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.

Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

Many viruses attach to target cells by binding to cell-surface glycans. To gain a better understanding of strategies used by viruses to engage carbohydrate receptors, we determined the crystal structures of reovirus attachment protein σ1 in complex with α-2,3-sialyllactose, α-2,6-sialyllactose, and α-2,8-di-siallylactose. All three oligosaccharides terminate in sialic acid, which serves as a receptor for the reovirus serotype studied here. The overall structure of σ1 resembles an elongated, filamentous trimer. It contains a globular head featuring a compact β-barrel, and a fibrous extension formed by seven repeating units of a triple β-spiral that is interrupted near its midpoint by a short α -helical coiled coil. The carbohydrate-binding site is located between β-spiral repeats two and three, distal from the head. In all three complexes, the terminal sialic acid forms almost all of the contacts with σ1 in an identical manner, while the remaining components of the oligosaccharides make little or no contacts. We used this structural information to guide mutagenesis studies to identify residues in σ1 that functionally engage sialic acid by assessing hemagglutination capacity and growth in murine erythroleukemia cells, which require sialic acid binding for productive infection. Our studies using σ1 mutant viruses reveal that residues 198, 202, 203, 204, and 205 are required for functional binding to sialic acid by reovirus. These findings provide insight into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to endow other trimeric proteins with carbohydrate-binding properties. Human reoviruses bind first with low affinity to a carbohydrate receptor that brings the virus in close proximity to the host cell. This interaction then facilitates high-affinity binding to a second receptor, the tight junction component junctional adhesion molecule-A (JAM-A). While all human reoviruses bind JAM-A, they differ in carbohydrate receptor specificity, and this difference may influence the distinct disease patterns of reovirus serotypes. We present here the structure of the attachment protein of type 3 reovirus in complex with carbohydrates that naturally occur on human cells. Our results show that the protein forms an elongated trimer, with the carbohydrate binding site being located close to the midpoint of the molecule in a fiber-like region. Our findings provide insights into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to introduce carbohydrate-binding properties into other trimeric proteins.