Synthesis of Homo- and Heteromultivalent Fucosylated and Sialylated Oligosaccharide Conjugates via Preactivated N-Methyloxyamine Precision Macromolecules and Their Binding to Polyomavirus Capsid Proteins.

Glycoconjugates are a versatile class of bioactive molecules that have found application as vaccines and antivirals and in cancer therapy. Their synthesis typically involves elaborate functionalization and use of protecting groups on the carbohydrate component in order to ensure efficient and selective conjugation. Alternatively, non-functionalized, non-protected carbohydrates isolated from biological sources or derived through biotechnological methods can be directly conjugated via N-methyloxyamine groups. In this study, we introduce such N-methyloxyamine groups into a variety of multivalent scaffolds─from small to oligomeric to polymeric scaffolds─making use of solid-phase polymer synthesis to assemble monodisperse sequence-defined macromolecules. These scaffolds are then successfully functionalized with different types of human milk oligosaccharides deriving a library of homo- and heteromultivalent glycoconjugates. Glycomacromolecules presenting oligosaccharide side chains with either α2,3- or α2,6-linked terminal sialic acid are used in a binding study with two types of polyomavirus capsid proteins showing that the multivalent presentation through the N-methyloxyamine-derived scaffolds increases the number of contacts with the protein. Overall, a straightforward route to derive glycoconjugates from complex oligosaccharides with high variability yet control in the multivalent scaffold is presented, and applicability of the derived structures is demonstrated.

The Protein Corona Does Not Influence Receptor-Mediated Targeting of Virus-like Particles.

Protein corona formation has been regarded as an obstacle to developing diagnostic and therapeutic nanoparticles for in vivo applications. Serum proteins that assemble around nanoparticles can hinder their targeting efficiency. Virus-based nanoparticles should be naturally predisposed to evade such barriers in host organisms. Here, we demonstrate that virus-like particles derived from mouse polyomavirus do not form a rich protein corona. These particles can be efficiently targeted to cells that overproduce transferrin receptors, e.g., cancer cells, by conjugating transferrin to the particle surface. In this study, we provide evidence that the interaction of virus-like particles with their newly assigned target receptor is not obstructed by serum proteins. The particles enter target cells via a clathrin-dependent endocytic pathway that is not naturally used by the virus. Our results support the notion that the natural properties of virus-like particles make them well-suited for development of nanosized theranostic tools resistant to detargeting by protein coronas.

Fish polyomaviruses belong to two distinct evolutionary lineages.

The Polyomaviridae is a diverse family of circular double-stranded DNA viruses. Polyomaviruses have been isolated from a wide array of animal hosts. An understanding of the evolutionary and ecological dynamics of these viruses is essential to understanding the pathogenicity of polyomaviruses. Using a high throughput sequencing approach, we identified a novel polyomavirus in an emerald notothen (Trematomus bernacchii) sampled in the Ross sea (Antarctica), expanding the known number of fish-associated polyomaviruses. Our analysis suggests that polyomaviruses belong to three main evolutionary clades; the first clade is made up of all recognized terrestrial polyomaviruses. The fish-associated polyomaviruses are not monophyletic, and belong to two divergent evolutionary lineages. The fish viruses provide evidence that the evolution of the key viral large T protein involves gain and loss of distinct domains.

Trichodysplasia spinulosa-Associated Polyomavirus Uses a Displaced Binding Site on VP1 to Engage Sialylated Glycolipids.

Trichodysplasia spinulosa-associated Polyomavirus (TSPyV) was isolated from a patient suffering from trichodysplasia spinulosa, a skin disease that can appear in severely immunocompromised patients. While TSPyV is one of the five members of the polyomavirus family that are directly linked to a human disease, details about molecular recognition events, the viral entry pathway, and intracellular trafficking events during TSPyV infection remain unknown. Here we have used a structure-function approach to shed light on the first steps of TSPyV infection. We established by cell binding and pseudovirus infection studies that TSPyV interacts with sialic acids during attachment and/or entry. Subsequently, we solved high-resolution X-ray structures of the major capsid protein VP1 of TSPyV in complex with three different glycans, the branched GM1 glycan, and the linear trisaccharides α2,3- and α2,6-sialyllactose. The terminal sialic acid of all three glycans is engaged in a unique binding site on TSPyV VP1, which is positioned about 18 Å from established sialic acid binding sites of other polyomaviruses. Structure-based mutagenesis of sialic acid-binding residues leads to reduction in cell attachment and pseudovirus infection, demonstrating the physiological relevance of the TSPyV VP1-glycan interaction. Furthermore, treatments of cells with inhibitors of N-, O-linked glycosylation, and glycosphingolipid synthesis suggest that glycolipids play an important role during TSPyV infection. Our findings elucidate the first molecular recognition events of cellular infection with TSPyV and demonstrate that receptor recognition by polyomaviruses is highly variable not only in interactions with sialic acid itself, but also in the location of the binding site.

Retargeting Polyomavirus-Like Particles to Cancer Cells by Chemical Modification of Capsid Surface.

Virus-like particles based on polyomaviruses (PVLPs) are promising delivery devices for various cargoes, including nucleic acids, imaging probes, and therapeutic agents. In biological environments, the major coat protein VP1 interacts with ubiquitously distributed sialic acid residues, and therefore PVLPs show a broad tropism. For selective targeting, appropriate engineering of the PVLP surface is needed. Here, we describe a chemical approach to retarget PVLPs to cancer cells displaying abnormally high levels of transferrin receptor. We created an array of transferrin molecules on the surface of PVLPs by combining a high-yielding bioconjugation approach with specific point modification of transferrin. This artificial surface protein architecture enables (i) suppression of natural VP1-specific interactions by blocking the surface conformational epitope on the VP1 protein, (ii) unusually high cellular uptake efficiency, and (iii) selective retargeting of PVLPs to osteosarcoma (U2OS) and lymphoblastoid leukemia (CCRF-CEM) cells.

Disulfide linkage and structure of highly stable yeast-derived virus-like particles of murine polyomavirus.

VP1 is the major coat protein of murine polyomavirus and forms virus-like particles (VLPs) in vitro. VLPs consist of 72 pentameric VP1 subunits held together by a terminal clamp structure that is further stabilized by disulfide bonds and chelation of calcium ions. Yeast-derived VLPs (yVLPs) assemble intracellularly in vivo during recombinant protein production. These in vivo assembled yVLPs differ in several properties from VLPs assembled in vitro from bacterially produced pentamers. We found several intermolecular disulfide linkages in yVLPs involving 5 of the 6 cysteines of VP1 (Cys(115)-Cys(20), Cys(12)-Cys(20), Cys(16)-Cys(16), Cys(12)/ Cys(16)-Cys(115), and Cys(274)-Cys(274)), indicating a highly coordinated disulfide network within the in vivo assembled particles involving the N-terminal region of VP1. Cryoelectron microscopy revealed structured termini not resolved in the published crystal structure of the bacterially expressed VLP that appear to clamp the pentameric subunits together. These structural features are probably the reason for the observed higher stability of in vivo assembled yVLPs compared with in vitro assembled bacterially expressed VLPs as monitored by increased thermal stability, higher resistance to trypsin cleavage, and a higher activation enthalpy of the disassembly reaction. This high stability is decreased following disassembly of yVLPs and subsequent in vitro reassembly, suggesting a role for cellular components in optimal assembly.

Construction of polyomavirus-derived pseudotype virus-like particles displaying a functionally active neutralizing antibody against hepatitis B virus surface antigen.

BACKGROUND: Virus-like particles (VLPs) can be efficiently produced by heterologous expression of viral structural proteins in yeast. Due to their repetitive structure, VLPs are extensively used for protein engineering and generation of chimeric VLPs with inserted foreign epitopes. Hamster polyomavirus VP1 represents a promising epitope carrier. However, insertion of large sized protein sequences may interfere with its self-assembly competence. The co-expression of polyomavirus capsid protein VP1 with minor capsid protein VP2 or its fusion protein may result in pseudotype VLPs where an intact VP1 protein mediates VLP formation. In the current study, the capacity of VP1 protein to self-assemble to VLPs and interact with the modified VP2 protein has been exploited to generate pseudotype VLPs displaying large-sized antibody molecules. RESULTS: Polyomavirus-derived pseudotype VLPs harbouring a surface-exposed functionally active neutralizing antibody specific to hepatitis B virus (HBV) surface antigen (HBsAg) have been generated. The pseudotype VLPs consisting of an intact hamster polyomavirus (HaPyV) major capsid protein VP1 and minor capsid protein VP2 fused with the anti-HBsAg molecule were efficiently produced in yeast Saccharomyces cerevisiae and purified by density-gradient centrifugation. Formation of VLPs was confirmed by electron microscopy. Two types of pseudotype VLPs were generated harbouring either the single-chain fragment variable (scFv) or Fc-engineered scFv on the VLP surface. The antigen-binding activity of the purified pseudotype VLPs was evaluated by ELISA and virus-neutralization assay on HBV-susceptible primary hepatocytes from Tupaia belangeri. Both types of the pseudotype VLPs were functionally active and showed a potent HBV-neutralizing activity comparable to that of the parental monoclonal antibody. The VP2-fused scFv molecules were incorporated into the VLPs with higher efficiency as compared to the VP2-fused Fc-scFv. However, the pseudotype VLPs with displayed VP2-fused Fc-scFv molecule showed higher antigen-binding activity and HBV-neutralizing capacity that might be explained by a better accessibility of the Fc-engineered scFv of the VLP surface. CONCLUSIONS: Polyomavirus-derived pseudotype VLPs harbouring multiple functionally active antibody molecules with virus-neutralizing capability may represent a novel platform for developing therapeutic tools with a potential application for post-exposure or therapeutic treatment of viral infections.

Crystallographic and glycan microarray analysis of human polyomavirus 9 VP1 identifies N-glycolyl neuraminic acid as a receptor candidate.

UNLABELLED: Human polyomavirus 9 (HPyV9) is a closely related homologue of simian B-lymphotropic polyomavirus (LPyV). In order to define the architecture and receptor binding properties of HPyV9, we solved high-resolution crystal structures of its major capsid protein, VP1, in complex with three putative oligosaccharide receptors identified by glycan microarray screening. Comparison of the properties of HPyV9 VP1 with the known structure and glycan-binding properties of LPyV VP1 revealed that both viruses engage short sialylated oligosaccharides, but small yet important differences in specificity were detected. Surprisingly, HPyV9 VP1 preferentially binds sialyllactosamine compounds terminating in 5-N-glycolyl neuraminic acid (Neu5Gc) over those terminating in 5-N-acetyl neuraminic acid (Neu5Ac), whereas LPyV does not exhibit such a preference. The structural analysis demonstrated that HPyV9 makes specific contacts, via hydrogen bonds, with the extra hydroxyl group present in Neu5Gc. An equivalent hydrogen bond cannot be formed by LPyV VP1. IMPORTANCE: The most common sialic acid in humans is 5-N-acetyl neuraminic acid (Neu5Ac), but various modifications give rise to more than 50 different sialic acid variants that decorate the cell surface. Unlike most mammals, humans cannot synthesize the sialic acid variant 5-N-glycolyl neuraminic acid (Neu5Gc) due to a gene defect. Humans can, however, still acquire this compound from dietary sources. The role of Neu5Gc in receptor engagement and in defining viral tropism is only beginning to emerge, and structural analyses defining the differences in specificity for Neu5Ac and Neu5Gc are still rare. Using glycan microarray screening and high-resolution protein crystallography, we have examined the receptor specificity of a recently discovered human polyomavirus, HPyV9, and compared it to that of the closely related simian polyomavirus LPyV. Our study highlights critical differences in the specificities of both viruses, contributing to an enhanced understanding of the principles that underlie pathogen selectivity for modified sialic acids.

Structure analysis of the major capsid proteins of human polyomaviruses 6 and 7 reveals an obstructed sialic acid binding site.

UNLABELLED: Human polyomavirus 6 (HPyV6) and HPyV7 are commonly found on human skin. We have determined the X-ray structures of their major capsid protein, VP1, at resolutions of 1.8 and 1.7 Å, respectively. In polyomaviruses, VP1 commonly determines antigenicity as well as cell-surface receptor specificity, and the protein is therefore linked to attachment, tropism, and ultimately, viral pathogenicity. The structures of HPyV6 and HPyV7 VP1 reveal uniquely elongated loops that cover the bulk of the outer virion surfaces, obstructing a groove that binds sialylated glycan receptors in many other polyomaviruses. In support of this structural observation, interactions of VP1 with α2,3- and α2,6-linked sialic acids could not be detected in solution by nuclear magnetic resonance spectroscopy. Single-cell binding studies indicate that sialylated glycans are likely not required for initial attachment to cultured human cells. Our findings establish distinct antigenic properties of HPyV6 and HPyV7 capsids and indicate that these two viruses engage nonsialylated receptors. IMPORTANCE: Eleven new human polyomaviruses, including the skin viruses HPyV6 and HPyV7, have been identified during the last decade. In contrast to better-studied polyomaviruses, the routes of infection, cell tropism, and entry pathways of many of these new viruses remain largely mysterious. Our high-resolution X-ray structures of major capsid proteins VP1 from HPyV6 and from HPyV7 reveal critical differences in surface morphology from those of all other known polyomavirus structures. A groove that engages specific sialic acid-containing glycan receptors in related polyomaviruses is obstructed, and VP1 of HPyV6 and HPyV7 does not interact with sialylated compounds in solution or on cultured human cells. A comprehensive comparison with other structurally characterized polyomavirus VP1 proteins enhances our understanding of molecular determinants that underlie receptor specificity, antigenicity, and, ultimately, pathogenicity within the polyomavirus family and highlight the need for structure-based analysis to better define phylogenetic relationships within the growing polyomavirus family and perhaps also for other viruses.

Structures of B-lymphotropic polyomavirus VP1 in complex with oligosaccharide ligands.

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.

Biophysical characterization of polyomavirus minor capsid proteins.

The murine polyomavirus encodes three structural proteins, VP1, VP2 and VP3, which together form the viral capsid. The outer shell of this capsid is composed of the major capsid protein VP1, the inner shell consists of VP2 and its N-terminally truncated form VP3. These two minor capsid proteins interact with their identical C-terminal part in the central cavity of VP1 pentamers, building the capsid assembly unit. While the VP1 structure and functions are well studied, VP2 and VP3 are poorly understood. In order to get a detailed insight into the structure and function of the minor capsid proteins, they were produced recombinantly in Escherichia coli as inclusion bodies and refolded in vitro. The success of refolding was strictly dependent on the presence of detergent in the refolding buffer. VP2 and VP3 are monomeric and their structures exhibit a high α-helical content. The function of both proteins could be monitored by complex formation with VP1. Furthermore, we could demonstrate a hemolytic activity of VP2/VP3 in vitro, which fits well into a postulated membrane interaction of VP2 during viral infection.

Polyomavirus large T antigen binds symmetrical repeats at the viral origin in an asymmetrical manner.

Polyomaviruses have repeating sequences at their origins of replication that bind the origin-binding domain of virus-encoded large T antigen. In murine polyomavirus, the central region of the origin contains four copies (P1 to P4) of the sequence G(A/G)GGC. They are arranged as a pair of inverted repeats with a 2-bp overlap between the repeats at the center. In contrast to simian virus 40 (SV40), where the repeats are nonoverlapping and all four repeats can be simultaneously occupied, the crystal structure of the four central murine polyomavirus sequence repeats in complex with the polyomavirus origin-binding domain reveals that only three of the four repeats (P1, P2, and P4) are occupied. Isothermal titration calorimetry confirms that the stoichiometry is the same in solution as in the crystal structure. Consistent with these results, mutation of the third repeat has little effect on DNA replication in vivo. Thus, the apparent 2-fold symmetry within the DNA repeats is not carried over to the protein-DNA complex. Flanking sequences, such as the AT-rich region, are known to be important for DNA replication. When the orientation of the central region was reversed with respect to these flanking regions, the origin was still able to replicate and the P3 sequence (now located at the P2 position with respect to the flanking regions) was again dispensable. This highlights the critical importance of the precise sequence of the region containing the pentamers in replication.

Biomimetic design of affinity peptide ligand for capsomere of virus-like particle.

Virus-like particle (VLP) of murine polyomavirus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV.

Merkel cell polyomavirus DNA sequences in the buffy coats of healthy blood donors.

Merkel cell polyomavirus (MCPyV), a DNA tumor virus, has been found to be associated with Merkel cell carcinoma and chronic lymphocytic leukemia. MCPyV sequences have also been detected in various normal tissues in tumor-affected patients. Immunologic studies have detected MCPyV antibodies in as many as 80% of healthy blood donors. This high seroprevalence suggests that MCPyV infection is widespread in humans. In our study, buffy coats, which were examined for MCPyV DNA Tag sequences, showed a prevalence of 22%. Viral DNA load was revealed in blood samples from 10 to 100 molecules/100 000 cells. DNA sequencing confirmed that polymerase chain reaction amplicons belong to the MCPyV strain, MKL-1. To interpret the putative role of MCPyV in chronic lymphocytic leukemia, we may infer that, during a long period of viral persistence in blood cells, this DNA tumor virus may generate mutants, which are able to participate as cofactors in the multistep process of cell transformation.

Structures of the major capsid proteins of the human Karolinska Institutet and Washington University polyomaviruses.

The Karolinska Institutet and Washington University polyomaviruses (KIPyV and WUPyV, respectively) are recently discovered human viruses that infect the respiratory tract. Although they have not yet been linked to disease, they are prevalent in populations worldwide, with initial infection occurring in early childhood. Polyomavirus capsids consist of 72 pentamers of the major capsid protein viral protein 1 (VP1), which determines antigenicity and receptor specificity. The WUPyV and KIPyV VP1 proteins are distant in evolution from VP1 proteins of known structure such as simian virus 40 or murine polyomavirus. We present here the crystal structures of unassembled recombinant WUPyV and KIPyV VP1 pentamers at resolutions of 2.9 and 2.55 Å, respectively. The WUPyV and KIPyV VP1 core structures fold into the same ß-sandwich that is a hallmark of all polyomavirus VP1 proteins crystallized to date. However, differences in sequence translate into profoundly different surface loop structures in KIPyV and WUPyV VP1 proteins. Such loop structures have not been observed for other polyomaviruses, and they provide initial clues about the possible interactions of these viruses with cell surface receptors.

A lipid receptor sorts polyomavirus from the endolysosome to the endoplasmic reticulum to cause infection.

The mechanisms by which receptors guide intracellular virus transport are poorly characterized. The murine polyomavirus (Py) binds to the lipid receptor ganglioside GD1a and traffics to the endoplasmic reticulum (ER) where it enters the cytosol and then the nucleus to initiate infection. How Py reaches the ER is unclear. We show that Py is transported initially to the endolysosome where the low pH imparts a conformational change that enhances its subsequent ER-to-cytosol membrane penetration. GD1a stimulates not viral binding or entry, but rather sorting of Py from late endosomes and/or lysosomes to the ER, suggesting that GD1a binding is responsible for ER targeting. Consistent with this, an artificial particle coated with a GD1a antibody is transported to the ER. Our results provide a rationale for transport of Py through the endolysosome, demonstrate a novel endolysosome-to-ER transport pathway that is regulated by a lipid, and implicate ganglioside binding as a general ER targeting mechanism.

Structural Basis and Evolution of Glycan Receptor Specificities within the Polyomavirus Family.

Asymptomatic infections with polyomaviruses in humans are common, but these small viruses can cause severe diseases in immunocompromised hosts. New Jersey polyomavirus (NJPyV) was identified via a muscle biopsy in an organ transplant recipient with systemic vasculitis, myositis, and retinal blindness, and human polyomavirus 12 (HPyV12) was detected in human liver tissue. The evolutionary origins and potential diseases are not well understood for either virus. In order to define their receptor engagement strategies, we first used nuclear magnetic resonance (NMR) spectroscopy to establish that the major capsid proteins (VP1) of both viruses bind to sialic acid in solution. We then solved crystal structures of NJPyV and HPyV12 VP1 alone and in complex with sialylated glycans. NJPyV employs a novel binding site for a α2,3-linked sialic acid, whereas HPyV12 engages terminal α2,3- or α2,6-linked sialic acids in an exposed site similar to that found in Trichodysplasia spinulosa-associated polyomavirus (TSPyV). Gangliosides or glycoproteins, featuring in mammals usually terminal sialic acids, are therefore receptor candidates for both viruses. Structural analyses show that the sialic acid-binding site of NJPyV is conserved in chimpanzee polyomavirus (ChPyV) and that the sialic acid-binding site of HPyV12 is widely used across the entire polyomavirus family, including mammalian and avian polyomaviruses. A comparison with other polyomavirus-receptor complex structures shows that their capsids have evolved to generate several physically distinct virus-specific receptor-binding sites that can all specifically engage sialylated glycans through a limited number of contacts. Small changes in each site may have enabled host-switching events during the evolution of polyomaviruses.IMPORTANCE Virus attachment to cell surface receptors is critical for productive infection. In this study, we have used a structure-based approach to investigate the cell surface recognition event for New Jersey polyomavirus (NJPyV) and human polyomavirus 12 (HPyV12). These viruses belong to the polyomavirus family, whose members target different tissues and hosts, including mammals, birds, fish, and invertebrates. Polyomaviruses are nonenveloped viruses, and the receptor-binding site is located in their capsid protein VP1. The NJPyV capsid features a novel sialic acid-binding site that is shifted in comparison to other structurally characterized polyomaviruses but shared with a closely related simian virus. In contrast, HPyV12 VP1 engages terminal sialic acids in a manner similar to the human Trichodysplasia spinulosa-associated polyomavirus. Our structure-based phylogenetic analysis highlights that even distantly related avian polyomaviruses possess the same exposed sialic acid-binding site. These findings complement phylogenetic models of host-virus codivergence and may also reflect past host-switching events.

Divalent Sialylated Precision Glycooligomers Binding to Polyomaviruses and the Effect of Different Linkers.

Divalent precision glycooligomers terminating in N-acetylneuraminic acid (Neu5Ac) or 3'-sialyllactose (3'-SL) with varying linkers between scaffold and the glycan portions are synthesized via solid phase synthesis for co-crystallization studies with the sialic acid-binding major capsid protein VP1 of human Trichodysplasia spinulosa-associated Polyomavirus. High-resolution crystal structures of complexes demonstrate that the compounds bind to VP1 depending on the favorable combination of carbohydrate ligand and linker. It is found that artificial linkers can replace portions of natural carbohydrate linkers as long as they meet certain requirements such as size or flexibility to optimize contact area between ligand and receptor binding sites. The obtained results will influence the design of future high affinity ligands based on the structures presented here, and they can serve as a blueprint to develop multivalent glycooligomers as inhibitors of viral adhesion.

Encapsulation of DNA and non-viral protein changes the structure of murine polyomavirus virus-like particles.

Asymmetrical-flow field flow fractionation with multiple-angle light scattering (AFFFF-MALS) was, for the first time, used to characterize the size of murine polyomavirus virus-like particles (MPV VLPs) packaged with either insect cell genomic DNA or non-viral protein. Encapsidation of both genomic DNA and non-viral protein were found to cause a contraction in VLP radii of gyration by approximately 1 nm. Non-viral protein packaged into VLPs consisted of a series of glutathione-S-transferase, His and S tags attached to the N-terminal end of the MPV structural protein VP2 (M(r) = 67108). Transmission electron microscopy analysis of MPV VLPs packaging non-viral protein suggested that VLPs grew in diameter by approximately 5 nm, highlighting the differences between this invasive technique and the relatively non-invasive AFFFF-MALS technique. Encapsulation of non-viral protein into MPV VLPs was found to prevent co-encapsidation of genomic DNA. Further investigation into why this occurred led to the discovery that encapsulation of non-viral protein alters the nuclear localization of MPV VLPs during in vivo assembly. VLPs were relocated away from the ring zone and the nuclear membrane towards the centre of the nucleus amongst the virogenic stroma. The change in nuclear localization away from the site where VLP assembly usually occurs is a likely reason why encapsidation of genomic DNA did not take place.

Purification of recombinant trichodysplasia spinulosa-associated polyomavirus VP1-derived virus-like particles using chromatographic techniques.

Trichodysplasia spinulosa-associated polyomavirus (TSPyV) has been linked to a rare and recently characterized skin disease occurring in immunocompromised patients. In analogy with other polyomaviruses, the major capsid protein VP1 of TSPyV can self-assemble into virus-like particles (VLPs). VLPs are increasingly applied for the vaccination and diagnostics. Mostly, non-scalable and labor intensive ultracentrifugation-based techniques are used for the purification of VLPs. In this work, we developed a purification procedure for TSPyV VP1 VLPs based on two chromatographic steps, ion-exchange monolith and core bead chromatography. Prior to chromatography, ammonium sulfate precipitation was used for the initial purification of TSPyV VP1 VLPs from yeast lysate. The VLPs were further purified using CIMmultus QA ion-exchange monolith in bind-elute mode. Most of TSPyV VP1 VLPs bound to the monolith and were subsequently eluted by a linear NaCl gradient. After ion-exchange monolith chromatography, the purity of TSPyV VP1 protein was about 75%. The final purification step of TSPyV VP1 VLPs was core bead chromatography using Capto Core 700 resin in flow-through mode. After core bead chromatography, 42% of TSPyV VP1 protein was recovered with a purity of 93%. The assembly of purified TSPyV VP1 protein into VLPs approximately 45-50 nm in diameter was confirmed by electron microscopy analysis. The purification procedure for TSPyV VP1 VLPs described here could be a scalable alternative to the conventional ultracentrifugation-based purification methods.