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About the Authors: Ursula Neu Affiliation: Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany ORCID logo http://orcid.org/0000-0001-6852-0879 Bernardo A. Mainou * E-mail: bernardo.mainou@emory.edu Affiliations Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, United States of America, Children’s Healthcare of Atlanta, Atlanta, Georgia, United States of America ORCID logo http://orcid.org/0000-0002-7721-183X Citation: Neu U, Mainou BA (2020) Virus interactions with bacteria: Interestingly, both gram-positive and gram-negative bacteria also provide protection from bleach treatment to Aichi, Mengo, and poliovirus [5]. [...]binding of HNoV to HBGAs protects the virus from heat stress [8]. Fecal transfer from mice bearing segmented filamentous bacteria also protects susceptible animals from infection with IAV, vesicular stomatitis virus, and reovirus. Surfactin, a cyclic lipopeptide with membrane disruptive properties produced by Bacillus subtilis [27], disrupts coronavirus virion integrity and impairs the infectivity of several enveloped viruses, including Chikungunya, Crimean–Congo hemorrhagic fever, Dugbe, Ebola, IAV, Mayaro, Nipah, Una, and Zika [28].

Viruses with membranes fuse them with cellular membranes, to transfer their genomes into cells at the beginning of infection. For Influenza virus, the membrane glycoprotein involved in fusion is the hemagglutinin (HA), the 3D structure of which is known from X-ray crystallographic studies. The soluble ectodomain fragments used in these studies lacked the “membrane anchor” portion of the molecule. Since this region has a role in membrane fusion, we have determined its structure by analyzing the intact, full-length molecule in a detergent micelle, using cryo-EM. We have also compared the structures of full-length HA−detergent micelles with full-length HA−Fab complex detergent micelles, to describe an infectivity-neutralizing monoclonal Fab that binds near the ectodomain membrane anchor junction. We determine a high-resolution HA structure which compares favorably in detail with the structure of the ectodomain seen by X-ray crystallography; we detect, clearly, all five carbohydrate side chains of HA; and we find that the ectodomain is joined to the membrane anchor by flexible, eight-residue-long, linkers. The linkers extend into the detergent micelle to join a central triple-helical structure that is a major component of the membrane anchor.

Simian virus 40 (SV40) has been a paradigm for understanding attachment and entry of nonenveloped viruses, viral DNA replication, and virus assembly, as well as for endocytosis pathways associated with caveolin and cholesterol. We find by glycan array screening that SV40 recognizes its ganglioside receptor GM1 with a quite narrow specificity, but isothermal titration calorimetry shows that individual binding sites have a relatively low affinity, with a millimolar dissociation constant. The high-resolution crystal structure of recombinantly produced SV40 capsid protein, VP1, in complex with the carbohydrate portion of GM1, reveals that the receptor is bound in a shallow solvent-exposed groove at the outer surface of the capsid. Through a complex network of interactions, VP1 recognizes a conformation of GM1 that is the dominant one in solution. Analysis of contacts provides a structural basis for the observed specificity and suggests binding mechanisms for additional physiologically relevant GM1 variants. Comparison with murine Polyomavirus (Polyoma) receptor complexes reveals that SV40 uses a different mechanism of sialic acid binding, which has implications for receptor binding of human polyomaviruses. The SV40-GM1 complex reveals a parallel to cholera toxin, which uses a similar cell entry pathway and binds GM1 in the same conformation.

Several bacterial toxins and viruses can deform membranes through multivalent binding to lipids for clathrin-independent endocytosis. However, it remains unclear, how membrane deformation and endocytic internalization are mechanistically linked. Here we show that many lipid-binding virions induce membrane deformation and clathrin-independent endocytosis, suggesting a common mechanism based on multivalent lipid binding by globular particles. We create a synthetic cellular system consisting of a lipid-anchored receptor in the form of GPI-anchored anti-GFP nanobodies and a multivalent globular binder exposing 180 regularly-spaced GFP molecules on its surface. We show that these globular, 40 nm diameter, particles bind to cells expressing the receptor, deform the plasma membrane upon adhesion and become endocytosed in a clathrin-independent manner. We explore the role of the membrane adhesion energy in endocytosis by using receptors with affinities varying over 7 orders of magnitude. Using this system, we find that once a threshold in adhesion energy is overcome to allow for membrane deformation, endocytosis occurs reliably. Multivalent, binding-induced membrane deformation by globular binders is thus sufficient for internalization to occur and we suggest it is the common, purely biophysical mechanism for lipid-binding mediated endocytosis of toxins and pathogens. Many bacterial toxins and viruses deform membranes prior to entering cells via clathrin independent endocytosis. Here the authors show that multivalent lipid binding by globular particles can exceed a threshold adhesion energy required for membrane deformation and that this is sufficient for internalization.

Structural and biochemical studies of scavenger decapping enzyme identify conformational changes induced by substrate binding to a second binding site that occur faster than catalytic turnover, such that high substrate concentrations inhibit enzyme activity. The scavenger decapping enzyme hydrolyzes the protective 5′ cap structure on short mRNA fragments that are generated from the exosomal degradation of mRNAs. From static crystal structures and NMR data, it is apparent that the dimeric enzyme has to undergo large structural changes to bind its substrate in a catalytically competent conformation. Here we studied the yeast enzyme and showed that the associated opening and closing motions can be orders of magnitude faster than the catalytic turnover rate. This excess of motion is induced by the binding of a second ligand to the enzyme, which occurs at high substrate concentrations. We designed a mutant that disrupted the allosteric pathway that links the second binding event to the dynamics and showed that this mutant enzyme is hyperactive. Our data reveal a unique mechanism of substrate inhibition in which motions that are required for catalytic activity also inhibit efficient turnover when they are present in excess.

Viruses within a family often vary in their cellular tropism and pathogenicity. In many cases, these variations are due to viruses switching their specificity from one cell surface receptor to another. The structural requirements that underlie such receptor switching are not well understood especially for carbohydrate-binding viruses, as methods capable of structure-specificity studies are only relatively recently being developed for carbohydrates. We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors. We show that the b-series gangliosides GD3, GD2, GD1b and GT1b all can serve as receptors for BKPyV. The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity. Comparison with the structure of simian virus 40 (SV40) VP1 bound to ganglioside GM1 identifies the amino acid at position 68 as a determinant of specificity. Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types.

The recently discovered human Merkel cell polyomavirus (MCPyV or MCV) causes the aggressive Merkel cell carcinoma (MCC) in the skin of immunocompromised individuals. Conflicting reports suggest that cellular glycans containing sialic acid (Neu5Ac) may play a role in MCPyV infectious entry. To address this question, we solved X-ray structures of the MCPyV major capsid protein VP1 both alone and in complex with several sialylated oligosaccharides. A shallow binding site on the apical surface of the VP1 capsomer recognizes the disaccharide Neu5Ac-α2,3-Gal through a complex network of interactions. MCPyV engages Neu5Ac in an orientation and with contacts that differ markedly from those observed in other polyomavirus complexes with sialylated receptors. Mutations in the Neu5Ac binding site abolish MCPyV infection, highlighting the relevance of the Neu5Ac interaction for MCPyV entry. Our study thus provides a powerful platform for the development of MCPyV-specific vaccines and antivirals. Interestingly, engagement of sialic acid does not interfere with initial attachment of MCPyV to cells, consistent with a previous proposal that attachment is mediated by a class of non-sialylated carbohydrates called glycosaminoglycans. Our results therefore suggest a model in which sialylated glycans serve as secondary, post-attachment co-receptors during MCPyV infectious entry. Since cell-surface glycans typically serve as primary attachment receptors for many viruses, we identify here a new role for glycans in mediating, and perhaps even modulating, post-attachment entry processes.

Murine polyomavirus (MuPyV) causes tumors of various origins in newborn mice and hamsters. Infection is initiated by attachment of the virus to ganglioside receptors at the cell surface. Single amino acid exchanges in the receptor-binding pocket of the major capsid protein VP1 are known to drastically alter tumorigenicity and spread in closely related MuPyV strains. The virus represents a rare example of differential receptor recognition directly influencing viral pathogenicity, although the factors underlying these differences remain unclear. We performed structural and functional analyses of three MuPyV strains with strikingly different pathogenicities: the low-tumorigenicity strain RA, the high-pathogenicity strain PTA, and the rapidly growing, lethal laboratory isolate strain LID. Using ganglioside deficient mouse embryo fibroblasts, we show that addition of specific gangliosides restores infectability for all strains, and we uncover a complex relationship between virus attachment and infection. We identify a new infectious ganglioside receptor that carries an additional linear [α-2,8]-linked sialic acid. Crystal structures of all three strains complexed with representative oligosaccharides from the three main pathways of ganglioside biosynthesis provide the molecular basis of receptor recognition. All strains bind to a range of sialylated glycans featuring the central [α-2,3]-linked sialic acid present in the established receptors GD1a and GT1b, but the presence of additional sialic acids modulates binding. An extra [α-2,8]-linked sialic acid engages a protein pocket that is conserved among the three strains, while another, [α-2,6]-linked branching sialic acid lies near the strain-defining amino acids but can be accommodated by all strains. By comparing electron density of the oligosaccharides within the binding pockets at various concentrations, we show that the [α-2,8]-linked sialic acid increases the strength of binding. Moreover, the amino acid exchanges have subtle effects on their affinity for the validated receptor GD1a. Our results indicate that both receptor specificity and affinity influence MuPyV pathogenesis.

B-Lymphotropic Polyomavirus (LPyV) serves as a paradigm of virus receptor binding and tropism, and is the closest relative of the recently discovered Human Polyomavirus 9 (HPyV9). LPyV infection depends on sialic acid on host cells, but the molecular interactions underlying LPyV-receptor binding were unknown. We find by glycan array screening that LPyV specifically recognizes a linear carbohydrate motif that contains α2,3-linked sialic acid. High-resolution crystal structures of the LPyV capsid protein VP1 alone and in complex with the trisaccharide ligands 3'-sialyllactose and 3'-sialyl-N-acetyl-lactosamine (3SL and 3SLN, respectively) show essentially identical interactions. Most contacts are contributed by the sialic acid moiety, which is almost entirely buried in a narrow, preformed cleft at the outer surface of the capsid. The recessed nature of the binding site on VP1 and the nature of the observed glycan interactions differ from those of related polyomaviruses and most other sialic acid-binding viruses, which bind sialic acid in shallow, more exposed grooves. Despite their different modes for recognition, the sialic acid binding sites of LPyV and SV40 are half-conserved, hinting at an evolutionary strategy for diversification of binding sites. Our analysis provides a structural basis for the observed specificity of LPyV for linear glycan motifs terminating in α2,3-linked sialic acid, and links the different tropisms of known LPyV strains to the receptor binding site. It also serves as a useful template for understanding the ligand-binding properties and serological crossreactivity of HPyV9.