Proteolytic Activities of Enterovirus 2A Do Not Depend on Its Interaction with SETD3.

Enterovirus 2Apro is a protease that proteolytically processes the viral polyprotein and cleaves several host proteins to antagonize host responses during enteroviral infection. Recently, the host protein actin histidine methyltransferase SET domain containing 3 (SETD3) was identified to interact with 2Apro and to be essential for virus replication. The role of SETD3 and its interaction with 2Apro remain unclear. In this study, we investigated the potential involvement of SETD3 in several functions of 2Apro. For this, we introduced the 2Apro from coxsackievirus B3 (CVB3) in a mutant of encephalomyocarditis virus (EMCV) containing an inactivated Leader protein (EMCV-Lzn) that is unable to shut down host mRNA translation, to trigger nucleocytoplasmic transport disorder (NCTD), and to suppress stress granule (SG) formation and type I interferon (IFN) induction. Both in wt HeLa cells and in HeLa SETD3 knockout (SETD3KO) cells, the virus containing active 2Apro (EMCV-2Apro) efficiently cleaved eukaryotic translation initiation factor 4 gamma (eIF4G) to shut off host mRNA translation, cleaved nucleoporins to trigger NCTD, and actively suppressed SG formation and IFN gene transcription, arguing against a role of SETD3 in these 2Apro-mediated functions. Surprisingly, we observed that the catalytic activity of enteroviral 2A is not crucial for triggering NCTD, as a virus containing an inactive 2Apro (EMCV-2Am) induced NCTD in both wt and SETD3KO cells, albeit delayed, challenging the idea that the NCTD critically depends on nucleoporin cleavage by this protease. Taken together, our results do not support a role of SETD3 in the proteolytic activities of enterovirus 2Apro.

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes a ring-shaped hexameric complex.

Enteroviruses are globally prevalent human pathogens responsible for many diseases. The nonstructural protein 2C is a AAA+ helicase and plays a key role in enterovirus replication. Drug repurposing screens identified 2C-targeting compounds such as fluoxetine and dibucaine, but how they inhibit 2C is unknown. Here, we present a crystal structure of the soluble and monomeric fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), revealing an allosteric binding site. To study the functional consequences of SFX binding, we engineered an adenosine triphosphatase (ATPase)­competent, hexameric 2C protein. Using this system, we show that SFX, dibucaine, HBB [2-(α-hydroxybenzyl)-benzimidazole], and guanidine hydrochloride inhibit 2C ATPase activity. Moreover, cryo­electron microscopy analysis demonstrated that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition. Collectively, these results provide important insights into 2C inhibition and a robust engineering strategy for structural, functional, and drug-screening analysis of 2C proteins.

Enterocytes, fibroblasts and myeloid cells synergize in anti-bacterial and anti-viral pathways with IL22 as the central cytokine.

IL22 is an important cytokine involved in the intestinal defense mechanisms against microbiome. By using ileum-derived organoids, we show that the expression of anti-microbial peptides (AMPs) and anti-viral peptides (AVPs) can be induced by IL22. In addition, we identified a bacterial and a viral route, both leading to IL22 production by T cells, but via different pathways. Bacterial products, such as LPS, induce enterocyte-secreted SAA1, which triggers the secretion of IL6 in fibroblasts, and subsequently IL22 in T cells. This IL22 induction can then be enhanced by macrophage-derived TNFα in two ways: by enhancing the responsiveness of T cells to IL6 and by increasing the expression of IL6 by fibroblasts. Viral infections of intestinal cells induce IFNß1 and subsequently IL7. IFNß1 can induce the expression of IL6 in fibroblasts and the combined activity of IL6 and IL7 can then induce IL22 expression in T cells. We also show that IL22 reduces the expression of viral entry receptors (e.g. ACE2, TMPRSS2, DPP4, CD46 and TNFRSF14), increases the expression of anti-viral proteins (e.g. RSAD2, AOS, ISG20 and Mx1) and, consequently, reduces the viral infection of neighboring cells. Overall, our data indicates that IL22 contributes to the innate responses against both bacteria and viruses.

Translation and Replication Dynamics of Single RNA Viruses.

RNA viruses are among the most prevalent pathogens and are a major burden on society. Although RNA viruses have been studied extensively, little is known about the processes that occur during the first several hours of infection because of a lack of sensitive assays. Here we develop a single-molecule imaging assay, virus infection real-time imaging (VIRIM), to study translation and replication of individual RNA viruses in live cells. VIRIM uncovered a striking heterogeneity in replication dynamics between cells and revealed extensive coordination between translation and replication of single viral RNAs. Furthermore, using VIRIM, we identify the replication step of the incoming viral RNA as a major bottleneck of successful infection and identify host genes that are responsible for inhibition of early virus replication. Single-molecule imaging of virus infection is a powerful tool to study virus replication and virus-host interactions that may be broadly applicable to RNA viruses.

Inhibition of the integrated stress response by viral proteins that block p-eIF2-eIF2B association.

Eukaryotic cells, when exposed to environmental or internal stress, activate the integrated stress response (ISR) to restore homeostasis and promote cell survival. Specific stress stimuli prompt dedicated stress kinases to phosphorylate eukaryotic initiation factor 2 (eIF2). Phosphorylated eIF2 (p-eIF2) in turn sequesters the eIF2-specific guanine exchange factor eIF2B to block eIF2 recycling, thereby halting translation initiation and reducing global protein synthesis. To circumvent stress-induced translational shutdown, viruses encode ISR antagonists. Those identified so far prevent or reverse eIF2 phosphorylation. We now describe two viral proteins-one from a coronavirus and the other from a picornavirus-that have independently acquired the ability to counteract the ISR at its very core by acting as a competitive inhibitor of p-eIF2-eIF2B interaction. This allows continued formation of the eIF2-GTP-Met-tRNAi ternary complex and unabated global translation at high p-eIF2 levels that would otherwise cause translational arrest. We conclude that eIF2 and p-eIF2 differ in their interaction with eIF2B to such effect that p-eIF2-eIF2B association can be selectively inhibited.

Identification of the Cell-Surface Protease ADAM9 as an Entry Factor for Encephalomyocarditis Virus.

Encephalomyocarditis virus (EMCV) is an animal pathogen and an important model organism, whose receptor requirements are poorly understood. Here, we employed a genome-wide haploid genetic screen to identify novel EMCV host factors. In addition to the previously described picornavirus receptors sialic acid and glycosaminoglycans, this screen unveiled important new host factors for EMCV. These factors include components of the fibroblast growth factor (FGF) signaling pathway, such as the potential receptors FGFR1 and ADAM9, a cell-surface metalloproteinase. By employing various knockout cells, we confirmed the importance of the identified host factors for EMCV infection. The largest reduction in infection efficiency was observed in cells lacking ADAM9. Pharmacological inhibition of the metalloproteinase activity of ADAM9 did not affect virus infection. Moreover, reconstitution of inactive ADAM9 in knockout cells restored susceptibility to EMCV, pointing to a proteinase-independent role of ADAM9 in mediating EMCV infection. Using neutralization assays with ADAM9-specific antiserum and soluble receptor proteins, we provided evidence for a role of ADAM9 in EMCV entry. Finally, binding assays showed that ADAM9 facilitates attachment of EMCV to the cell surface. Together, our findings reveal a role for ADAM9 as a novel receptor or cofactor for EMCV.IMPORTANCE EMCV is an animal pathogen that causes acute viral infections, usually myocarditis or encephalitis. It is thought to circulate mainly among rodents, from which it is occasionally transmitted to other animal species, including humans. EMCV causes fatal outbreaks of myocarditis and encephalitis in pig farms and zoos, making it an important veterinary pathogen. Although EMCV has been widely used as a model to study mechanisms of viral disease in mice, little is known about its entry mechanism. Here, we employ a haploid genetic screen for EMCV host factors and identify an essential role for ADAM9 in EMCV entry.

Bypassing pan-enterovirus host factor PLA2G16.

Enteroviruses are a major cause of human disease. Adipose-specific phospholipase A2 (PLA2G16) was recently identified as a pan-enterovirus host factor and potential drug target. In this study, we identify a possible mechanism of PLA2G16 evasion by employing a dual glycan receptor-binding enterovirus D68 (EV-D68) strain. We previously showed that this strain does not strictly require the canonical EV-D68 receptor sialic acid. Here, we employ a haploid screen to identify sulfated glycosaminoglycans (sGAGs) as its second glycan receptor. Remarkably, engagement of sGAGs enables this virus to bypass PLA2G16. Using cryo-EM analysis, we reveal that, in contrast to sialic acid, sGAGs stimulate genome release from virions via structural changes that enlarge the putative openings for genome egress. Together, we describe an enterovirus that can bypass PLA2G16 and identify additional virion destabilization as a potential mechanism to circumvent PLA2G16.

Molecular basis for the acid-initiated uncoating of human enterovirus D68.

Enterovirus D68 (EV-D68) belongs to a group of enteroviruses that contain a single positive-sense RNA genome surrounded by an icosahedral capsid. Like common cold viruses, EV-D68 mainly causes respiratory infections and is acid-labile. The molecular mechanism by which the acid-sensitive EV-D68 virions uncoat and deliver their genome into a host cell is unknown. Using cryoelectron microscopy (cryo-EM), we have determined the structures of the full native virion and an uncoating intermediate [the A (altered) particle] of EV-D68 at 2.2- and 2.7-Å resolution, respectively. These structures showed that acid treatment of EV-D68 leads to particle expansion, externalization of the viral protein VP1 N termini from the capsid interior, and formation of pores around the icosahedral twofold axes through which the viral RNA can exit. Moreover, because of the low stability of EV-D68, cryo-EM analyses of a mixed population of particles at neutral pH and following acid treatment demonstrated the involvement of multiple structural intermediates during virus uncoating. Among these, a previously undescribed state, the expanded 1 ("E1") particle, shows a majority of internal regions (e.g., the VP1 N termini) to be ordered as in the full native virion. Thus, the E1 particle acts as an intermediate in the transition from full native virions to A particles. Together, the present work delineates the pathway of EV-D68 uncoating and provides the molecular basis for the acid lability of EV-D68 and of the related common cold viruses.

Role of enhanced receptor engagement in the evolution of a pandemic acute hemorrhagic conjunctivitis virus.

Acute hemorrhagic conjunctivitis (AHC) is a painful, contagious eye disease, with millions of cases in the last decades. Coxsackievirus A24 (CV-A24) was not originally associated with human disease, but in 1970 a pathogenic "variant" (CV-A24v) emerged, which is now the main cause of AHC. Initially, this variant circulated only in Southeast Asia, but it later spread worldwide, accounting for numerous AHC outbreaks and two pandemics. While both CV-A24 variant and nonvariant strains still circulate in humans, only variant strains cause AHC for reasons that are yet unknown. Since receptors are important determinants of viral tropism, we set out to map the CV-A24 receptor repertoire and establish whether changes in receptor preference have led to the increased pathogenicity and rapid spread of CV-A24v. Here, we identify ICAM-1 as an essential receptor for both AHC-causing and non-AHC strains. We provide a high-resolution cryo-EM structure of a virus-ICAM-1 complex, which revealed critical ICAM-1-binding residues. These data could help identify a possible conserved mode of receptor engagement among ICAM-1-binding enteroviruses and rhinoviruses. Moreover, we identify a single capsid substitution that has been adopted by all pandemic CV-A24v strains and we reveal that this adaptation enhances the capacity of CV-A24v to bind sialic acid. Our data elucidate the CV-A24v receptor repertoire and point to a role of enhanced receptor engagement in the adaptation to the eye, possibly enabling pandemic spread.

Betacoronavirus Adaptation to Humans Involved Progressive Loss of Hemagglutinin-Esterase Lectin Activity.

Human beta1-coronavirus (ß1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal ß1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. ß1CoV receptor binding is typically controlled by attachment/fusion spike protein S and receptor-binding/receptor-destroying hemagglutinin-esterase protein HE. We show that following OC43's introduction into humans, HE-mediated receptor binding was selected against and ultimately lost through progressive accumulation of mutations in the HE lectin domain. Consequently, virion-associated receptor-destroying activity toward multivalent glycoconjugates was reduced and altered such that some clustered receptor populations are no longer cleaved. Loss of HE lectin function was also observed for another respiratory human coronavirus, HKU1. This thus appears to be an adaptation to the sialoglycome of the human respiratory tract and for replication in human airways. The findings suggest that the dynamics of virion-glycan interactions contribute to host tropism. Our observations are relevant also to other human respiratory viruses of zoonotic origin, particularly influenza A virus.

Enterovirus D68 receptor requirements unveiled by haploid genetics.

Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory disease and is associated with cases of paralysis, especially among children. Heretofore, information on host factor requirements for EV-D68 infection is scarce. Haploid genetic screening is a powerful tool to reveal factors involved in the entry of pathogens. We performed a genome-wide haploid screen with the EV-D68 prototype Fermon strain to obtain a comprehensive overview of cellular factors supporting EV-D68 infection. We identified and confirmed several genes involved in sialic acid (Sia) biosynthesis, transport, and conjugation to be essential for infection. Moreover, by using knockout cell lines and gene reconstitution, we showed that both α2,6- and α2,3-linked Sia can be used as functional cellular EV-D68 receptors. Importantly, the screen did not reveal a specific protein receptor, suggesting that EV-D68 can use multiple redundant sialylated receptors. Upon testing recent clinical strains, we identified strains that showed a similar Sia dependency, whereas others could infect cells lacking surface Sia, indicating they can use an alternative, nonsialylated receptor. Nevertheless, these Sia-independent strains were still able to bind Sia on human erythrocytes, raising the possibility that these viruses can use multiple receptors. Sequence comparison of Sia-dependent and Sia-independent EV-D68 strains showed that many changes occurred near the canyon that might allow alternative receptor binding. Collectively, our findings provide insights into the identity of the EV-D68 receptor and suggest the possible existence of Sia-independent viruses, which are essential for understanding tropism and disease.

Complexity and Diversity of the Mammalian Sialome Revealed by Nidovirus Virolectins.

Sialic acids (Sias), 9-carbon-backbone sugars, are among the most complex and versatile molecules of life. As terminal residues of glycans on proteins and lipids, Sias are key elements of glycotopes of both cellular and microbial lectins and thus act as important molecular tags in cell recognition and signaling events. Their functions in such interactions can be regulated by post-synthetic modifications, the most common of which is differential Sia-O-acetylation (O-Ac-Sias). The biology of O-Ac-Sias remains mostly unexplored, largely because of limitations associated with their specific in situ detection. Here, we show that dual-function hemagglutinin-esterase envelope proteins of nidoviruses distinguish between a variety of closely related O-Ac-Sias. By using soluble forms of hemagglutinin-esterases as lectins and sialate-O-acetylesterases, we demonstrate differential expression of distinct O-Ac-sialoglycan populations in an organ-, tissue- and cell-specific fashion. Our findings indicate that programmed Sia-O-acetylation/de-O-acetylation may be critical to key aspects of cell development, homeostasis, and/or function.

Attachment of mouse hepatitis virus to O-acetylated sialic acid is mediated by hemagglutinin-esterase and not by the spike protein.

The members of Betacoronavirus phylocluster A possess two types of surface projections, one comprised of the spike protein (S) and the other of hemagglutinin-esterase (HE). Purportedly, these viruses bind to O-acetylated sialic acids (O-Ac-Sias) primarily through S, with HE serving merely as receptor-destroying enzyme. Here, we show that, in apparent contrast to human and ungulate host range variants of Betacoronavirus-1, murine coronaviruses actually bind to O-Ac-Sias via HE exclusively. Apparently, expansion of group A betacoronaviruses into new hosts and niches was accompanied by changes in HE ligand and substrate preference and in the roles of HE and S in Sia receptor usage.

Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution.

The hemagglutinin-esterases (HEs) are a family of viral envelope glycoproteins that mediate reversible attachment to O-acetylated sialic acids by acting both as lectins and as receptor-destroying enzymes (RDEs). Related HEs occur in influenza C, toro-, and coronaviruses, apparently as a result of relatively recent lateral gene transfer events. Here, we report the crystal structure of a coronavirus (CoV) HE in complex with its receptor. We show that CoV HE arose from an influenza C-like HE fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer, whereas remnants of the fusion domain were adapted to establish novel monomer-monomer contacts. Whereas the structural design of the RDE-acetylesterase domain remained unaltered, the HE receptor-binding domain underwent remodeling to such extent that the ligand is now bound in opposite orientation. This is surprising, because the architecture of the HEF site was preserved in influenza A HA over a much larger evolutionary distance, a switch in receptor specificity and extensive antigenic variation notwithstanding. Apparently, HA and HEF are under more stringent selective constraints than HE, limiting their exploration of alternative binding-site topologies. We attribute the plasticity of the CoV HE receptor-binding site to evolutionary flexibility conferred by functional redundancy between HE and its companion spike protein S. Our findings offer unique insights into the structural and functional consequences of independent protein evolution after interviral gene exchange and open potential avenues to broad-spectrum antiviral drug design.

Torovirus non-discontinuous transcription: mutational analysis of a subgenomic mRNA promoter.

Toroviruses (order Nidovirales) are enveloped positive-strand RNA viruses of mammals. The prototype torovirus, equine torovirus strain Berne (Berne virus [BEV]), uses two different transcription strategies to produce a 3'-coterminal nested set of subgenomic (sg) mRNAs. Its mRNA 2 carries a leader sequence derived from the 5' end of the genome and is produced via discontinuous transcription. The remaining three sg mRNAs, 3 to 5, are colinear with the 3' end of the genome and are made via non-discontinuous RNA synthesis. Their synthesis is supposedly regulated by short conserved sequence motifs, 5'-ACN3-4CUUUAGA-3', within the noncoding intergenic regions that precede the M, HE, and N genes (A. L. van Vliet, S. L. Smits, P. J. Rottier, and R. J. de Groot, EMBO J. 21:6571-6580, 2002). We have now studied the--for nidoviruses unusual--non-discontinuous transcription mechanism in further detail by probing the role of the postulated transcription-regulating sequences (TRSs). To this end, we constructed a synthetic defective interfering (DI) RNA, carrying a 24-nucleotide segment of the intergenic region between the HE and N genes. We demonstrate that this DI RNA, when introduced into BEV-infected cells, directs the synthesis of a sg DI RNA species; in fact, a 16-nucleotide cassette containing the TRS already proved sufficient. Synthesis of this sg DI RNA, like that of mRNAs 3 to 5 of the standard virus, initiated at the 5'-most adenylate of the TRS. An extensive mutational analysis of the TRS is presented. Our results provide first and formal experimental evidence that the conserved motifs within the BEV intergenic sequences indeed drive sg RNA synthesis.

Nidovirus sialate-O-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes.

Many viruses achieve reversible attachment to sialic acid (Sia) by encoding envelope glycoproteins with receptor-binding and receptor-destroying activities. Toroviruses and group 2 coronaviruses bind to O-acetylated Sias, presumably via their spike proteins (S), whereas other glycoproteins, the hemagglutinin-esterases (HE), destroy Sia receptors by de-O-acetylation. Here, we present a comprehensive study of these enzymes. Sialate-9-O-acetylesterases specific for 5-N-acetyl-9-O-acetylneuraminic acid, described for bovine and human coronaviruses, also occur in equine coronaviruses and in porcine toroviruses. Bovine toroviruses, however, express novel sialate-9-O-acetylesterases, which prefer the di-O-acetylated substrate 5-N-acetyl-7(8),9-di-O-acetylneuraminic acid. Whereas most rodent coronaviruses express sialate-4-O-acetylesterases, the HE of murine coronavirus DVIM cleaves 9-O-acetylated Sias. Under the premise that HE specificity reflects receptor usage, we propose that two types of Sias serve as initial attachment factors for coronaviruses in mice. There are striking parallels between orthomyxo- and nidovirus biology. Reminiscent of antigenic shifts in orthomyxoviruses, rodent coronaviruses exchanged S and HE sequences through recombination to extents not appreciated before. As for orthomyxovirus reassortants, the fitness of nidovirus recombinant offspring probably depends both on antigenic properties and on compatibility of receptor-binding and receptor-destroying activities.

Envelope gene sequences encoding variable regions 3 and 4 are involved in macrophage tropism of feline immunodeficiency virus.

The envelope is of cardinal importance for the entry of feline immunodeficiency virus (FIV) into its host cells, which consist of cells of the immune system including macrophages. To characterize the envelope glycoprotein determinants involved in macrophage tropism, chimeric infectious molecular clones were constructed containing envelope gene sequences from isolates that had been propagated in peripheral blood mononuclear cells (PBMC). The progeny virus was examined for growth in PBMC and bone marrow-derived macrophages and viruses with different replication kinetics in macrophages were selected. Envelope-chimeric viruses revealed that nucleotide sequences encoding variable regions 3 and 4 of the surface glycoprotein, SU, are involved in macrophage tropism of FIV. To assess the biological importance of this finding, the phenotypes of envelope proteins of viruses derived from bone marrow, brain, lymph node and PBMC of an experimentally FIV-infected, healthy cat were examined. Since selection during propagation had to be avoided, provirus envelope gene sequences were amplified directly and cloned into an infectious molecular clone of FIV strain Petaluma. The viruses obtained were examined for their replication properties. Of 15 clones tested, 13 clones replicated both in PBMC and macrophages, two (brain-derived clones) replicated in PBMC only and none replicated in Crandell feline kidney cells or astrocytes. These results indicate that dual tropism for PBMC and macrophages is a common feature of FIV variants present in vivo.