Welsh, O. L. | bioRxiv Science

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 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↗