Stefan Taube

Ganglioside-Linked Terminal Sialic Acid Moieties on Murine Macrophages Function as Attachment Receptors for Murine Noroviruses

ABSTRACT Noroviruses are the major cause of nonbacterial gastroenteritis in humans. However, little is known regarding the norovirus life cycle, including cell binding and entry. In contrast to human noroviruses, the recently discovered murine norovirus 1 (MNV-1) readily infects murine macrophages and dendritic cells in culture. Many viruses, including the related feline calicivirus, use terminal sialic acids (SA) as receptors for infection. Therefore, we tested whether SA moieties play a role during MNV-1 infection of murine macrophages. Competition with SA-binding lectins and neuraminidase treatment led to a reduction in MNV-1 binding and infection in cultured and primary murine macrophages, suggesting a role for SA during the initial steps of the MNV-1 life cycle. Because SA moieties can be attached to glycolipids (i.e., gangliosides), we next determined whether MNV-1 uses gangliosides during infection. The gangliosides GD1a, GM1, and asialo-GM1 (GA1) are natural components of murine macrophages. MNV-1 bound to ganglioside GD1a, which is characterized by an SA on the terminal galactose, but not to GM1 or asialo-GM1 in an enzyme-linked immunosorbent assay. The depletion of gangliosides using an inhibitor of glycosylceramide synthase (d-threo-P4) led to a reduction of MNV-1 binding and infection in cultured and primary murine macrophages. This defect was specifically rescued by the addition of GD1a. A similar phenotype was observed for MNV field strains WU11 (GV/WU11/2005/USA) and S99 (GV/Berlin/2006/DE). In conclusion, our data indicate that MNV can use terminal SA on gangliosides as attachment receptors during binding to murine macrophages.

A Mouse Model for Human Norovirus

ABSTRACT Human noroviruses (HuNoVs) cause significant morbidity and mortality worldwide. However, despite substantial efforts, a small-animal model for HuNoV has not been described to date. Since “humanized” mice have been successfully used to study human-tropic pathogens in the past, we challenged BALB/c mice deficient in recombination activation gene (Rag) 1 or 2 and common gamma chain (γc) (Rag-γc) engrafted with human CD34+ hematopoietic stem cells, nonengrafted siblings, and immunocompetent wild-type controls with pooled stool isolates from patients positive for HuNoV. Surprisingly, both humanized and nonhumanized BALB/c Rag-γc-deficient mice supported replication of a GII.4 strain of HuNoV, as indicated by increased viral loads over input. In contrast, immunocompetent wild-type BALB/c mice were not infected. An intraperitoneal route of infection and the BALB/c genetic background were important for facilitating a subclinical HuNoV infection of Rag-γc-deficient mice. Expression of structural and nonstructural proteins was detected in cells with macrophage-like morphology in the spleens and livers of BALB/c Rag-γc-deficient mice, confirming the ability of HuNoV to replicate in a mouse model. In summary, HuNoV replication in BALB/c Rag-γc-deficient mice is dependent on the immune-deficient status of the host but not on the presence of human immune cells and provides the first genetically manipulable small-animal model for studying HuNoV infection. IMPORTANCE Human noroviruses are a significant cause of viral gastroenteritis worldwide, resulting in significant morbidity and mortality. Antivirals and vaccines are currently not available, in part due to the inability to study these viruses in a genetically manipulable, small-animal model. Herein, we report the first mouse model for human noroviruses. This model will accelerate our understanding of human norovirus biology and provide a useful resource for evaluating antiviral therapies. Human noroviruses are a significant cause of viral gastroenteritis worldwide, resulting in significant morbidity and mortality. Antivirals and vaccines are currently not available, in part due to the inability to study these viruses in a genetically manipulable, small-animal model. Herein, we report the first mouse model for human noroviruses. This model will accelerate our understanding of human norovirus biology and provide a useful resource for evaluating antiviral therapies.

A Functional Toll-Like Receptor 8 Variant Is Associated with HIV Disease Restriction

BACKGROUND Toll-like receptors (TLRs) play an important role in the innate immune response to pathogens. TLR8 has been found to recognize RNA derived from various viruses, including human immunodeficiency virus (HIV). Presently, very little is known about the influence of TLR8 genetic variation on susceptibility to and progression of HIV disease. METHODS AND RESULTS We genotyped a population of 782 HIV-positive adults and 550 healthy control subjects for 3 nonsynonymous TLR8 single-nucleotide polymorphisms. We found that the presence of the most frequent TLR8 polymorphism, TLR8 A1G (rs3764880), confers a significantly protective effect regarding progression of the disease. In overexpression assays, we demonstrated that this receptor variant displays impaired NF-kappaB activation in vitro. Furthermore, we analyzed different cell types obtained from individuals differing in their TLR8 genotype and assessed their response to TLR8 ligands in vitro. The presence of the mutated receptor variant was associated with modulation of cytokine secretion profiles and lipid mediator synthesis patterns in monocytes and neutrophils. CONCLUSIONS This first report of a functional TLR8 variant associated with a different clinical course of an RNA viral disease may have implications for the individual risk assessment of patients infected with HIV and other RNA viruses as well as for future HIV vaccine development.

High-Resolution X-Ray Structure and Functional Analysis of the Murine Norovirus 1 Capsid Protein Protruding Domain

ABSTRACT Murine noroviruses (MNV) are closely related to the human noroviruses (HuNoV), which cause the majority of nonbacterial gastroenteritis. Unlike HuNoV, MNV grow in culture and in a small-animal model that represents a tractable model to study norovirus biology. To begin a detailed investigation of molecular events that occur during norovirus binding to cells, the crystallographic structure of the murine norovirus 1 (MNV-1) capsid protein protruding (P) domain has been determined. Crystallization of the bacterially expressed protein yielded two different crystal forms (Protein Data Bank identifiers [PDB ID], 3LQ6 and 3LQE). Comparison of the structures indicated a large degree of structural mobility in loops on the surface of the P2 subdomain. Specifically, the A′-B′ and E′-F′ loops were found in open and closed conformations. These regions of high mobility include the known escape mutation site for the neutralizing antibody A6.2 and an attenuation mutation site, which arose after serial passaging in culture and led to a loss in lethality in STAT1−/− mice, respectively. Modeling of a Fab fragment and crystal structures of the P dimer into the cryoelectron microscopy three-dimensional (3D) image reconstruction of the A6.2/MNV-1 complex indicated that the closed conformation is most likely bound to the Fab fragment and that the antibody contact is localized to the A′-B′ and E′-F′ loops. Therefore, we hypothesize that these loop regions and the flexibility of the P domains play important roles during MNV-1 binding to the cell surface.

Disruption of the Human Gut Microbiota following Norovirus Infection

The gut microbiota, the collection of all bacterial members in the intestinal tract, plays a key role in health. Disruption of the indigenous microbiota by a variety of stressors, including antibiotic therapy and intestinal infections, is associated with multiple health problems. We sought to determine if infection with Norovirus disrupts the gut microbiota. Barcoded pyrosequencing of the 16S rRNA-encoding gene was used to characterize the stool microbiota in Norovirus-infected human patients (n = 38). While the microbiota in most infected patients (n = 31) resembled that seen in uninfected healthy controls, a minority of patients (n = 7) possessed a significantly altered microbiota characterized by reduced relative numbers of Bacteriodetes and a corresponding increase in Proteobacteria. In these patients, the increase in Proteobacteria was due to a single operational taxonomic unit (OTU) of Escherichia coli. We cultured E. coli from Norovirus-infected patients and characterized them using PCR-ribotyping and virulence factor analysis. Multiple ribotypes were encountered, but none possessed typical virulence factors commonly carried by enteropathogenic E. coli strains. Microbiota disruption and elevated Proteobacteria were not significantly correlated to patient age, gender, sampling time following illness onset, or overall gut inflammation. These results demonstrate that some patients have a disrupted microbiota following Norovirus infection, and therefore may be at elevated risk for long-term health complications.

Murine Noroviruses Bind Glycolipid and Glycoprotein Attachment Receptors in a Strain-Dependent Manner

ABSTRACT Human norovirus infections are the most common cause of acute nonbacterial gastroenteritis in humans worldwide, and glycan binding plays an important role in the susceptibility to these infections. However, due to the lack of an efficient cell culture system or small animal model for human noroviruses, little is known about the biological role of glycan binding during infection. Murine noroviruses (MNV) are also enteric viruses that bind to cell surface glycans, but in contrast to their human counterparts, they can be grown in tissue culture and a small animal host. In this study, we determined glycan-binding specificities of the MNV strains MNV-1 and CR3 in vitro, identified molecular determinants of glycan binding, and analyzed infection in vivo. We showed that unlike MNV-1, CR3 binding to murine macrophages was resistant to neuraminidase treatment and glycosphingolipid depletion. Both strains depended on N-linked glycoproteins for binding, while only MNV-1 attachment to macrophages was sensitive to O-linked glycoprotein depletion. In vivo, CR3 showed differences in tissue tropism compared to MNV-1 by replicating in the large intestine. Mapping of a glycan-binding site in the MNV-1 capsid by reverse genetics identified a region topologically similar to the histo-blood group antigen (HBGA)-binding sites of the human norovirus strain VA387. The recombinant virus showed distinct changes in tissue tropism compared to wild-type virus. Taken together, our data demonstrate that MNV strains evolved multiple strategies to bind different glycan receptors on the surface of murine macrophages and that glycan binding contributes to tissue tropism in vivo.

High-resolution cryo-electron microscopy structures of MNV-1 and RHDV reveals marked flexibility in the receptor binding domains.

ABSTRACT Our previous structural studies on intact, infectious murine norovirus 1 (MNV-1) virions demonstrated that the receptor binding protruding (P) domains are lifted off the inner shell of the virus. Here, the three-dimensional (3D) reconstructions of recombinant rabbit hemorrhagic disease virus (rRHDV) virus-like particles (VLPs) and intact MNV-1 were determined to ∼8-Å resolution. rRHDV also has a raised P domain, and therefore, this conformation is independent of infectivity and genus. The atomic structure of the MNV-1 P domain was used to interpret the MNV-1 reconstruction. Connections between the P and shell domains and between the floating P domains were modeled. This observed P-domain flexibility likely facilitates virus-host receptor interactions.

Glycosphingolipids as Receptors for Non-Enveloped Viruses

Glycosphingolipids are ubiquitous molecules composed of a lipid and a carbohydrate moiety. Their main functions are as antigen/toxin receptors, in cell adhesion/recognition processes, or initiation/modulation of signal transduction pathways. Microbes take advantage of the different carbohydrate structures displayed on a specific cell surface for attachment during infection. For some viruses, such as the polyomaviruses, binding to gangliosides determines the internalization pathway into cells. For others, the interaction between microbe and carbohydrate can be a critical determinant for host susceptibility. In this review, we summarize the role of glycosphingolipids as receptors for members of the non-enveloped calici-, rota-, polyoma- and parvovirus families.

Murine norovirus-1 entry into permissive macrophages and dendritic cells is pH-independent.

Murine norovirus (MNV) is a recently discovered mouse pathogen. Unlike the fastidious human noroviruses that cause the overwhelming majority of non-bacterial gastroenteritis worldwide, MNV readily infects cells in culture. Its replication in primary murine macrophages and dendritic cells and their derived cell lines allows the study of norovirus cell entry for the first time. In this study we determined the role of pH during MNV-1 infection since the low pH environment of endosomes often triggers uncoating of viruses. We demonstrated that MNV-1 viral titers by plaque assay and expression of the non-structural protein VPg by immunofluorescence were not affected by pH in cultured and primary macrophages and dendritic cells in the presence of two known endosome acidification inhibitors, bafilomycin A1 and chloroquine. These data indicate that MNV-1 enters permissive cells in a pH-independent manner.

Generation of recombinant Norovirus-like particles (VLP) in the human endothelial kidney cell line 293T

Summary.Noroviruses (NVs) are the major cause of non-bacterial outbreaks of gastroenteritis. Here we report a new alternative system to generate recombinant NV virus-like particles (rNV-VLP) in a human endothelial kidney cell line (HEK). Transfecting HEK-293T cells with an expression vector coding for the ORF-2 gene lead to the expression of the viral structural protein VP1 which spontaneously assembled into virus-like particles (VLP), as shown by electron microscopy. The transfected cells did not show a cytopathic effect and released rNV-VLP into the culture medium. The HEK-293T cell derived particles were morphologically indistinguishable to the rNV-VLP produced from baculovirus and the Venezuelan equine encephalitis virus (VEE)-replicon. The produced particles were stable for at least 2.5 months at 4 °C.