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Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

ABSTRACT: The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

SUBMITTER: Chappell JD

PROVIDER: S-EPMC191253 | biostudies-other | 1997 Mar

REPOSITORIES: biostudies-other

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Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

Chappell J D JD Gunn V L VL Wetzel J D JD Baer G S GS Dermody T S TS

Journal of virology 19970301 3

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function r ...[more]

PMID: 9032313

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Project description: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 ?1 viral attachment protein, which is composed of head, body, and tail domains. To identify ?1 sequences that contribute to tropism for specific cell types in the CNS, we engineered a panel of viruses expressing chimeric ?1 proteins in which discrete ?1 domains have been reciprocally exchanged. Parental and chimeric ?1 viruses were compared for replication, tropism, and disease induction following intracranial inoculation of newborn mice. Viruses expressing T1 ?1 head sequences infect the ependyma, produce relatively lower titers in the brain, and do not cause significant disease. In contrast, viruses expressing T3 ?1 head sequences efficiently infect neurons, replicate to relatively higher titers in the brain, and cause a lethal encephalitis. Additionally, T3 ?1 head-expressing viruses display enhanced infectivity of cultured primary cortical neurons compared with T1 ?1 head-expressing viruses. These results indicate that T3 ?1 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 ?1 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 ?1 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.

Project description:Mammalian orthoreovirus attachment to target cells is mediated by the outer capsid protein ?1, which projects from the virion surface. The ?1 protein is a homotrimer consisting of a filamentous tail, which is partly inserted into the virion; a body domain constructed from ?-spiral repeats; and a globular head with receptor-binding properties. The ?1 tail is predicted to form an ?-helical coiled coil. Although ?1 undergoes a conformational change during cell entry, the nature of this change and its contributions to viral replication are unknown. Electron micrographs of ?1 molecules released from virions identified three regions of flexibility, including one at the midpoint of the molecule, that may be involved in its structural rearrangement. To enable a detailed understanding of essential ?1 tail organization and properties, we determined high-resolution structures of the reovirus type 1 Lang (T1L) and type 3 Dearing (T3D) ?1 tail domains. Both molecules feature extended ?-helical coiled coils, with T1L ?1 harboring central chloride ions. Each molecule displays a discontinuity (stutter) within the coiled coil and an unexpectedly seamless transition to the body domain. The transition region features conserved interdomain interactions and appears rigid rather than highly flexible. Functional analyses of reoviruses containing engineered ?1 mutations suggest that conserved residues predicted to stabilize the coiled-coil-to-body junction are essential for ?1 folding and encapsidation, whereas central chloride ion coordination and the stutter are dispensable for efficient replication. Together, these findings enable modeling of full-length reovirus ?1 and provide insight into the stabilization of a multidomain virus attachment protein.IMPORTANCE While it is established that different conformational states of attachment proteins of enveloped viruses mediate receptor binding and membrane fusion, less is understood about how such proteins mediate attachment and entry of nonenveloped viruses. The filamentous reovirus attachment protein ?1 binds cellular receptors; contains regions of predicted flexibility, including one at the fiber midpoint; and undergoes a conformational change during cell entry. Neither the nature of the structural change nor its contribution to viral infection is understood. We determined crystal structures of large ?1 fragments for two different reovirus serotypes. We observed an unexpectedly tight transition between two domains spanning the fiber midpoint, which allows for little flexibility. Studies of reoviruses with engineered changes near the ?1 midpoint suggest that the stabilization of this region is critical for function. Together with a previously determined structure, we now have a complete model of the full-length, elongated reovirus ?1 attachment protein.

Project description:Structural metastability of viral capsids is pivotal for viruses to survive in harsh environments and to undergo timely conformational changes required for cell entry. Mammalian orthoreovirus (reovirus) is a model to study capsid metastability. Following initial disassembly of the reovirus particle mediated by proteases, a metastable intermediate called the infectious subvirion particle (ISVP) is generated. Using a σ1 monoreassortant virus, we recently showed that σ1 properties affect its encapsidation on particles and the metastability of ISVPs. How metastability is impacted by σ1 and whether the lower encapsidation level of σ1 is connected to this property is unknown. To define a correlation between encapsidation of σ1 and ISVP stability, we generated mutant viruses with single amino acid polymorphisms in σ1 or those that contain chimeric σ1 molecules composed of σ1 portions from type 1 and type 3 reovirus strains. We found that under most conditions where σ1 encapsidation on the particle was lower, ISVPs displayed lower stability. Characterization of mutant viruses selected for enhanced stability via a forward genetic approach also revealed that in some cases, σ1 properties influence stability without influencing σ1 encapsidation. These data indicate that σ1 can also influence ISVP stability independent of its level of incorporation. Together, our work reveals an underappreciated effect of the σ1 attachment protein on the properties of the reovirus capsid. IMPORTANCE Reovirus particles are comprised of eight proteins. Among them, the reovirus σ1 protein functions engages cellular receptors. σ1 also influences the stability of an entry intermediate called ISVP. Here, we sought to define the basis of the link between σ1 properties and stability of ISVPs. Using variety of mutant strains, we determined that when virus preparations contain particles with a high amount of encapsidated σ1, ISVP stability is higher. Additionally, we identified portions of σ1 that impact its encapsidation and consequently the stability of ISVPs. We also determined that in some cases, σ1 properties alter stability of ISVPs without affecting encapsidation. This work highlights that proteins of these complex particles are arranged in an intricate, interconnected manner such that changing the properties of these proteins has a profound impact on the remainder of the particle.

Dataset Information

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

Publications

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

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