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Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germ line ablation of NF-κB subunit p50. It is not known whether the proapoptotic function of NF-κB is mediated by neural-cell-intrinsic (neural-intrinsic) processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neural cells using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural cell NF-κB p65 (Nsp65(−/−)) survived. While viral loads in brains of WT and Nsp65(−/−) mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brains of WT mice displayed increased immunoreactivity for cleaved caspase-3, a marker of apoptosis, relative to Nsp65(−/−) mice. These data suggest that neural-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected brain cortices of WT and Nsp65(−/−) mice suggests that NF-κB activation in neuronal cells upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations. IMPORTANCE Viral encephalitis contributes to illness and death in children and adults worldwide and has limited treatment options. Identifying common host factors upregulated by neurotropic viruses can enhance an understanding of virus-induced neuropathogenesis and aid in development of therapeutics. Although many neurotropic viruses activate NF-κB during infection, mechanisms by which NF-κB regulates viral neuropathogenesis and contributes to viral encephalitis are not well understood. We established mice in which NF-κB expression is ablated in neural tissue to study the function of NF-κB in reovirus neurovirulence and identify genes activated by NF-κB in response to reovirus infection in the central nervous system. Encephalitis following reovirus infection was dampened in mice lacking neural cell NF-κB. Reovirus induced a chemokine profile in the brain that was dependent on NF-κB signaling and was similar to chemokine profiles elicited by other neurotropic viruses. These data suggest common underlying mechanisms of encephalitis caused by neurotropic viruses and potentially shared therapeutic targets.

Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs This has been attributed to the proofreading activity of the viral 3'-5' exoribonuclease (ExoN) β-d-N4-Hydroxycytidine (NHC) (EIDD-1931;Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC50] = 0 17 μM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0 56 μM) with minimal cytotoxicity NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease However, no antivirals have been approved to treat these infections Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-d-N4-hydroxycytidine (NHC), against two divergent CoVs Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections [ABSTRACT FROM AUTHOR] Copyright of Journal of Virology is the property of American Society for Microbiology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission However, users may print, download, or email articles for individual use This abstract may be abridged No warranty is given about the accuracy of the copy Users should refer to the original published version of the material for the full abstract (Copyright applies to all Abstracts )

The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-d-N4-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections., Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3′-5′ exoribonuclease (ExoN). β-d-N4-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC50] = 0.17 μM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0.56 μM) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication. IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-d-N4-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.

Viruses in the family Polydnaviridae are symbiotically associated with parasitoid wasps. Wasps inject polydnaviruses (PDVs) when laying an egg into their insect host, and expression of viral gene products causes several physiological alterations, including immunosuppression, that allow the wasp's progeny to develop. As with other PDVs, most Microplitis demolitor bracovirus (MdBV) genes are related variants that form gene families. The largest MdBV gene family includes 13 members that encode predicted proteins related to protein tyrosine phosphatases (PTPs). Sequence analysis during the present study indicated that five PTP family members (PTP-H2, -H3, -N1, and -N2) have fully conserved catalytic domains, whereas other family members exhibited replacements, deletions, or rearrangements of amino acids considered essential for tyrosine phosphatase activity. Expression studies indicated that most MdBV PTP genes are expressed in virus-infected host insects, with transcript abundance usually being highest in hemocytes. MdBV-infected hemocytes also exhibited higher levels of tyrosine phosphatase activity than noninfected hemocytes. We produced expression constructs for four of the most abundantly expressed PTP family members and conducted functional studies with hemocyte-like Drosophila S2 cells. These experiments suggested that recombinant PTP-H2 and PTP-H3 are functional tyrosine phosphatases whereas PTP-H1 and PTP-J1 are not. PTP-H2 and -H3 localized to focal adhesions in S2 cells, and coexpression with another MdBV gene product, Glc1.8, resulted in complete inhibition of phagocytosis.

Mammalian reoviruses display serotype-specific patterns of tropism and disease in the murine central nervous system (CNS) attributable to polymorphisms in viral attachment protein σ1. While all reovirus serotypes use junctional adhesion molecule-A as a cellular receptor, they differ in their utilization of carbohydrate coreceptors. This observation raises the possibility that carbohydrate binding by σ1 influences reovirus pathology in the CNS. In this study, we sought to define the function of carbohydrate binding in reovirus neuropathogenesis. Newborn mice were inoculated intramuscularly with wild-type strain type 3 Dearing (T3D) and T3D-σ1R202W, a point mutant T3D derivative that does not bind sialic acid (SA). Infected mice were monitored for survival, and viral loads at the sites of primary and secondary replication were quantified. Fewer mice inoculated with the wild-type virus survived in comparison to those inoculated with the mutant virus. The wild-type virus also produced higher titers in the spinal cord and brain at late times postinoculation but lower titers in the liver in comparison to those produced by the mutant virus. In addition, the wild-type virus was more virulent and produced higher titers in the brain than the mutant following intracranial inoculation. These animal infectivity studies suggest that T3D-σ1R202W harbors a defect in neural growth. Concordantly, compared with the wild-type virus, the mutant virus displayed a decreased capacity to infect and replicate in primary cultures of cortical neurons, a property dependent on cell surface SA. These results suggest that SA binding enhances the kinetics of reovirus replication in neural tissues and highlight a functional role for sialylated glycans as reovirus coreceptors in the CNS.

In mouse embryonic fibroblasts (MEFs), the bovine rotavirus (UK strain) but not the simian rhesus rotavirus (RRV) robustly triggers beta interferon (IFN-β) secretion, resulting in an IFN-dependent restriction of replication. We now find that both rotavirus strains trigger antiviral transcriptional responses early during infection and that both transcriptional responses and IFN-β secretion are completely abrogated in MAVS/IPS-1 −/− MEFs. Replication of UK virus could be rescued in MAVS/IPS-1 −/− MEFs, and synthesis of viral RNA significantly increased early during virus infection. UK virus induced IFN-β secretion and transcription of IFN-stimulated genes (ISGs) in both RIG-I −/− and MDA-5 −/− MEFs, and neither receptor was essential by itself for the antiviral response to UK rotavirus. However, when receptors RIG-I and MDA-5 were depleted using RNA interference, we found that both contribute to the magnitude of the IFN response. IRF3 was found to be essential for MAVS/IPS-1-directed ISG transcription and IFN-β secretion during rotavirus infection. Interestingly, absence of the double-stranded RNA-dependent protein kinase PKR led to a profound defect in the capacity of host cells to secrete IFN-β in response to virus. Both PKR and IRF3 restricted the early replication of UK as indicated by significant increases in viral RNA in fibroblasts lacking either gene. Despite the loss in IFN-β secretion in PKR −/− MEFs, we did not observe decreased IRF3- or NF-κB-dependent early ISG transcription in these cells. Levels of transcripts encoding IFN-α4, IFN-α5, and IFN-β were high in infected PKR −/− MEFs, indicating that during rotavirus infection, PKR functions at a stage between IFN gene transcription and subsequent IFN-β secretion. These findings reveal that activation of the antiviral response by rotavirus is dependent on MAVS/IPS-1 and IRF3 and involves both RIG-I and MDA-5 and that IFN-β secretion during rotavirus infection is regulated by PKR.