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

ABSTRACT: 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.

SUBMITTER: Sutherland DM

PROVIDER: S-EPMC6232475 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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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.

Project description:BACKGROUND:Neurotropic virus-based tracers have been extensively applied in mapping and manipulation of neural circuits. However, their neurotropic and neurotoxic properties remain to be fully characterized. METHODS:Through neural circuit tracing, we systematically compared the neurotropism discrepancy among different multi-trans-synaptic and mono-synaptic retrograde viral tracers including pseudorabies virus (PRV), rabies virus (RV), and the newly engineered retro adeno-associated virus (rAAV2-retro) tracers. The (single-cell) RNA sequencing analysis was utilized for seeking possible attribution to neurotropism discrepancy and comparing cell toxicity caused by viral infection between glycoprotein-deleted RV (RV-?G) and rAAV2-retro. Viral toxicity induced microglia activation and neuronal protein change were evaluated by immunohistochemistry. RESULTS:Multi-trans-synaptic retrograde viral tracers, PRV and RV, exhibit differential neurotropism when they were used for central neural circuit tracing from popliteal lymph nodes. Mono-synaptic retrograde tracers, including RV-?G and rAAV2-retro, displayed discrepant neurotropic property, when they were applied to trace the inputs of lateral hypothalamic area and medial preoptic nucleus. rAAV2-retro demonstrated preference in cerebral cortex, whereas RV-?G prefers to label basal ganglia and hypothalamus. Remarkably, we detected a distinct preference for specific cortical layer of rAAV2-retro in layer 5 and RV-?G in layer 6 when they were injected into dorsal lateral geniculate nucleus to label corticothalamic neurons in primary visual cortex. Complementation of TVA receptor gene in RV-resistant neurons enabled EnvA-pseudotyped RV infection, supporting receptors attribution to viral neurotropism. Furthermore, both RV-?G and rAAV2-retro exerted neurotoxic influence at the injection sites and retrogradely labeled sites, while the changes were more profound for RV-?G infection. Finally, we demonstrated a proof-of-concept strategy for more comprehensive high-order circuit tracing of a specific target nucleus by combining rAAV2-retro, RV, and rAAV tracers. CONCLUSIONS:Different multi-trans-synaptic and mono-synaptic retrograde viral tracers exhibited discrepant neurotropism within certain brain regions, even cortical layer preference. More neurotoxicity was observed under RV-?G infection as compared with rAAV2-retro. By combining rAAV2-retro, RV, and rAAV tracers, high-order circuit tracing can be achieved. Our findings provide important reference for appropriate application of viral tracers to delineate the landscape and dissect the function of neural network.

Project description:Molecular dynamics simulations were performed using the recently determined crystal structure of the reovirus attachment protein, sigma1. These studies were conducted to improve an understanding of two unique features of sigma1 structure: the protonation state of Asp(345), which is buried in the sigma1 trimer interface, and the flexibility of the protein at a defined region below the receptor-binding head domain. Three copies of aspartic acids Asp(345) and Asp(346) cluster in a solvent-inaccessible and hydrophobic region at the sigma1 trimer interface. These residues are hypothesized to mediate conformational changes in sigma1 during viral attachment or cell entry. Our results indicate that protonation of Asp(345) is essential to the integrity of the trimeric structure seen by x-ray crystallography, whereas deprotonation induces structural changes that destabilize the trimer interface. This finding was confirmed by electrostatic calculations using the finite difference Poisson-Boltzmann method. Earlier studies show that sigma1 can exist in retracted and extended conformations on the viral surface. Since protonated Asp(345) is necessary to form a stable, extended trimer, our results suggest that protonation of Asp(345) may allow for a structural transition from a partially detrimerized molecule to the fully formed trimer seen in the crystal structure. Additional studies were conducted to quantify the previously observed flexibility of sigma1 at a defined region below the receptor-binding head domain. Increased mobility was observed for three polar residues (Ser(291), Thr(292), and Ser(293)) located within an insertion between the second and third beta-spiral repeats of the crystallized portion of the sigma1 tail. These amino acids interact with water molecules of the solvent bulk and are responsible for oscillating movement of the head of approximately 50 degrees during 5 ns of simulations. This flexibility may facilitate viral attachment and also function in cell entry and disassembly. These findings provide new insights about the conformational dynamics of sigma1 that likely underlie the initiation of the reovirus infectious cycle.

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

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