The Nogo Receptor NgR1 Mediates Infection by Mammalian Reovirus

SummaryNeurotropic viruses, including mammalian reovirus, must disseminate from an initial site of replication to the central nervous system (CNS), often binding multiple receptors to facilitate systemic spread. Reovirus engages junctional adhesion molecule A (JAM-A) to disseminate hematogenously. However, JAM-A is dispensable for reovirus replication in the CNS. We demonstrate that reovirus binds Nogo receptor NgR1, a leucine-rich repeat protein expressed in the CNS, to infect neurons. Expression of NgR1 confers reovirus binding and infection of nonsusceptible cells. Incubating reovirus virions with soluble NgR1 neutralizes infectivity. Blocking NgR1 on transfected cells or primary cortical neurons abrogates reovirus infection. Concordantly, reovirus infection is ablated in primary cortical neurons derived from NgR1 null mice. Reovirus virions bind to soluble JAM-A and NgR1, while infectious disassembly intermediates (ISVPs) bind only to JAM-A. These results suggest that reovirus uses different capsid components to bind distinct cell-surface molecules, engaging independent receptors to facilitate spread and tropism

Altered Glycan Expression on Breast Cancer Cells Facilitates Infection by T3 Seroptype Oncolytic Reovirus

[Image: see text] Breast cancer is the most common cancer in women. Although current therapies have increased survival rates for some breast cancer types, other aggressive invasive breast cancers remain difficult to treat. As the onset of breast cancer is often associated with the appearance of extracellular markers, these could be used to better target therapeutic agents. Here, we demonstrated by nanobiophysical approaches that overexpression of α-sialylated glycans in breast cancer provides an opportunity to combat cancer cells with oncolytic reoviruses. Notably, a correlation between cellular glycan expression and the mechanical properties of reovirus attachment and infection is observed in a serotype-dependent manner. Furthermore, we enhance the infectivity of reoviruses in malignant cells by the coinjection of α-sialylated glycans. In conclusion, this study supports both the use of reoviruses as an oncolytic agent in nanomedicine and the role of α-sialylated glycans as adjuvants in oncolysis, offering new perspective in oncolytic cancer therapy

Reovirus uses macropinocytosis-mediated entry and fast axonal transport to infect neurons.

Several barriers protect the central nervous system (CNS) from pathogen invasion. Yet viral infections of the CNS are common and often debilitating. Understanding how neurotropic viruses co-opt host machinery to overcome challenges to neuronal entry and transmission is important to combat these infections. Neurotropic reovirus disseminates through neural routes and invades the CNS to cause lethal encephalitis in newborn animals. To define mechanisms of reovirus neuronal entry and directional transport, we used primary neuron cultures, which reproduce in vivo infection patterns displayed by different reovirus serotypes. Treatment of neurons with small-molecule inhibitors of different endocytic uptake pathways allowed us to discover that the cellular machinery mediating macropinocytosis is required for reovirus neuronal entry. This mechanism of reovirus entry differs from clathrin-mediated endocytosis, which is used by reovirus to invade non-neuronal cells. Analysis of reovirus transport and release from isolated soma or axonal termini of neurons cultivated in microfluidic devices indicates that reovirus is capable of retrograde but only limited anterograde neuronal transmission. The dynamics of retrograde reovirus movement are consistent with fast axonal transport coordinated by dynein along microtubules. Further analysis of viral transport revealed that multiple virions are transported together in axons within non-acidified vesicles. Reovirus-containing vesicles acidify after reaching the soma, where disassembly of virions and release of the viral core into the cytoplasm initiates replication. These results define mechanisms of reovirus neuronal entry and transport and establish a foundation to identify common host factors used by neuroinvasive viruses. Furthermore, our findings emphasize consideration of cell type-specific entry mechanisms in the tailored design of neurotropic viruses as tracers, oncolytic agents, and delivery vectors

Function, Architecture, and Biogenesis of Reovirus Replication Neoorganelles

Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Reoviruses are common pathogens of mammals that have been linked to celiac disease and show promise for oncolytic applications. These viruses form nonenveloped, double-shelled virions that contain ten segments of double-stranded RNA. Replication organelles in reovirus-infected cells are nucleated by viral nonstructural proteins µNS and σNS. Both proteins partition the endoplasmic reticulum to form the matrix of these structures. The resultant membranous webs likely serve to anchor viral RNA–protein complexes for the replication of the reovirus genome and the assembly of progeny virions. Ongoing studies of reovirus replication organelles will advance our knowledge about the strategies used by viruses to commandeer host biosynthetic pathways and may expose new targets for therapeutic intervention against diverse families of pathogenic viruses

Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus.

Mammalian orthoreovirus (reovirus) infects most mammals and is associated with celiac disease in humans. In mice, reovirus infects the intestine and disseminates systemically to cause serotype-specific patterns of disease in the brain. To identify receptors conferring reovirus serotype-dependent neuropathogenesis, we conducted a genome-wide CRISPRa screen and identified paired immunoglobulin-like receptor B (PirB) as a receptor candidate. Ectopic expression of PirB allowed reovirus binding and infection. PirB extracelluar D3D4 region is required for reovirus attachment and infectivity. Reovirus binds to PirB with nM affinity as determined by single molecule force spectroscopy. Efficient reovirus endocytosis requires PirB signaling motifs. In inoculated mice, PirB is required for maximal replication in the brain and full neuropathogenicity of neurotropic serotype 3 (T3) reovirus. In primary cortical neurons, PirB expression contributes to T3 reovirus infectivity. Thus, PirB is an entry receptor for reovirus and contributes to T3 reovirus replication and pathogenesis in the murine brain

Paired immunoglobulin-like receptor B is an entry receptor for mammalian orthoreovirus

Abstract Mammalian orthoreovirus (reovirus) infects most mammals and is associated with celiac disease in humans. In mice, reovirus infects the intestine and disseminates systemically to cause serotype-specific patterns of disease in the brain. To identify receptors conferring reovirus serotype-dependent neuropathogenesis, we conducted a genome-wide CRISPRa screen and identified paired immunoglobulin-like receptor B (PirB) as a receptor candidate. Ectopic expression of PirB allowed reovirus binding and infection. PirB extracelluar D3D4 region is required for reovirus attachment and infectivity. Reovirus binds to PirB with nM affinity as determined by single molecule force spectroscopy. Efficient reovirus endocytosis requires PirB signaling motifs. In inoculated mice, PirB is required for maximal replication in the brain and full neuropathogenicity of neurotropic serotype 3 (T3) reovirus. In primary cortical neurons, PirB expression contributes to T3 reovirus infectivity. Thus, PirB is an entry receptor for reovirus and contributes to T3 reovirus replication and pathogenesis in the murine brain

NgR1 binding to reovirus reveals an unusual bivalent interaction and a new viral attachment protein.

Nogo-66 receptor 1 (NgR1) binds a variety of structurally dissimilar ligands in the adult central nervous system to inhibit axon extension. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron outgrowth, making NgR1 an important therapeutic target for diverse neurological conditions such as spinal crush injuries and Alzheimer's disease. Human NgR1 serves as a receptor for mammalian orthoreovirus (reovirus), but the mechanism of virus-receptor engagement is unknown. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that central NgR1 surfaces form a bridge between two copies of viral capsid protein σ3, establishing that σ3 serves as a receptor ligand for reovirus. This unusual binding interface produces high-avidity interactions between virus and receptor to prime early entry steps. These studies refine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components