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Science Advances Jun 2023Numerous viruses use specialized surface molecules called fusogens to enter host cells. Many of these viruses, including the severe acute respiratory syndrome...
Numerous viruses use specialized surface molecules called fusogens to enter host cells. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can infect the brain and are associated with severe neurological symptoms through poorly understood mechanisms. We show that SARS-CoV-2 infection induces fusion between neurons and between neurons and glia in mouse and human brain organoids. We reveal that this is caused by the viral fusogen, as it is fully mimicked by the expression of the SARS-CoV-2 spike (S) protein or the unrelated fusogen p15 from the baboon orthoreovirus. We demonstrate that neuronal fusion is a progressive event, leads to the formation of multicellular syncytia, and causes the spread of large molecules and organelles. Last, using Ca imaging, we show that fusion severely compromises neuronal activity. These results provide mechanistic insights into how SARS-CoV-2 and other viruses affect the nervous system, alter its function, and cause neuropathology.
Topics: Animals; Humans; Mice; COVID-19; SARS-CoV-2; Neurons; Brain; Neuroglia
PubMed: 37285437
DOI: 10.1126/sciadv.adg2248 -
Cells May 2022Mammalian orthoreoviruses (ReoV) are non-enveloped viruses with segmented double-stranded RNA genomes. In humans, ReoV are generally considered non-pathogenic, although... (Review)
Review
Mammalian orthoreoviruses (ReoV) are non-enveloped viruses with segmented double-stranded RNA genomes. In humans, ReoV are generally considered non-pathogenic, although members of this family have been proven to cause mild gastroenteritis in young children and may contribute to the development of inflammatory conditions, including Celiac disease. Because of its low pathogenic potential and its ability to efficiently infect and kill transformed cells, the ReoV strain Type 3 Dearing (T3D) is clinical trials as an oncolytic agent. ReoV manifests its oncolytic effects in large part by infecting tumor cells and activating programmed cell death pathways (PCDs). It was previously believed that apoptosis was the dominant PCD pathway triggered by ReoV infection. However, new studies suggest that ReoV also activates other PCD pathways, such as autophagy, pyroptosis, and necroptosis. Necroptosis is a caspase-independent form of PCD reliant on receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and its substrate, the pseudokinase mixed-lineage kinase domain-like protein (MLKL). As necroptosis is highly inflammatory, ReoV-induced necroptosis may contribute to the oncolytic potential of this virus, not only by promoting necrotic lysis of the infected cell, but also by inflaming the surrounding tumor microenvironment and provoking beneficial anti-tumor immune responses. In this review, we summarize our current understanding of the ReoV replication cycle, the known and potential mechanisms by which ReoV induces PCD, and discuss the consequences of non-apoptotic cell death-particularly necroptosis-to ReoV pathogenesis and oncolysis.
Topics: Animals; Apoptosis; Cell Death; Child; Child, Preschool; Humans; Mammals; Necroptosis; Necrosis; Protein Kinases
PubMed: 35681452
DOI: 10.3390/cells11111757 -
Nature Microbiology Aug 2022Pangolins are the most trafficked wild animal in the world according to the World Wildlife Fund. The discovery of SARS-CoV-2-related coronaviruses in Malayan pangolins...
Pangolins are the most trafficked wild animal in the world according to the World Wildlife Fund. The discovery of SARS-CoV-2-related coronaviruses in Malayan pangolins has piqued interest in the viromes of these wild, scaly-skinned mammals. We sequenced the viromes of 161 pangolins that were smuggled into China and assembled 28 vertebrate-associated viruses, 21 of which have not been previously reported in vertebrates. We named 16 members of Hunnivirus, Pestivirus and Copiparvovirus pangolin-associated viruses. We report that the L-protein has been lost from all hunniviruses identified in pangolins. Sequences of four human-associated viruses were detected in pangolin viromes, including respiratory syncytial virus, Orthopneumovirus, Rotavirus A and Mammalian orthoreovirus. The genomic sequences of five mammal-associated and three tick-associated viruses were also present. Notably, a coronavirus related to HKU4-CoV, which was originally found in bats, was identified. The presence of these viruses in smuggled pangolins identifies these mammals as a potential source of emergent pathogenic viruses.
Topics: Animals; COVID-19; Chiroptera; Humans; Mammals; Pangolins; SARS-CoV-2
PubMed: 35918420
DOI: 10.1038/s41564-022-01181-1 -
Proceedings of the National Academy of... May 2023Mammalian orthoreoviruses (reoviruses) serve as potential triggers of celiac disease and have oncolytic properties, making these viruses potential cancer therapeutics....
Mammalian orthoreoviruses (reoviruses) serve as potential triggers of celiac disease and have oncolytic properties, making these viruses potential cancer therapeutics. Primary attachment of reovirus to host cells is mainly mediated by the trimeric viral protein, σ1, which engages cell-surface glycans, followed by high-affinity binding to junctional adhesion molecule-A (JAM-A). This multistep process is thought to be accompanied by major conformational changes in σ1, but direct evidence is lacking. By combining biophysical, molecular, and simulation approaches, we define how viral capsid protein mechanics influence virus-binding capacity and infectivity. Single-virus force spectroscopy experiments corroborated by in silico simulations show that GM2 increases the affinity of σ1 for JAM-A by providing a more stable contact interface. We demonstrate that conformational changes in σ1 that lead to an extended rigid conformation also significantly increase avidity for JAM-A. Although its associated lower flexibility impairs multivalent cell attachment, our findings suggest that diminished σ1 flexibility enhances infectivity, indicating that fine-tuning of σ1 conformational changes is required to successfully initiate infection. Understanding properties underlying the nanomechanics of viral attachment proteins offers perspectives in the development of antiviral drugs and improved oncolytic vectors.
Topics: Animals; Capsid Proteins; Reoviridae; Orthoreovirus; Viral Proteins; Virus Attachment; Antibodies, Viral; Mammals
PubMed: 37186838
DOI: 10.1073/pnas.2220741120 -
Viruses Feb 2021RNAs with methylated cap structures are present throughout multiple domains of life. Given that cap structures play a myriad of important roles beyond translation, such... (Review)
Review
RNAs with methylated cap structures are present throughout multiple domains of life. Given that cap structures play a myriad of important roles beyond translation, such as stability and immune recognition, it is not surprising that viruses have adopted RNA capping processes for their own benefit throughout co-evolution with their hosts. In fact, that RNAs are capped was first discovered in a member of the family, , before these findings were translated to other domains of life. This review revisits long-past knowledge and recent studies on RNA capping among members of to help elucidate the perplex processes of RNA capping and functions of RNA cap structures during infection. The review brings to light the many uncertainties that remain about the precise capping status, enzymes that facilitate specific steps of capping, and the functions of RNA caps during replication.
Topics: Animals; Humans; RNA Caps; RNA Processing, Post-Transcriptional; RNA, Viral; Reoviridae; Reoviridae Infections
PubMed: 33668598
DOI: 10.3390/v13020294 -
Nature Communications May 2023Mammalian orthoreovirus (reovirus) infects most mammals and is associated with celiac disease in humans. In mice, reovirus infects the intestine and disseminates...
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.
Topics: Animals; Humans; Mice; Antibodies, Viral; Orthoreovirus, Mammalian; Receptors, Immunologic; Reoviridae Infections; Receptors, Virus
PubMed: 37147336
DOI: 10.1038/s41467-023-38327-6 -
Viruses Jun 2021Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence...
Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and "genomeless" top component particles. Whether reovirus virions or top component particles package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the RNA content of enriched reovirus particles. Reovirus virions exclusively packaged viral double-stranded RNA. In contrast, reovirus top component particles contained similar proportions but reduced amounts of viral double-stranded RNA and were selectively enriched for numerous host RNA species, especially short, non-polyadenylated transcripts. Host RNA selection was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains and were not selected solely by the viral RNA polymerase. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is differentially selective and may contribute to or result from inefficient viral RNA packaging.
Topics: Animals; Cell Line; Genome, Viral; Host Microbial Interactions; Mice; RNA, Double-Stranded; RNA-Seq; Reoviridae; Viral Genome Packaging; Viral Proteins; Virion
PubMed: 34201386
DOI: 10.3390/v13061096 -
Viruses Jan 2021Mammalian orthoreovirus (reovirus), a dsRNA virus with a multilayered capsid, serves as a model system for studying the entry of similar viruses. The outermost layer of... (Review)
Review
Mammalian orthoreovirus (reovirus), a dsRNA virus with a multilayered capsid, serves as a model system for studying the entry of similar viruses. The outermost layer of this capsid undergoes processing to generate a metastable intermediate. The metastable particle undergoes further remodeling to generate an entry-capable form that delivers the genome-containing inner capsid, or core, into the cytoplasm. In this review, we highlight capsid proteins and the intricacies of their interactions that control the stability of the capsid and consequently impact capsid structural changes that are prerequisites for entry. We also discuss a novel proviral role of host membranes in promoting capsid conformational transitions. Current knowledge gaps in the field that are ripe for future investigation are also outlined.
Topics: Animals; Capsid Proteins; Cell Line; Mice; Orthoreovirus, Mammalian; Protein Domains; Proteolysis; Reoviridae Infections; Virion; Virus Internalization
PubMed: 33494426
DOI: 10.3390/v13020153 -
BioRxiv : the Preprint Server For... Aug 2023Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral...
Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization.
PubMed: 37693509
DOI: 10.1101/2023.08.29.555250 -
Bioactive Materials Dec 2023Effective oral drugs and vaccines require high delivery efficiency across the gastrointestinal epithelia and protection of medically effective payloads (i.e.,...
Effective oral drugs and vaccines require high delivery efficiency across the gastrointestinal epithelia and protection of medically effective payloads (i.e., immunogens) against gastric damage. In this study, hollowed nanocarriers (NCs: silica nanospheres and gold nanocages) with poly-l-lysine (PLL) coating and mammalian orthoreovirus cell attachment protein σ1 functionalization (NC-PLL-σ1) were explored as functional oral drug delivery vehicles (ODDVs). The transport of these ODDVs to mucosal lymphoid tissues could be facilitated by microfold cells (M-cells) mediated transcytosis (via σ1-α2-3-linked sialic acids adherence) across gastrointestinal epithelia. PLL coating provided protection and slow-release of rhodamine 6 G (R6G), a model payload. The transport effectiveness of these ODDVs was tested on intestinal organoid monolayers . When compared with other experimental groups, the fully functionalized ODDV system (with PLL-σ1) demonstrated two significant advantages: a significantly higher transport efficiency (198% over blank control at 48 h); and protection of payloads which led to both better transport efficiency and extended-release of payloads (61% over uncoated carriers at 48 h). In addition, it was shown that the M cell presence in intestinal organoid monolayers (modulated by Rank L stimulation) was a determining factor on the transport efficiency of the ODDVs: more M-cells (induced by higher Rank L) in the organoid monolayers led to higher transport efficiency for ODDV-delivered model payload (R6G). The fully functionalized ODDVs showed great potential as effective oral delivery vehicles for drugs and vaccines.
PubMed: 37560199
DOI: 10.1016/j.bioactmat.2023.07.014