Biology subjects

Dermody, T. S.

Publications and source records attributed to Dermody, T. S..

6 recordsLinked to original sources

bioRxiv2021

Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis

Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-{beta}-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection. Author summaryGenetic screens are useful strategies to identify host factors required for viral infection. NPC1 was identified in independent CRISPR and RNA interference screens as a putative host factor required for reovirus replication. We discovered that NPC1-mediated cholesterol transport is dispensable for reovirus attachment, internalization, and disassembly but required for penetration of the viral disassembly intermediate from late endosomes into the cytoplasm. These findings pinpoint an essential function for cholesterol in the entry of reovirus and raise the possibility that cholesterol homeostasis regulates the entry of other viruses that penetrate late endosomes to initiate replication.

cell biology↗

bioRxiv2021

The human neuronal receptor NgR1 bridges reovirus capsid proteins to initiate infection

Human Nogo-66 receptor 1 (NgR1) is a receptor for mammalian orthoreoviruses (reoviruses), but the mechanism of virus-receptor engagement is unknown. NgR1 binds a variety of structurally dissimilar ligands in the adult central nervous system (CNS) to inhibit axon outgrowth. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron regrowth, making NgR1 an important therapeutic target for diverse conditions such as spinal crush injuries and Alzheimer disease. 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 NgR1 sequences in a central concave region of the molecule establish a bridge between two copies of the viral capsid protein, {sigma}3. This unusual binding interface produces high-avidity interactions between virus and receptor and likely primes early entry steps. NgR1 sequences engaged by reovirus also are required for NgR1 binding to ligands expressed by neurons and oligodendrocytes. These studies redefine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.

microbiology↗

bioRxiv2021

The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis

Engagement of host receptors is essential for viruses to enter target cells and initiate infection. Expression patterns of receptors in turn dictate host and tissue tropism and disease pathogenesis during infection. Mammalian orthoreovirus (reovirus) displays serotype-dependent patterns of tropism in the murine central nervous system (CNS) that are dictated by viral attachment protein {sigma}1. However, the receptor that mediates reovirus CNS tropism is unknown. Two proteinaceous receptors have been identified for reovirus, junctional adhesion molecule-A (JAM-A) and Nogo 66 receptor 1 (NgR1). Engagement of JAM-A is required for reovirus hematogenous dissemination but is dispensable for neural spread. To determine whether NgR1 functions in reovirus neuropathogenesis, we compared virus replication and disease following inoculation of wild-type (WT) and NgR1-/- mice. Genetic ablation of NgR1 did not alter replication of neurotropic reovirus strain T3SA- in the intestine and transmission to the brain following peroral inoculation. Viral titers in neural tissues following intramuscular inoculation, which provides access to neural dissemination routes, also were comparable in WT and NgR1-/- mice, suggesting that NgR1 is dispensable for reovirus neural spread to the CNS. The absence of both NgR1 and JAM-A also did not alter replication, neural tropism, and virulence of T3SA- following direct intracranial inoculation. In agreement with these findings, we found that the human but not the murine homolog of NgR1 functions as a receptor and confers efficient reovirus binding and infection of nonsusceptible cells in vitro. These results eliminate functions for JAM-A and NgR1 in shaping CNS tropism in mice and suggest that other receptors, yet to be identified, support this function. IMPORTANCEThe CNS presents a range of barriers to pathogen invasion. Yet neurotropic viruses have evolved strategies to breach these barriers and establish infection by engagement of host factors that allow navigation to the CNS and neural cell entry. Human NgR1 was identified as a reovirus receptor in an RNA interference screen and is expressed in CNS neurons in a pattern overlapping with reovirus tropism. Using mice genetically lacking NgR1 expression, and following different routes of inoculation, we discovered that murine NgR1 is dispensable for reovirus dissemination to the CNS, tropism and replication in the brain, and resultant disease. Concordant with these results, expression of human but not murine NgR1 confers reovirus binding and infection of nonsusceptible cells in vitro. These results point to species-specific use of alternate receptors by reovirus. A detailed understanding of species- and tissue-specific factors that dictate viral tropism will inform development of interventions and targeted gene delivery and therapeutic viral vectors.

microbiology↗

bioRxiv2021

The multi-functional reovirus σ3 protein is a virulence factor that suppresses stress granule formation to allow viral replication and myocardial injury

The mammalian orthoreovirus double-stranded (ds) RNA binding protein {sigma}3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of {sigma}3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of {sigma}3 binding to dsRNA during viral infection remains largely unknown. To identify functions of {sigma}3 dsRNA-binding activity during reovirus infection, we engineered a panel of 13 {sigma}3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of {sigma}3. Two recombinant viruses expressing these {sigma}3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are produced at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not form SGs at late times post-infection. These findings indicate that {sigma}3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that {sigma}3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.

microbiology↗

bioRxiv2020

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, {sigma}1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are unknown. 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 identified as required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains differing in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for 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. Infectivity of T3SA+ was diminished to levels 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. IMPORTANCEAttachment 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, that 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.

molecular biology↗

bioRxiv2020

Reovirus σ1 conformational flexibility modulates the efficiency of host cell attachment

Reovirus attachment protein {sigma}1 is a trimeric molecule containing tail, body, and head domains. During infection, {sigma}1 engages sialylated glycans and junctional adhesion molecule-A (JAM-A), triggering uptake into the endocytic compartment, where virions are proteolytically converted to infectious subvirion particles (ISVPs). Further disassembly allows {sigma}1 release and escape of transcriptionally active reovirus cores into the cytosol. Electron microscopy has revealed a distinct conformational change in {sigma}1 from a compact form on virions to an extended form on ISVPs. To determine the importance of {sigma}1 conformational mobility, we used reverse genetics to introduce cysteine mutations that can crosslink {sigma}1 by establishing disulfide bonds between structurally adjacent sites in the tail, body, and head domains. We detected phenotypic differences among the engineered viruses. A mutant with a cysteine pair in the head domain replicates with enhanced kinetics, forms large plaques, and displays increased avidity for JAM-A relative to the parental virus, mimicking properties of ISVPs. However, unlike ISVPs, particles containing cysteine mutations that crosslink the head domain uncoat and transcribe viral positive-sense RNA with kinetics similar to the parental virus and are sensitive to ammonium chloride. Together, these data suggest that {sigma}1 conformational flexibility modulates the efficiency of reovirus host cell attachment. IMPORTANCENonenveloped virus entry is an incompletely understood process. For reovirus, the functional significance of conformational rearrangements in the attachment protein, {sigma}1, that occur during entry and particle uncoating are unknown. We engineered and characterized reoviruses containing cysteine mutations that crosslink {sigma}1 monomers in non-reducing conditions. We found that the introduction of a cysteine pair in the receptor-binding domain of {sigma}1 yielded a virus that replicates with faster kinetics than the parental virus and forms larger plaques. Using functional assays, we found that crosslinking the {sigma}1 receptor-binding domain modulates reovirus attachment but not uncoating or transcription. These data suggest that {sigma}1 conformational rearrangements mediate the efficiency of reovirus host cell attachment.

microbiology↗