Arno L. W. van Vliet

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.

Enterovirus D68 receptor requirements unveiled by haploid genetics.

Significance Enterovirus D68 (EV-D68) is an emerging pathogen that recently caused a large outbreak of severe respiratory disease in the United States and is associated with cases of paralysis. Little is known about EV-D68 host factor requirements. Here, using a genome-wide knockout approach, we identified several genes in sialic acid (Sia) biology as being essential for infection. We also showed that not only α2,6-linked Sia, which mainly occurs in the upper respiratory tract, but also α2,3-linked Sia, which mainly occurs in the lower respiratory tract, can serve as the receptor. Moreover, we identified recent EV-D68 isolates that can use an alternative, nonsialylated receptor. Our findings are essential to understand tropism and pathogenesis of EV-D68 as well as the potential of using Sia-targeting inhibitors to treat EV-D68 infections. 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.

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.

Attachment of Mouse Hepatitis Virus to O-Acetylated Sialic Acid Is Mediated by Hemagglutinin-Esterase and Not by the Spike Protein

ABSTRACT 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.

Evaluation of subunit vaccines against feline immunodeficiency virus infection

Subunit vaccines prepared against feline immunodeficiency virus (FIV) infection were evaluated in two trials. First, cats were immunized with bacterial expression products of an envelope fragment that contained the V3 neutralization domain of the FIV surface protein fused to either galactokinase (K-SU3) or glutathione-S-transferase (G-SU3). Quantitative and qualitative differences in the humoral immune response were observed with three adjuvants of which Quil A was the best in terms of total and virus neutralizing antibody. Notwithstanding the responses induced, 19 of 20 immunized cats did not resist challenge and became infected. To determine whether priming with a live viral vector would confer protection, cats were inoculated oronasally and subcutaneously with a feline herpesvirus (FHV) mutant expressing the FIV env gene; two booster immunizations followed using the K-SU3 product in either Quil A or a mineral oill Al(OH)3 adjuvant. FIV-specific antibody responses were only weak, and the vaccinates did not withstand challenge with a low dose of homologous virus.

Torovirus Non-Discontinuous Transcription: Mutational Analysis of a Subgenomic mRNA Promoter

ABSTRACT 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.

Monoclonal antibodies to immunodominant and neutralizing domains of the envelope surface protein of feline immunodeficiency virus

Hybridomas secreting monoclonal antibodies (MAbs) specific for the surface protein (SU) of feline immunodeficiency virus were generated. Four MAbs were obtained which could be assigned to two groups based on their neutralization and competition behaviour. Using SU protein fragments expressed in Escherichia coli the antigenic site recognized by one of the MAbs (2H11) could be mapped to the c terminus. The neutralizing MAb 1E1 did not bind to any of the SU protein fragments and was directed to a conformational epitope. Binding of the MAb 1E1 to native SU protein could be blocked with a rabbit serum raised against the SU3 fragment (amino acids 361 to 445). These data indicate that at least part of the epitope is located on this SU3 domain. In competition experiments most sera of naturally infected cats were able to inhibit binding of the MAbs. This shows the conserved and immunodominant nature of the epitopes involved.

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

Significance Acute hemorrhagic conjunctivitis (AHC) is a painful and highly contagious infection of the eye, with reported incidence rates of up to 48%. No drugs or vaccines are available for treatment or prevention of AHC. Coxsackievirus A24 variant (CV-A24v) is the main etiological agent of AHC, being responsible for >10 million AHC cases worldwide during the last decades. We have identified the CV-A24v protein receptor and determined the high-resolution structure of the virus–receptor complex. Furthermore, we found that an adaptation which enhances binding to the receptor sialic acid may have contributed to the pathogenicity and pandemic nature of CV-A24v. These findings highlight the importance of sialic acid for viruses with ocular tropism, such as influenza A virus and several adenoviruses. 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.

Summary Human beta1-coronavirus (β1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal β1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. β1CoV 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.