heparitin-sulfate and Enterovirus-Infections

Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease outbreaks with neurological complications and deaths. We previously isolated an EV-A71 variant in the stool, cerebrospinal fluid, and blood of an immunocompromised patient who had a leucine-to-arginine substitution on the VP1 capsid protein, resulting in increased heparin sulfate binding. We show here that this mutation increases the virus's pathogenicity in orally infected mice with depleted B cells, which mimics the patient's immune status, and increases susceptibility to neutralizing antibodies. However, a double mutant with even greater heparin sulfate affinity is not pathogenic, suggesting that increased heparin sulfate affinity may trap virions in peripheral tissues and reduce neurovirulence. This research sheds light on the increased pathogenicity of variant with heparin sulfate (HS)-binding ability in individuals with decreased B cell immunity.

Topics: Animals; Antigens, Viral; Enterovirus; Enterovirus A, Human; Enterovirus Infections; Heparin; Heparitin Sulfate; Humans; Mice

While infections by enterovirus A71 (EV-A71) are generally self-limiting, they can occasionally lead to serious neurological complications and death. No licensed therapies against EV-A71 currently exist. Using anti-virus-induced cytopathic effect assays, 3,4-dicaffeoylquinic acid (3,4-DCQA) from Ilex kaushue extracts was found to exert significant anti-EV-A71 activity, with a broad inhibitory spectrum against different EV-A71 genotypes. Time-of-drug-addition assays revealed that 3,4-DCQA affects the initial phase (entry step) of EV-A71 infection by directly targeting viral particles and disrupting viral attachment to host cells. Using resistant virus selection experiments, we found that 3,4-DCQA targets the glutamic acid residue at position 98 (E98) and the proline residue at position 246 (P246) in the 5-fold axis located within the VP1 structural protein. Recombinant viruses harboring the two mutations were resistant to 3,4-DCQA-elicited inhibition of virus attachment and penetration into human rhabdomyosarcoma (RD) cells. Finally, we showed that 3,4-DCQA specifically inhibited the attachment of EV-A71 to the host receptor heparan sulfate (HS), but not to the scavenger receptor class B member 2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL1). Molecular docking analysis confirmed that 3,4-DCQA targets the 5-fold axis to form a stable structure with the E98 and P246 residues through noncovalent and van der Waals interactions. The targeting of E98 and P246 by 3,4-DCQA was found to be specific; accordingly, HS binding of viruses carrying the K242A or K244A mutations in the 5-fold axis was successfully inhibited by 3,4-DCQA.The clinical utility of 3,4-DCQA in the prevention or treatment of EV-A71 infections warrants further scrutiny.

Topics: Antiviral Agents; Cell Line, Tumor; Chlorogenic Acid; Enterovirus A, Human; Enterovirus Infections; Heparitin Sulfate; Humans; Ilex; Molecular Docking Simulation; Plant Extracts; Plants, Medicinal

As an important neurotropic enterovirus, enterovirus 71 (EV71) is occasionally associated with severe neurological diseases and high mortality rates in infants and young children. Understanding the interaction between host factors and EV71 will play a vital role in developing antivirals and optimizing vaccines. Here, we performed a genome-wide CRISPR-Cas9 knockout screen and revealed that scavenger receptor class B member 2 (SCARB2), solute carrier family 35 member B2 (SLC35B2), and beta-1,3-glucuronyltransferase 3 (B3GAT3) are essential in facilitating EV71 replication. Subsequently, the exploration of molecular mechanisms suggested that the knockout of SLC35B2 or B3GAT3, not SCARB2, led to a remarkable decrease in the binding of EV71 to cells and internalization into cells. Furthermore, we found that the infection efficiency for EV71 was positively correlated with the level of host cell sulfation, not simply with the amount of heparan sulfate, suggesting that an unidentified sulfated protein(s) must contribute to EV71 infection. In support of this idea, we screened possible sulfated proteins among the proteinous receptors for EV71 and confirmed that SCARB2 could uniquely interact with both tyrosyl protein sulfotransferases in humans. We then performed mass spectrometric analysis of SCARB2, identifying five sites with tyrosine sulfation. The function verification test indicated that there were more than five tyrosine-sulfated sites on SCARB2. Finally, we constructed a model for EV71 entry in which both heparan sulfate and SCARB2 are regulated by SLC35B2 and act cooperatively to support viral binding, internalization, and uncoating. Taken together, this is the first time that we performed the pooled CRISPR-Cas9 genetic screening to investigate the interplay of host cells and EV71. Furthermore, we found that a novel host factor, SLC35B2, played a dual role in regulating the overall sulfation comprising heparan sulfate sulfation and protein tyrosine sulfation, which are critical for EV71 entry.

Topics: Enterovirus A, Human; Enterovirus Infections; Glucuronosyltransferase; HeLa Cells; Heparitin Sulfate; Humans; Lysosomal Membrane Proteins; Receptors, Scavenger; Sulfate Transporters; Sulfotransferases; Tyrosine

Enterovirus A71 (EV-A71) is a causative agent of life-threatening neurological diseases in young children. EV-A71 is highly infectious but it remains unclear how the virus disseminates from primary entry sites-the mucosa of the respiratory tract or the intestine-to secondary replication sites-skin or brain. Here, we investigated the role of dendritic cells (DCs) in EV-A71 dissemination. DCs reside in the mucosa of the airway and gut, and migrate to lymphoid tissues upon activation and, therefore, could facilitate EV-A71 dissemination to secondary replication sites. Monocyte-derived DCs were not permissive to different genotypes of EV-A71 but, notably, coculture with EV-A71-susceptiblle RD99 cells led to very efficient infection of RD99 cells. Notably, EV-A71 transmission of DCs to RD99 was independent of viral replication as a replication inhibitor did not affect transmission. Soluble heparin blocked EV-A71 transmission by DCs to RD99 cells, in contrast to antibodies against known attachment receptor DC-SIGN. These results strongly suggest that DCs might be a first target for EV-A71 and involved in viral dissemination via heparan sulfates and heparin derivatives might be an effective treatment to attenuate dissemination.

Topics: Antigens, Viral; Dendritic Cells; Enterovirus; Enterovirus A, Human; Enterovirus Infections; Heparin; Heparitin Sulfate; Humans; Sulfates; Virus Replication

Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine,

Topics: Animals; Antiviral Agents; Capsid Proteins; Cell Line; Cinnamates; Depsides; Disease Models, Animal; Drug Evaluation, Preclinical; Enterovirus A, Human; Enterovirus Infections; Heparitin Sulfate; Humans; Jurkat Cells; Membrane Glycoproteins; Mice; Mutation; Plant Extracts; Protein Binding; Rosmarinic Acid; Salvia miltiorrhiza; Static Electricity; Virulence Factors

Enterovirus 71 (EV71) is the major causative pathogen of human hand, foot, and mouth disease (hHFMD) and has evolved to use various cellular receptors for infection. However, the relationship between receptor preference and EV71 virulence has not been fully revealed. By using reverse genetics, we identified that a single E98K mutation in VP1 is responsible for rapid viral replication in vitro. The E98K mutation enhanced binding of EV71-GZCII to cells in a heparan sulfate (HS)-dependent manner, and it attenuated the virulence of EV71-GZCII in BALB/c mice, indicating that the HS-binding property is negatively associated with viral virulence. HS is widely expressed in vascular endothelial cells in different mouse tissues, and weak colocalization of HS with scavenger receptor B2 (SCARB2) was detected. The cGZCII-98K virus bound more efficiently to mouse tissue homogenates, and the cGZCII-98K virus titers in mouse tissues and blood were much lower than the cGZCII virus titers. Together, these findings suggest that the enhanced adsorption of the cGZCII-98K virus, which likely occurs through HS, is unable to support the efficient replication of EV71 in vivo. Our study confirmed the role of HS-binding sites in EV71 infection and highlighted the importance of the HS receptor in EV71 pathogenesis.

Topics: Amino Acid Substitution; Animals; Capsid Proteins; Cell Line; Chlorocebus aethiops; Enterovirus A, Human; Enterovirus Infections; HeLa Cells; Heparitin Sulfate; Humans; Mice; Mice, Inbred BALB C; Mutation; Vero Cells; Viral Load; Virulence; Virus Attachment; Virus Replication

Enterovirus A71 (EV-A71) is a non-polio neurotropic enterovirus with pandemic potential. There are no antiviral agents approved to prevent or treat EV-A71 infections. We here report on the molecular mechanism by which a novel class of tryptophan dendrimers inhibits (at low nanomolar to high picomolar concentration) EV-A71 replication in vitro. A lead compound in the series (MADAL385) prevents binding and internalization of the virus but does not, unlike classical capsid binders, stabilize the particle. By means of resistance selection, reverse genetics and cryo-EM, we map the binding region of MADAL385 to the 5-fold vertex of the viral capsid and demonstrate that a single molecule binds to each vertex. By interacting with this region, MADAL385 prevents the interaction of the virus with its cellular receptors PSGL1 and heparan sulfate, thereby blocking the attachment of EV-A71 to the host cells.

Topics: Antiviral Agents; Capsid; Capsid Proteins; Dendrimers; Enterovirus; Enterovirus Infections; HeLa Cells; Heparitin Sulfate; Humans; Membrane Glycoproteins; Protein Conformation; Tryptophan; Virus Replication

Enterovirus 71 (EV71) is a major etiological agent of hand, foot, and mouth disease, for which there is no antiviral therapy. We have developed densely sulfated disaccharide heparan sulfate (HS) analogues that are potent small molecule inhibitors of EV71 infection, binding to the viral capsid and acting as decoy receptors to block early events of virus replication. The simplified structures, more potent than defined HS disaccharides and with no significant anticoagulant activity, offer promise as anti-EV71 agents.

Topics: Antiviral Agents; Cell Line; Dose-Response Relationship, Drug; Enterovirus A, Human; Enterovirus Infections; Heparitin Sulfate; Humans; Somatomedins; Virus Attachment; Virus Replication

Topics: Amino Acid Substitution; Animals; Antibodies, Neutralizing; Antibodies, Viral; Capsid Proteins; Chlorocebus aethiops; COS Cells; Enterovirus A, Human; Enterovirus Infections; Heparitin Sulfate; Macaca fascicularis; Male; Vero Cells; Virulence

Infection by enterovirus 71 (EV71) is affected by cell surface receptors, including the human scavenger receptor B2 (hSCARB2), which are required for viral uncoating, and attachment receptors, such are heparan sulfate (HS), which bind virus but do not support uncoating. Amino acid residue 145 of the capsid protein VP1 affects viral binding to HS and virulence in mice. However, the contribution of this amino acid to pathogenicity in humans is not known. We produced EV71 having glycine (VP1-145G) or glutamic acid (VP1-145E) at position 145. VP1-145G, but not VP1-145E, enhanced viral infection in cell culture in an HS-dependent manner. However, VP1-145G virus showed an attenuated phenotype in wild-type suckling mice and in a transgenic mouse model expressing hSCARB2, while VP1-145E virus showed a virulent phenotype in both models. Thus, the HS-binding property and

Topics: Amino Acid Substitution; Animals; Capsid Proteins; Cell Line; Central Nervous System; Disease Models, Animal; Endothelial Cells; Enterovirus A, Human; Enterovirus Infections; Glycine; Heparitin Sulfate; Humans; Lysosomal Membrane Proteins; Mice; Mice, Transgenic; Mutation; Receptors, Scavenger; Viral Load; Virus Attachment

Enterovirus A71 (EV-A71) is a neurotropic enterovirus that uses heparan sulfate as an attachment receptor. The molecular determinants of EV-A71-heparan sulfate interaction are unknown. With In silico heparin docking and mutagenesis of all possible lysine residues in VP1, we identified that K162, K242 and K244 are responsible for heparin interaction and inhibition. EV-A71 mutants with K242A and K244A rapidly acquired compensatory mutations, T100K or E98A, and Q145R-T237N respectively, which restored the heparin-binding phenotype. Both VP1-98 and VP1-145 modulates heparin binding. Heparin-binding phenotype was completely abolished with VP1-E98-E145, but was restored by an E98K or E145Q substitution. During cell culture adaptation, EV-A71 rapidly acquired K98 or Q/G145 to restore the heparin-binding phenotype. Together with next-generation sequencing analysis, our results implied that EV-A71 has high genetic plasticity by modulating positively-charged residues at the five-fold axis during in vitro heparin adaptation. Our finding has impact on EV-A71 vaccine production, evolutionary studies and pathogenesis.

Topics: Amino Acid Sequence; Base Sequence; Enterovirus A, Human; Enterovirus Infections; Heparitin Sulfate; Humans; Molecular Sequence Data; Mutation; Protein Binding; Receptors, Virus; Viral Proteins

NF449, a sulfated compound derived from the antiparasitic drug suramin, was previously reported to inhibit infection by enterovirus A71 (EV-A71). In the current work, we found that NF449 inhibits virus attachment to target cells, and specifically blocks virus interaction with two identified receptors--the P-selectin ligand, PSGL-1, and heparan sulfate glycosaminoglycan--with no effect on virus binding to a third receptor, the scavenger receptor SCARB2. We also examined a number of commercially available suramin analogues, and newly synthesized derivatives of NF449; among these, NF110 and NM16, like NF449, inhibited virus attachment at submicromolar concentrations. PSGL-1 and heparan sulfate, but not SCARB2, are both sulfated molecules, and their interaction with EV-A71 is thought to involve positively charged capsid residues, including a conserved lysine at VP1-244, near the icosahedral 5-fold vertex. We found that mutation of VP1-244 resulted in resistance to NF449, suggesting that this residue is involved in NF449 interaction with the virus capsid. Consistent with this idea, NF449 and NF110 prevented virus interaction with monoclonal antibody MA28-7, which specifically recognizes an epitope overlapping VP1-244 at the 5-fold vertex. Based on these observations we propose that NF449 and related compounds compete with sulfated receptor molecules for a binding site at the 5-fold vertex of the EV-A71 capsid.

Topics: Antiviral Agents; Benzenesulfonates; Binding Sites; Capsid; Enterovirus A, Human; Enterovirus Infections; HeLa Cells; Heparitin Sulfate; Humans; Jurkat Cells; Membrane Glycoproteins; Models, Molecular; Molecular Sequence Data; Suramin; Virus Attachment

Infections caused by Echovirus 5 (E5), an enterovirus of the Picornaviridae family, have been associated with fever, rashes and sporadic cases of aseptic meningitis. To elucidate the receptor usage of this virus, the significance of a previously proposed integrin binding arginine-glycine-aspartic acid (RGD) motif found in the VP3 capsid protein was investigated, as well as the capacity of E5 to interact with heparan sulfate on the cell surface. Using the prototype strain E5 Noyce (E5N), an E5N mutant where the aspartic acid of the RGD motif has been substituted to a glutamic acid and clinical E5 isolates, the RGD motif of VP3 was found to be non-essential and hence not involved in integrin receptor binding. However, E5N and clinical E5 isolates interact with heparan sulfate at the cell surface, as demonstrated by virus replication inhibition assays using heparin and heparinase III, and studies of E5 interactions at the cell surface measured by real-time PCR analysis. In conclusion, E5 utilizes heparan sulfate as a cellular receptor, but the RGD motif of VP3 is not essential for E5 infectivity.

Topics: Animals; Cell Line; Chlorocebus aethiops; Cricetinae; Cricetulus; Enterovirus B, Human; Enterovirus Infections; Heparitin Sulfate; Humans; Molecular Sequence Data; Receptors, Virus; Viral Structural Proteins; Virus Attachment

Recently, it was demonstrated that the coxsackievirus B3 variant PD (CVB3 PD) is able to infect coxsackievirus-adenovirus receptor (CAR)-lacking cells by using heparan sulfates (HS) as additional receptors (A. E. Zautner, U. Korner, A. Henke, C. Badorff, and M. Schmidtke, J. Virol. 77:10071-10077, 2003). For this study, competition experiments with growth factors binding to known HS sequences as well as with specifically desulfated heparins were performed with Chinese hamster ovary cells (CHO-K1) to determine the structural requirements of HS for interaction with CVB3. Hepatocyte growth factor interacting with HS sequences containing [IdUA-GlcNSO(3)(6OSO(3))](n), but not basic fibroblast growth factor binding to [HexUA-GlcNSO(3)-HexUA-GlcNSO(3)-IdUA(2OSO(3))](n), was shown to compete effectively with CVB3 PD for cell surface HS. Whereas unmodified heparin and 2-O-desulfated heparin strongly inhibited the CVB3 PD-induced cytopathic effect, the antiviral activity was markedly reduced after N-, O- and 6-O-desulfation of heparin. Taken together, these results indicate that 6-O- and N-sulfation of GlcNAc of HS is crucial for HS interaction with CVB3 PD and that the disaccharide [IdUA-GlcNSO(3)(6OSO(3))](n) is involved in viral binding. Results from experiments with various inhibitors of endocytic pathways suggest that HS-mediated virus internalization is pH dependent. Despite the fact that CVB3 PD initiates infection about four times slower by making use of HS as a receptor than by using CAR, the time required for a complete viral life cycle in Chinese hamster ovary cells was independent of the utilized receptor.

Topics: Animals; Anticoagulants; CHO Cells; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Cricetinae; Cricetulus; Cytopathogenic Effect, Viral; Drug Antagonism; Drug Synergism; Endocytosis; Enterovirus B, Human; Enterovirus Infections; Fibroblast Growth Factors; Heparin; Heparitin Sulfate; Hepatocyte Growth Factor; Humans; Hydrogen-Ion Concentration; Receptors, Virus; Time Factors