The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV) in cell culture. This cross-neutralizing antibody targets a communal epitope on these viruses and may offer potential for prevention and treatment of COVID-19.
SUMMARY Many RNA viruses remodel intracellular membranes to generate specialized sites for RNA replication. How membranes are remodeled and what properties make them conducive for replication are unknown. Here we show how RNA viruses can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid micro-environment is essential for both enteroviral and flaviviral RNA replication; PI4KIIIβ inhibition interferes with this process; and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication.
Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1 through S1 Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1 We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1 When multivalently displayed on nanoparticles, S1 or S1 bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5--glycolylation and (7,)9--acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus-Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.
Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.
Human betacoronaviruses OC43 and HKU1 are endemic respiratory pathogens and, while related, originated from independent zoonotic introductions. OC43 is in fact a host-range variant of the species Betacoronavirus-1, and more closely related to bovine coronavirus (BCoV)-its presumptive ancestor-and porcine hemagglutinating encephalomyelitis virus (PHEV). The β1-coronaviruses (β1CoVs) and HKU1 employ glycan-based receptors carrying 9-Oacetylated sialic acid (9-O-Ac-Sia). Receptor binding is mediated by spike protein S, the main determinant of coronavirus host specificity. For BCoV, a crystal structure for the receptor-binding domain S1 A is available and for HKU1 a cryoelectron microscopy structure of the complete S ectodomain. However, the location of the receptorbinding site (RBS), arguably the single-most important piece of information, is unknown. Here we solved the 3.0-Å crystal structure of PHEV S1 A . We then took a comparative structural analysis approach to map the β1CoV S RBS, using the general design of 9-O-Ac-Siabinding sites as blueprint, backed-up by automated ligand docking, structure-guided mutagenesis of OC43, BCoV, and PHEV S1 A , and infectivity assays with BCoV-S-pseudotyped vesicular stomatitis viruses. The RBS is not exclusive to OC43 and related animal viruses, but is apparently conserved and functional also in HKU1 S1 A . The binding affinity of the HKU1 S RBS toward short sialoglycans is significantly lower than that of OC43, which we attribute to differences in local architecture and accessibility, and which may be indicative for differences between the two viruses in receptor finespecificity. Our findings challenge reports that would map the OC43 RBS elsewhere in S1 A and that of HKU1 in domain S1 B .coronavirus | spike | 9-O-acetylated sialic acid | OC43 | HKU1 C oronaviruses (CoVs; order Nidovirales, family Coronaviridae) are enveloped positive-strand RNA viruses of mammals and birds. So far, four coronaviruses of zoonotic origin are known to have successfully breached the species barrier to become true human pathogens (1-6). These viruses-NL63, 229E, HKU1, and OC43-are persistently maintained in the human population through continuous circulation. Remarkably, the latter two both belong to a single minor clade, "lineage A," in the genus Betacoronavirus. Although generally associated with common colds, HKU1 and OC43 may cause severe and sometimes fatal pulmonary infections in the frail (7, 8), and in rare instances, OC43 may cause lethal encephalitis (9). OC43 and HKU1 are distinct viruses that entered the human population independently to seemingly follow convergent evolutionary trajectories in their adaptation to the novel host (10). OC43 is in fact more related to coronaviruses of ruminants, horses, dogs, rabbits, and swine, with which it has been united in a single species, Betacoronavirus-1.Lineage A betacoronaviruses like HKU1 and OC43 differ from other CoVs in that their virions possess two types of surface projections, both of which are involved in attachment: large 20-nm peplomer...
The genus Enterovirus (EV) of the family Picornaviridae includes poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. These diverse viruses cause a variety of diseases, including non-specific febrile illness, hand-foot-and-mouth disease, neonatal sepsis-like disease, encephalitis, paralysis and respiratory diseases. In recent years, several non-polio enteroviruses (NPEVs) have emerged as serious public health concerns. These include EV-A71, which has caused epidemics of hand-foot-and-mouth disease in Southeast Asia, and EV-D68, which recently caused a large outbreak of severe lower respiratory tract disease in North America. Infections with these viruses are associated with severe neurological complications. For decades, most research has focused on poliovirus, but in recent years, our knowledge of NPEVs has increased considerably. In this Review, we summarize recent insights from enterovirus research with a special emphasis on NPEVs. We discuss virion structures, host-receptor interactions, viral uncoating and the recent discovery of a universal enterovirus host factor that is involved in viral genome release. Moreover, we briefly explain the mechanisms of viral genome replication, virion assembly and virion release, and describe potential targets for antiviral therapy. We reflect on how these recent discoveries may help the development of antiviral therapies and vaccines.
Porcine deltacoronavirus (PDCoV), identified in 2012, is a common enteropathogen of swine with worldwide distribution. The source and evolutionary history of this virus is, however, unknown. PDCoV belongs to the genus that comprises predominantly avian CoV. Phylogenetic analysis suggests that PDCoV originated relatively recently from a host-switching event between birds and mammals. Insight into receptor engagement by PDCoV may shed light into such an exceptional phenomenon. Here we report that PDCoV employs host aminopeptidase N (APN) as an entry receptor and interacts with APN via domain B of its spike (S) protein. Infection of porcine cells with PDCoV was drastically reduced by APN knockout and rescued after reconstitution of APN expression. In addition, we observed that PDCoV efficiently infects cells of unusual broad species range, including human and chicken. Accordingly, PDCoV S was found to target the phylogenetically conserved catalytic domain of APN. Moreover, transient expression of porcine, feline, human, and chicken APN renders cells susceptible to PDCoV infection. Binding of PDCoV to an interspecies conserved site on APN may facilitate direct transmission of PDCoV to nonreservoir species, including humans, potentially reflecting the mechanism that enabled a virus, ancestral to PDCoV, to breach the species barrier between birds and mammals. The APN cell surface protein is also used by several members of the genus. Hence, our data constitute the second identification of CoVs from different genera that use the same receptor, implying that CoV receptor selection is subjected to specific restrictions that are still poorly understood.
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