry.

The entry of coronaviruses is initiated by spike recognition of host cellular receptors, involving proteinaceous and/or glycan receptors. Recently, TMPRSS2 was identified as the proteinaceous receptor for HCoV-HKU1 alongside sialoglycan as a glycan receptor. However, the underlying mechanisms for viral entry remain unknown. Here, we investigated the HCoV-HKU1C spike in the inactive, glycan-activated, and functionally anchored states, revealing that sialoglycan binding induces a conformational change of the NTD and promotes the neighboring RBD of the spike to open for TMPRSS2 recognition, exhibiting a synergistic mechanism for the entry of HCoV-HKU1. The RBD of HCoV-HKU1 features an insertion subdomain that recognizes TMPRSS2 through three previously undiscovered interfaces. Furthermore, structural investigation of HCoV-HKU1A in combination with mutagenesis and binding assays confirms a conserved receptor recognition pattern adopted by HCoV-HKU1. These studies advance our understanding of the complex viral-host interactions during entry, laying the groundwork for developing new therapeutics against coronavirus-associated diseases.

Structural basis of TMPRSS2 zymogen activation and recognition by the HKU1 seasonal coronavirus.

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.

Structure, receptor recognition, and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein.

The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single-nucleotide polymorphisms might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among ɑ-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.

Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion.

Recent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism.

Coronaviral ORF6 protein mediates inter-organelle contacts and modulates host cell lipid flux for virus production.

Lipid droplets (LDs) form inter-organelle contacts with the endoplasmic reticulum (ER) that promote their biogenesis, while LD contacts with mitochondria enhance ß-oxidation of contained fatty acids. Viruses have been shown to take advantage of lipid droplets to promote viral production, but it remains unclear whether they also modulate the interactions between LDs and other organelles. Here, we showed that coronavirus ORF6 protein targets LDs and is localized to the mitochondria-LD and ER-LD contact sites, where it regulates LD biogenesis and lipolysis. At the molecular level, we find that ORF6 inserts into the LD lipid monolayer via its two amphipathic helices. ORF6 further interacts with ER membrane proteins BAP31 and USE1 to mediate ER-LDs contact formation. Additionally, ORF6 interacts with the SAM complex in the mitochondrial outer membrane to link mitochondria to LDs. In doing so, ORF6 promotes cellular lipolysis and LD biogenesis to reprogram host cell lipid flux and facilitate viral production.

Structural basis for mouse receptor recognition by bat SARS2-like coronaviruses.

The animal origin of SARS-CoV-2 remains elusive, lacking a plausible evolutionary narrative that may account for its emergence. Its spike protein resembles certain segments of BANAL-236 and RaTG13, two bat coronaviruses considered possible progenitors of SARS-CoV-2. Additionally, its spike contains a furin motif, a common feature of rodent coronaviruses. To explore the possible involvement of rodents in the emergence of SARS-CoV-2 spike, we examined the crystal structures of the spike receptor-binding domains (RBDs) of BANAL-236 and RaTG13 each complexed with mouse receptor ACE2. Both RBDs have residues at positions 493 and 498 that align well with two virus-binding hotspots on mouse ACE2. Our biochemical evidence supports that both BANAL-236 and RaTG13 spikes can use mouse ACE2 as their entry receptor. These findings point to a scenario in which these bat coronaviruses may have coinfected rodents, leading to a recombination of their spike genes and a subsequent acquisition of a furin motif in rodents, culminating in the emergence of SARS-CoV-2.

Evolution of coronavirus frameshifting elements: Competing stem networks explain conservation and variability.

The frameshifting RNA element (FSE) in coronaviruses (CoVs) regulates the programmed -1 ribosomal frameshift (-1 PRF) mechanism common to many viruses. The FSE is of particular interest as a promising drug candidate. Its associated pseudoknot or stem loop structure is thought to play a large role in frameshifting and thus viral protein production. To investigate the FSE structural evolution, we use our graph theory-based methods for representing RNA secondary structures in the RNA-As-Graphs (RAG) framework to calculate conformational landscapes of viral FSEs with increasing sequence lengths for representative 10 Alpha and 13 Beta-CoVs. By following length-dependent conformational changes, we show that FSE sequences encode many possible competing stems which in turn favor certain FSE topologies, including a variety of pseudoknots, stem loops, and junctions. We explain alternative competing stems and topological FSE changes by recurring patterns of mutations. At the same time, FSE topology robustness can be understood by shifted stems within different sequence contexts and base pair coevolution. We further propose that the topology changes reflected by length-dependent conformations contribute to tuning the frameshifting efficiency. Our work provides tools to analyze virus sequence/structure correlations, explains how sequence and FSE structure have evolved for CoVs, and provides insights into potential mutations for therapeutic applications against a broad spectrum of CoV FSEs by targeting key sequence/structural transitions.

Crystal structure and biochemical activity of the macrodomain from rubella virus p150.

Rubella virus encodes a nonstructural polyprotein with RNA polymerase, methyltransferase, and papain-like cysteine protease activities, along with a putative macrodomain of unknown function. Macrodomains bind ADP-ribose adducts, a post-translational modification that plays a key role in host-virus conflicts. Some macrodomains can also remove the mono-ADP-ribose adduct or degrade poly-ADP-ribose chains. Here, we report high-resolution crystal structures of the macrodomain from rubella virus nonstructural protein p150, with and without ADP-ribose binding. The overall fold is most similar to macroD-type macrodomains from various nonviral species. The specific composition and structure of the residues that coordinate ADP-ribose in the rubella virus macrodomain are most similar to those of macrodomains from alphaviruses. Isothermal calorimetry shows that the rubella virus macrodomain binds ADP-ribose in solution. Enzyme assays show that the rubella virus macrodomain can hydrolyze both mono- and poly-ADP-ribose adducts. Site-directed mutagenesis identifies Asn39 and Cys49 required for mono-ADP-ribosylhydrolase (de-MARylation) activity.IMPORTANCERubella virus remains a global health threat. Rubella infections during pregnancy can cause serious congenital pathology, for which no antiviral treatments are available. Our work demonstrates that, like alpha- and coronaviruses, rubiviruses encode a mono-ADP-ribosylhydrolase with a structurally conserved macrodomain fold to counteract MARylation by poly (ADP-ribose) polymerases (PARPs) in the host innate immune response. Our structural data will guide future efforts to develop novel antiviral therapeutics against rubella or infections with related viruses.

CovInter: interaction data between coronavirus RNAs and host proteins.

Coronavirus has brought about three massive outbreaks in the past two decades. Each step of its life cycle invariably depends on the interactions among virus and host molecules. The interaction between virus RNA and host protein (IVRHP) is unique compared to other virus-host molecular interactions and represents not only an attempt by viruses to promote their translation/replication, but also the host's endeavor to combat viral pathogenicity. In other words, there is an urgent need to develop a database for providing such IVRHP data. In this study, a new database was therefore constructed to describe the interactions between coronavirus RNAs and host proteins (CovInter). This database is unique in (a) unambiguously characterizing the interactions between virus RNA and host protein, (b) comprehensively providing experimentally validated biological function for hundreds of host proteins key in viral infection and (c) systematically quantifying the differential expression patterns (before and after infection) of these key proteins. Given the devastating and persistent threat of coronaviruses, CovInter is highly expected to fill the gap in the whole process of the 'molecular arms race' between viruses and their hosts, which will then aid in the discovery of new antiviral therapies. It's now free and publicly accessible at: https://idrblab.org/covinter/.

Porcine deltacoronavirus accessory protein NS6 harnesses VPS35-mediated retrograde trafficking to facilitate efficient viral infection.

IMPORTANCE: Retrograde transport has been reported to be closely associated with normal cellular biological processes and viral replication. As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has attracted considerable attention. However, whether retrograde transport is associated with PDCoV infection remains unclear. Our present study demonstrates that retromer protein VPS35 acts as a critical host factor that is required for PDCoV infection. Mechanically, VPS35 interacts with PDCoV NS6, mediating the retrograde transport of NS6 from endosomes to the Golgi and preventing it from lysosomal degradation. Recombinant PDCoVs with an NS6 deletion display resistance to VPS35 deficiency. Our work reveals a novel evasion mechanism of PDCoV that involves the manipulation of the retrograde transport pathway by VPS35, providing new insight into the mechanism of PDCoV infection.

HDAC6 Degrades nsp8 of Porcine Deltacoronavirus through Deacetylation and Ubiquitination to Inhibit Viral Replication.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that has the potential to infect humans. Histone deacetylase 6 (HDAC6) is a unique type IIb cytoplasmic deacetylase with both deacetylase activity and ubiquitin E3 ligase activity, which mediates a variety of cellular processes by deacetylating histone and nonhistone substrates. In this study, we found that ectopic expression of HDAC6 significantly inhibited PDCoV replication, while the reverse effects could be observed after treatment with an HDAC6-specific inhibitor (tubacin) or knockdown of HDAC6 expression by specific small interfering RNA. Furthermore, we demonstrated that HDAC6 interacted with viral nonstructural protein 8 (nsp8) in the context of PDCoV infection, resulting in its proteasomal degradation, which was dependent on the deacetylation activity of HDAC6. We further identified the key amino acid residues lysine 46 (K46) and K58 of nsp8 as acetylation and ubiquitination sites, respectively, which were required for HDAC6-mediated degradation. Through a PDCoV reverse genetics system, we confirmed that recombinant PDCoV with a mutation at either K46 or K58 exhibited resistance to the antiviral activity of HDAC6, thereby exhibiting higher replication compared with wild-type PDCoV. Collectively, these findings contribute to a better understanding of the function of HDAC6 in regulating PDCoV infection and provide new strategies for the development of anti-PDCoV drugs. IMPORTANCE As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has sparked tremendous attention. Histone deacetylase 6 (HDAC6) is a critical deacetylase with both deacetylase activity and ubiquitin E3 ligase activity and is extensively involved in many important physiological processes. However, little is known about the role of HDAC6 in the infection and pathogenesis of coronaviruses. Our present study demonstrates that HDAC6 targets PDCoV-encoded nonstructural protein 8 (nsp8) for proteasomal degradation through the deacetylation at the lysine 46 (K46) and the ubiquitination at K58, suppressing viral replication. Recombinant PDCoV with a mutation at K46 and/or K58 of nsp8 displayed resistance to the antiviral activity of HDAC6. Our work provides significant insights into the role of HDAC6 in regulating PDCoV infection, opening avenues for the development of novel anti-PDCoV drugs.

Human Coronavirus: Host-Pathogen Interaction.

Human coronavirus (HCoV) infection causes respiratory diseases with mild to severe outcomes. In the last 15 years, we have witnessed the emergence of two zoonotic, highly pathogenic HCoVs: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Replication of HCoV is regulated by a diversity of host factors and induces drastic alterations in cellular structure and physiology. Activation of critical signaling pathways during HCoV infection modulates the induction of antiviral immune response and contributes to the pathogenesis of HCoV. Recent studies have begun to reveal some fundamental aspects of the intricate HCoV-host interaction in mechanistic detail. In this review, we summarize the current knowledge of host factors co-opted and signaling pathways activated during HCoV infection, with an emphasis on HCoV-infection-induced stress response, autophagy, apoptosis, and innate immunity. The cross talk among these pathways, as well as the modulatory strategies utilized by HCoV, is also discussed.

Coronavirus induces diabetic macrophage-mediated inflammation via SETDB2.

COVID-19 induces a robust, extended inflammatory "cytokine storm" that contributes to an increased morbidity and mortality, particularly in patients with type 2 diabetes (T2D). Macrophages are a key innate immune cell population responsible for the cytokine storm that has been shown, in T2D, to promote excess inflammation in response to infection. Using peripheral monocytes and sera from human patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and a murine hepatitis coronavirus (MHV-A59) (an established murine model of SARS), we identified that coronavirus induces an increased Mφ-mediated inflammatory response due to a coronavirus-induced decrease in the histone methyltransferase, SETDB2. This decrease in SETDB2 upon coronavirus infection results in a decrease of the repressive trimethylation of histone 3 lysine 9 (H3K9me3) at NFkB binding sites on inflammatory gene promoters, effectively increasing inflammation. Mφs isolated from mice with a myeloid-specific deletion of SETDB2 displayed increased pathologic inflammation following coronavirus infection. Further, IFNß directly regulates SETDB2 in Mφs via JaK1/STAT3 signaling, as blockade of this pathway altered SETDB2 and the inflammatory response to coronavirus infection. Importantly, we also found that loss of SETDB2 mediates an increased inflammatory response in diabetic Mϕs in response to coronavirus infection. Treatment of coronavirus-infected diabetic Mφs with IFNß reversed the inflammatory cytokine production via up-regulation of SETDB2/H3K9me3 on inflammatory gene promoters. Together, these results describe a potential mechanism for the increased Mφ-mediated cytokine storm in patients with T2D in response to COVID-19 and suggest that therapeutic targeting of the IFNß/SETDB2 axis in T2D patients may decrease pathologic inflammation associated with COVID-19.

Transmissible Gastroenteritis Virus Nucleocapsid Protein Interacts with Na+/H+ Exchanger 3 To Reduce Na+/H+ Exchanger Activity and Promote Piglet Diarrhea.

Transmissible gastroenteritis virus (TGEV) is member of the family Coronaviridae and mainly causes acute diarrhea. TGEV infection is characterized by vomiting, watery diarrhea, and severe dehydration, resulting in high mortality rates in neonatal piglets. TGEV infection symptoms are related to an imbalance of sodium absorption in small intestinal epithelial cells; however, the etiology of sodium imbalance diarrhea caused by TGEV remains unclear. In this study, we performed transcriptomic analysis of intestinal tissues from infected and healthy piglets and observed that the expression of NHE3, encoding Na+/H+ exchanger 3 (NHE3), the main exchanger of electroneutral sodium in intestinal epithelial cells, was significantly reduced upon TGEV infection. We also showed that specific inhibition of intestinal NHE3 activity could lead to the development of diarrhea in piglets. Furthermore, we revealed an interaction between TGEV N protein and NHE3 near the nucleus. The binding of TGEV N to NHE3 directly affected the expression and activity of NHE3 on the cell surface and affected cellular electrolyte absorption, leading to diarrhea. Molecular docking and computer-aided screening techniques were used to screen for the blocker of the interaction between TGEV N and NHE3, which identified irinotecan. We then demonstrated that irinotecan was effective in relieving TGEV-induced diarrhea in piglets. These findings provide new insights into the mechanism of TGEV-induced sodium imbalance diarrhea and could lead to the design of novel antiviral strategies against TGEV. IMPORTANCE A variety of coronaviruses have been found to cause severe diarrhea in hosts, including TGEV; however, the pathogenic mechanism is not clear. Therefore, prompt determination of the mechanism and identification of efficient therapeutic agents are required, both for public health reasons and for economic development. In this study, we demonstrated that NHE3 is the major expressed protein of NHEs in the intestine, and its expression decreased by nearly 70% after TGEV infection. Also, specific inhibition of intestinal NHE3 resulted in severe diarrhea in piglets. This demonstrated that NHE3 plays an important role in TGEV-induced diarrhea. In addition, we found that TGEV N directly regulates NHE3 expression and activity through protein-protein interaction, which is essential to promote diarrhea. Molecular docking and other techniques demonstrated that irinotecan could block the interaction and diarrhea caused by TGEV. Thus, our results provide a basis for the development of novel therapeutic agents against TGEV and guidance for the development of drugs for other diarrhea-causing coronaviruses.

Novel modulators of p53-signaling encoded by unknown genes of emerging viruses.

The p53 transcription factor plays a key role both in cancer and in the cell-intrinsic response to infections. The ORFEOME project hypothesized that novel p53-virus interactions reside in hitherto uncharacterized, unknown, or hypothetical open reading frames (orfs) of human viruses. Hence, 172 orfs of unknown function from the emerging viruses SARS-Coronavirus, MERS-Coronavirus, influenza, Ebola, Zika (ZIKV), Chikungunya and Kaposi Sarcoma-associated herpesvirus (KSHV) were de novo synthesized, validated and tested in a functional screen of p53 signaling. This screen revealed novel mechanisms of p53 virus interactions and two viral proteins KSHV orf10 and ZIKV NS2A binding to p53. Originally identified as the target of small DNA tumor viruses, these experiments reinforce the notion that all viruses, including RNA viruses, interfere with p53 functions. These results validate this resource for analogous systems biology approaches to identify functional properties of uncharacterized viral proteins, long non-coding RNAs and micro RNAs.

CoV3D: a database of high resolution coronavirus protein structures.

SARS-CoV-2, the etiologic agent of COVID-19, exemplifies the general threat to global health posed by coronaviruses. The urgent need for effective vaccines and therapies is leading to a rapid rise in the number of high resolution structures of SARS-CoV-2 proteins that collectively reveal a map of virus vulnerabilities. To assist structure-based design of vaccines and therapeutics against SARS-CoV-2 and other coronaviruses, we have developed CoV3D, a database and resource for coronavirus protein structures, which is updated on a weekly basis. CoV3D provides users with comprehensive sets of structures of coronavirus proteins and their complexes with antibodies, receptors, and small molecules. Integrated molecular viewers allow users to visualize structures of the spike glycoprotein, which is the major target of neutralizing antibodies and vaccine design efforts, as well as sets of spike-antibody complexes, spike sequence variability, and known polymorphisms. In order to aid structure-based design and analysis of the spike glycoprotein, CoV3D permits visualization and download of spike structures with modeled N-glycosylation at known glycan sites, and contains structure-based classification of spike conformations, generated by unsupervised clustering. CoV3D can serve the research community as a centralized reference and resource for spike and other coronavirus protein structures, and is available at: https://cov3d.ibbr.umd.edu.

PAGER-CoV: a comprehensive collection of pathways, annotated gene-lists and gene signatures for coronavirus disease studies.

PAGER-CoV (http://discovery.informatics.uab.edu/PAGER-CoV/) is a new web-based database that can help biomedical researchers interpret coronavirus-related functional genomic study results in the context of curated knowledge of host viral infection, inflammatory response, organ damage, and tissue repair. The new database consists of 11 835 PAGs (Pathways, Annotated gene-lists, or Gene signatures) from 33 public data sources. Through the web user interface, users can search by a query gene or a query term and retrieve significantly matched PAGs with all the curated information. Users can navigate from a PAG of interest to other related PAGs through either shared PAG-to-PAG co-membership relationships or PAG-to-PAG regulatory relationships, totaling 19 996 993. Users can also retrieve enriched PAGs from an input list of COVID-19 functional study result genes, customize the search data sources, and export all results for subsequent offline data analysis. In a case study, we performed a gene set enrichment analysis (GSEA) of a COVID-19 RNA-seq data set from the Gene Expression Omnibus database. Compared with the results using the standard PAGER database, PAGER-CoV allows for more sensitive matching of known immune-related gene signatures. We expect PAGER-CoV to be invaluable for biomedical researchers to find molecular biology mechanisms and tailored therapeutics to treat COVID-19 patients.

Coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors.

Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5'-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type-infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.

Coronavirus hemagglutinin-esterase and spike proteins coevolve for functional balance and optimal virion avidity.

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.