Sequence polymorphisms in the reovirus σ1 attachment protein modulate encapsidation efficiency and replication in mice.

Many functions of viral attachment proteins are established, but less is known about the biological importance of viral attachment protein encapsidation efficiency. The mammalian orthoreovirus (reovirus) σ1 attachment protein forms filamentous trimers that incorporate into pentamers of the λ2 capsid protein. Reovirus strains vary in the efficiency of σ1 encapsidation onto progeny virions, which influences viral stability during entry into cells and the efficacy of tumor cell lysis. While the role of σ1 encapsidation has been evaluated in studies using cultured cells, the contribution of attachment protein encapsidation efficiency to viral infection in animals is less clear. Polymorphisms in reovirus σ1 at residues 22 and 249 have been implicated in viral dissemination in mice and susceptibility to proteolysis in the murine intestine, respectively. To determine whether these residues contribute to σ1 encapsidation efficiency, we engineered σ1 mutant viruses with single- and double-residue substitutions at sites 22 and 249. We found that substitutions at these sites alter the encapsidation of σ1 and that reoviruses encapsidating higher amounts of σ1 bind cells more avidly and have a modest replication advantage in a cell-type-specific manner relative to low σ1-encapsidating reoviruses. Furthermore, we found that a high σ1-encapsidating reovirus replicates and disseminates more efficiently in mice relative to a low σ1-encapsidating reovirus. These findings provide evidence of a relationship between viral attachment protein encapsidation efficiency and viral replication in cell culture and animal hosts. IMPORTANCE: Viral attachment proteins can serve multiple functions during viral replication, including attachment to host cells, cell entry and disassembly, and modulation of host immune responses. The relationship between viral attachment protein encapsidation efficiency and viral replication in cells and animals is poorly understood. We engineered and characterized a panel of reoviruses that differ in the capacity to encapsidate the σ1 attachment protein. We found that strains encapsidating σ1 with higher efficiency bind cells more avidly and replicate and spread more efficiently in mice relative to those encapsidating σ1 with lower efficiency. These results highlight a function for σ1 attachment protein capsid abundance in viral replication in cells and animals, which may inform future use of reovirus as an oncolytic therapeutic.

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

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.

Cytidine Monophosphate N-Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection.

Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.

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.

Reovirus Neurotropism and Virulence Are Dictated by Sequences in the Head Domain of the Viral Attachment Protein.

Viral capsid components that bind cellular receptors mediate critical functions in viral tropism and disease pathogenesis. Mammalian orthoreoviruses (reoviruses) spread systemically in newborn mice to cause serotype-specific disease in the central nervous system (CNS). Serotype 1 (T1) reovirus infects ependymal cells to cause nonlethal hydrocephalus, whereas serotype 3 (T3) reovirus infects neurons to cause fulminant and lethal encephalitis. This serotype-dependent difference in tropism and concomitant disease is attributed to the σ1 viral attachment protein, which is composed of head, body, and tail domains. To identify σ1 sequences that contribute to tropism for specific cell types in the CNS, we engineered a panel of viruses expressing chimeric σ1 proteins in which discrete σ1 domains have been reciprocally exchanged. Parental and chimeric σ1 viruses were compared for replication, tropism, and disease induction following intracranial inoculation of newborn mice. Viruses expressing T1 σ1 head sequences infect the ependyma, produce relatively lower titers in the brain, and do not cause significant disease. In contrast, viruses expressing T3 σ1 head sequences efficiently infect neurons, replicate to relatively higher titers in the brain, and cause a lethal encephalitis. Additionally, T3 σ1 head-expressing viruses display enhanced infectivity of cultured primary cortical neurons compared with T1 σ1 head-expressing viruses. These results indicate that T3 σ1 head domain sequences promote infection of neurons, likely by interaction with a neuron-specific receptor, and dictate tropism in the CNS and induction of encephalitis.IMPORTANCE Viral encephalitis is a serious and often life-threatening inflammation of the brain. Mammalian orthoreoviruses are promising oncolytic therapeutics for humans but establish virulent, serotype-dependent disease in the central nervous system (CNS) of many young mammals. Serotype 1 reoviruses infect ependymal cells and produce hydrocephalus, whereas serotype 3 reoviruses infect neurons and cause encephalitis. Reovirus neurotropism is hypothesized to be dictated by the filamentous σ1 viral attachment protein. However, it is not apparent how this protein mediates disease. We discovered that sequences forming the most virion-distal domain of T1 and T3 σ1 coordinate infection of either ependyma or neurons, respectively, leading to mutually exclusive patterns of tropism and disease in the CNS. These studies contribute new knowledge about how reoviruses target cells for infection in the brain and inform the rational design of improved oncolytic therapies to mitigate difficult-to-treat tumors of the CNS.