Discovery of 2-Amide-3-methylester Thiophenes that Target SARS-CoV-2 Mac1 and Repress Coronavirus Replication, Validating Mac1 as an Antiviral Target.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed. Here, we describe small-molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation-mediated innate immune responses. Three high-throughput screening hits had the same 2-amide-3-methylester thiophene scaffold. We studied the compound binding mode using X-ray crystallography, allowing us to design analogues. Compound 27 (MDOLL-0229) had an IC50 of 2.1 µM and was selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human proteins. The improved potency allowed testing of its effect on virus replication, and indeed, 27 inhibited replication of both murine hepatitis virus (MHV) prototypes CoV and SARS-CoV-2. Sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1-targeted small molecule demonstrated to inhibit coronavirus replication in a cell model.

Insight into the role of copper-based materials against the coronaviruses MHV-3, a model for SARS-CoV-2, during the COVID-19 pandemic.

Coating high-touch surfaces with inorganic agents, such as metals, appears to be a promising long-term disinfection strategy. However, there is a lack of studies exploring the effectiveness of copper-based products against viruses. In this study, we evaluated the cytotoxicity and virucidal effectiveness of products and materials containing copper against mouse hepatitis virus (MHV-3), a surrogate model for SARS-CoV-2. The results demonstrate that pure CuO and Cu possess activity against the enveloped virus at very low concentrations, ranging from 0.001 to 0.1% (w/v). A greater virucidal efficacy of CuO was found for nanoparticles, which showed activity even against viruses that are more resistant to disinfection such as feline calicivirus (FCV). Most of the evaluated products, with concentrations of Cu or CuO between 0.003 and 15% (w/v), were effective against MHV-3. Cryomicroscopy images of an MHV-3 sample exposed to a CuO-containing surface showed extensive damage to the viral capsid, presumably due to the direct or indirect action of copper ions.

2-Bromopalmitate depletes lipid droplets to inhibit viral replication.

The global impact of emerging viral infections emphasizes the urgent need for effective broad-spectrum antivirals. The cellular organelle, lipid droplet (LD), is utilized by many types of viruses for replication, but its reduction does not affect cell survival. Therefore, LD is a potential target for developing broad-spectrum antivirals. In this study, we found that 2-bromopalmitate (2 BP), a previously defined palmitoylation inhibitor, depletes LD across all studied cell lines and exerts remarkable antiviral effects on different coronaviruses. We comprehensively utilized 2 BP, alongside other palmitoylation inhibitors such as cerulenin and 2-fluoro palmitic acid (2-FPA), as well as the enhancer palmostatin B and evaluated their impact on LD and the replication of human coronaviruses (hCoV-229E, hCoV-Oc43) and murine hepatitis virus (MHV-A59) at non-cytotoxic concentrations. While cerulenin and 2-FPA exhibited moderate inhibition of viral replication, 2 BP exhibited a much stronger suppressive effect on MHV-A59 replication, although they share similar inhibitory effects on palmitoylation. As expected, palmostatin B significantly enhanced viral replication, it failed to rescue the inhibitory effects of 2 BP, whereas it effectively counteracted the effects of cerulenin and 2-FPA. This suggests that the mechanism that 2 BP used to inhibit viral replication is beyond palmitoylation inhibition. Further investigations unveil that 2 BP uniquely depletes LDs, a phenomenon not exhibited by 2-FPA and cerulenin. Importantly, the depletion of LDs was closely associated with the inhibition of viral replication because the addition of oleic acid to 2 BP significantly rescued LD depletion and its inhibitory effects on MHV-A59. Our findings indicate that the inhibitory effects of 2 BP on viral replication primarily stem from LD disruption rather than palmitoylation inhibition. Intriguingly, fatty acid (FA) assays demonstrated that 2 BP reduces the FA level in mitochondria while concurrently increasing FA levels in the cytoplasm. These results highlight the crucial role of LDs in viral replication and uncover a novel biological activity of 2 BP. These insights contribute to the development of broad-spectrum antiviral strategies. IMPORTANCE: In our study, we conducted a comparative investigation into the antiviral effects of palmitoylation inhibitors including 2-bromopalmitate (2-BP), 2-fluoro palmitic acid (2-FPA), and cerulenin. Surprisingly, we discovered that 2-BP has superior inhibitory effects on viral replication compared to 2-FPA and cerulenin. However, their inhibitory effects on palmitoylation were the same. Intrigued by this finding, we delved deeper into the underlying mechanism of 2-BP's potent antiviral activity, and we unveiled a novel biological activity of 2-BP: depletion of lipid droplets (LDs). Importantly, we also highlighted the crucial role of LDs in viral replication. Our insights shed new light on the antiviral mechanism of LD depletion paving the way for the development of broad-spectrum antiviral strategies by targeting LDs.

Retinoic Acid-Mediated Inhibition of Mouse Coronavirus Replication Is Dependent on IRF3 and CaMKK.

The ongoing COVID-19 pandemic has revealed the shortfalls in our understanding of how to treat coronavirus infections. With almost 7 million case fatalities of COVID-19 globally, the catalog of FDA-approved antiviral therapeutics is limited compared to other medications, such as antibiotics. All-trans retinoic acid (RA), or activated vitamin A, has been studied as a potential therapeutic against coronavirus infection because of its antiviral properties. Due to its impact on different signaling pathways, RA's mechanism of action during coronavirus infection has not been thoroughly described. To determine RA's mechanism of action, we examined its effect against a mouse coronavirus, mouse hepatitis virus strain A59 (MHV). We demonstrated that RA significantly decreased viral titers in infected mouse L929 fibroblasts and RAW 264.7 macrophages. The reduced viral titers were associated with a corresponding decrease in MHV nucleocapsid protein expression. Using interferon regulatory factor 3 (IRF3) knockout RAW 264.7 cells, we demonstrated that RA-induced suppression of MHV required IRF3 activity. RNA-seq analysis of wildtype and IRF3 knockout RAW cells showed that RA upregulated calcium/calmodulin (CaM) signaling proteins, such as CaM kinase kinase 1 (CaMKK1). When treated with a CaMKK inhibitor, RA was unable to upregulate IRF activation during MHV infection. In conclusion, our results demonstrate that RA-induced protection against coronavirus infection depends on IRF3 and CaMKK.

Viral Inactivation with Emphasis on SARS-CoV-2 Using Physical and Chemical Disinfectants.

BACKGROUND: Recently, an outbreak of a novel human coronavirus SARS-CoV-2 has become a world health concern leading to severe respiratory tract infections in humans. Virus transmission occurs through person-to-person contact, respiratory droplets, and contaminated hands or surfaces. Accordingly, we aim at reviewing the literature on all information available about the persistence of coronaviruses, including human and animal coronaviruses, on inanimate surfaces and inactivation strategies with biocides employed for chemical and physical disinfection. METHOD: A comprehensive search was systematically conducted in main databases from 1998 to 2020 to identify various viral disinfectants associated with HCoV and methods for control and prevention of this newly emerged virus. RESULTS: The analysis of 62 studies shows that human coronaviruses such as severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome (MERS) coronavirus or endemic human coronaviruses (HCoV), canine coronavirus (CCV), transmissible gastroenteritis virus (TGEV), and mouse hepatitis virus (MHV) can be efficiently inactivated by physical and chemical disinfectants at different concentrations (70, 80, 85, and 95%) of 2-propanol (70 and 80%) in less than or equal to 60 s and 0.5% hydrogen peroxide or 0.1% sodium hypochlorite within 1 minute. Additionally, glutaraldehyde (0.5-2%), formaldehyde (0.7-1%), and povidone-iodine (0.1-0.75%) could readily inactivate coronaviruses. Moreover, dry heat at 56°C, ultraviolet light dose of 0.2 to 140 J/cm2, and gamma irradiation could effectively inactivate coronavirus. The WHO recommends the use of 0.1% sodium hypochlorite solution or an ethanol-based disinfectant with an ethanol concentration between 62% and 71%. CONCLUSION: The results of the present study can help researchers, policymakers, health decision makers, and people perceive and take the correct measures to control and prevent further transmission of COVID-19. Prevention and decontamination will be the main ways to stop the ongoing outbreak of COVID-19.

A Biosafety Level 2 Mouse Model for Studying Betacoronavirus-Induced Acute Lung Damage and Systemic Manifestations.

The emergence of life-threatening zoonotic diseases caused by betacoronaviruses, including the ongoing coronavirus disease 19 (COVID-19) pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus murine hepatitis coronavirus 3 (MHV-3) develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic, and a high virus burden could be detected in multiple organs along with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the proinflammatory cytokines interleukin 1 beta (IL-1ß), IL-6, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor (TNF), thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line with this, TNF blockage also diminished the infection-mediated release of proinflammatory cytokines and virus replication of human epithelial lung cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower-cost, biosafety level 2 (BSL2) in vivo platform for evaluating the respiratory and multiorgan involvement of betacoronavirus infections. IMPORTANCE Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race toward the characterization of SARS-CoV-2 infection in other animals (e.g., hamsters, cats, ferrets, bats, and monkeys), as well as adaptation of the mouse model, by modifying either the host or the virus. In the present study, we utilized a natural pathogen of mice, MHV, as a prototype to model betacoronavirus-induced acute lung injure and multiorgan involvement under biosafety level 2 conditions. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops severe disease, which includes acute lung damage and respiratory distress that precede systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Manipulation of the unfolded protein response: A pharmacological strategy against coronavirus infection.

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.

GABAA-Receptor Agonists Limit Pneumonitis and Death in Murine Coronavirus-Infected Mice.

There is an urgent need for new approaches to limit the severity of coronavirus infections. Many cells of the immune system express receptors for the neurotransmitter γ-aminobutyric acid (GABA), and GABA-receptor (GABA-R) agonists have anti-inflammatory effects. Lung epithelial cells also express GABA-Rs, and GABA-R modulators have been shown to limit acute lung injuries. There is currently, however, no information on whether GABA-R agonists might impact the course of a viral infection. Here, we assessed whether clinically applicable GABA-R agonists could be repurposed for the treatment of a lethal coronavirus (murine hepatitis virus 1, MHV-1) infection in mice. We found that oral GABA administration before, or after the appearance of symptoms, very effectively limited MHV-1-induced pneumonitis, severe illness, and death. GABA treatment also reduced viral load in the lungs, suggesting that GABA-Rs may provide a new druggable target to limit coronavirus replication. Treatment with the GABAA-R-specific agonist homotaurine, but not the GABAB-R-specific agonist baclofen, significantly reduced the severity of pneumonitis and death rates in MHV-1-infected mice, indicating that the therapeutic effects were mediated primarily through GABAA-Rs. Since GABA and homotaurine are safe for human consumption, they are promising candidates to help treat coronavirus infections.

Glycyrrhizic Acid Nanoparticles as Antiviral and Anti-inflammatory Agents for COVID-19 Treatment.

COVID-19 has been diffusely pandemic around the world, characterized by massive morbidity and mortality. One of the remarkable threats associated with mortality may be the uncontrolled inflammatory processes, which were induced by SARS-CoV-2 in infected patients. As there are no specific drugs, exploiting safe and effective treatment strategies is an instant requirement to dwindle viral damage and relieve extreme inflammation simultaneously. Here, highly biocompatible glycyrrhizic acid (GA) nanoparticles (GANPs) were synthesized based on GA. In vitro investigations revealed that GANPs inhibit the proliferation of the murine coronavirus MHV-A59 and reduce proinflammatory cytokine production caused by MHV-A59 or the N protein of SARS-CoV-2. In an MHV-A59-induced surrogate mouse model of COVID-19, GANPs specifically target areas with severe inflammation, such as the lungs, which appeared to improve the accumulation of GANPs and enhance the effectiveness of the treatment. Further, GANPs also exert antiviral and anti-inflammatory effects, relieving organ damage and conferring a significant survival advantage to infected mice. Such a novel therapeutic agent can be readily manufactured into feasible treatment for COVID-19.

A high-throughput drug screening strategy against coronaviruses.

The emergence and re-emergence of coronaviruses (CoV) continually cause circulating epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. The resultant disease, coronavirus disease 2019 (COVID-19), has rapidly developed into a worldwide pandemic, leading to severe health and economic burdens. Although the recently announced vaccines against COVID-19 has rekindled hope, there is still a major challenge to urgently meet the global need for rapid treatment of the pandemic. Given the urgency of the CoV outbreak, we propose a strategy to screen potential broad-spectrum drugs against CoV in a high-throughput manner, particularly against SARS-CoV-2. Since the essential functional domains of CoV are extensively homologous, the availability of two types of mild CoV, HCoV-OC43 and MHV, should provide a valuable tool for the rapid identification of promising drugs against CoV without the drawbacks of level three biological confinements. The luciferase reporter gene is introduced into HCoV-OC43 and MHV to indicate viral activity, and hence the antiviral efficiency of screened drugs can be quantified by luciferase activity. Compounds with antiviral activity against both HCoV-OC43 and MHV are further evaluated in SARS-CoV-2 after structural optimizations. This system allows large-scale compounds to be screened to search for broad-spectrum drugs against CoV in a high-throughput manner, providing potential alternatives for clinical management of SARS-CoV-2 or other CoV.

Translation of Mycobacterium Survival Strategy to Develop a Lipo-peptide based Fusion Inhibitor*.

The entry of enveloped virus requires the fusion of viral and host cell membranes. An effective fusion inhibitor aiming at impeding such membrane fusion may emerge as a broad-spectrum antiviral agent against a wide range of viral infections. Mycobacterium survives inside the phagosome by inhibiting phagosome-lysosome fusion with the help of a coat protein coronin 1. Structural analysis of coronin 1 and other WD40-repeat protein suggest that the trp-asp (WD) sequence is placed at distorted ß-meander motif (more exposed) in coronin 1. The unique structural feature of coronin 1 was explored to identify a simple lipo-peptide sequence (myr-WD), which effectively inhibits membrane fusion by modulating the interfacial order, water penetration, and surface potential. The mycobacterium inspired lipo-dipeptide was successfully tested to combat type 1 influenza virus (H1N1) and murine coronavirus infections as a potential broad-spectrum antiviral agent.

Microglia depletion exacerbates demyelination and impairs remyelination in a neurotropic coronavirus infection.

Microglia are considered both pathogenic and protective during recovery from demyelination, but their precise role remains ill defined. Here, using an inhibitor of colony stimulating factor 1 receptor (CSF1R), PLX5622, and mice infected with a neurotropic coronavirus (mouse hepatitis virus [MHV], strain JHMV), we show that depletion of microglia during the time of JHMV clearance resulted in impaired myelin repair and prolonged clinical disease without affecting the kinetics of virus clearance. Microglia were required only during the early stages of remyelination. Notably, large deposits of extracellular vesiculated myelin and cellular debris were detected in the spinal cords of PLX5622-treated and not control mice, which correlated with decreased numbers of oligodendrocytes in demyelinating lesions in drug-treated mice. Furthermore, gene expression analyses demonstrated differential expression of genes involved in myelin debris clearance, lipid and cholesterol recycling, and promotion of oligodendrocyte function. The results also demonstrate that microglial functions affected by depletion could not be compensated by infiltrating macrophages. Together, these results demonstrate that microglia play key roles in debris clearance and in the initiation of remyelination following infection with a neurotropic coronavirus but are not necessary during later stages of remyelination.

The autoimmune response elicited by mouse hepatitis virus (MHV-A59) infection is modulated by liver tryptophan-2,3-dioxygenase (TDO).

In a previous work we demonstrated that inhibition of mouse indoleamine 2,3-dioxygenase (IDO) by methyltryptophan (MT) exacerbated the pathological actions of mouse hepatitis virus (MHV-A59) infection, suggesting that tryptophan (TRP) catabolism was involved in viral effects. Since there is a second enzyme that dioxygenates TRP, tryptophan-2, 3-dioxygenase (TDO), which is mainly located in liver, we decided to study its role in our model of MHV-infection. Results showed that in vivo TDO inhibition by LM10, a derivative of 3-(2-(pyridyl) ethenyl) indole, resulted in a decrease of anti- MHV Ab titers induced by the virus infection. Besides, a reduction of some alarmin release, i.e, uric acid and high-mobility group box1 protein (HMGB1), was observed. Accordingly, since alarmin liberation was related to the expression of autoantibodies (autoAb) to fumarylacetoacetate hydrolase (FAH), these autoAb also diminished. Moreover, PCR results indicated that TDO inhibition did not abolish viral replication. Furthermore, histological liver examination did not reveal strong pathologies, whereas mouse survival was hundred percent in control as well as in MHV-infected mice treated with LM10. Data presented in this work indicate that in spite of the various TDO actions already described, specific TDO blockage could also restrain some MHV actions, mainly suppressing autoimmune reactions. Such results should prompt further experiments with various viruses to confirm the possible use of a TDO inhibitor such as LM-10 to treat either viral infections or even autoimmune diseases triggered by a viral infection.

Small-Molecule Antiviral ß-d-N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance.

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). ß-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, ß-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.

The crystal structure of main protease from mouse hepatitis virus A59 in complex with an inhibitor.

Mouse hepatitis virus A59 (MHV-A59) is a representative member of the genus betacoronavirus within the subfamily Coronavirinae, which infects the liver, brain and respiratory tract. Through different inoculation routes, MHV-A59 can provide animal models for encephalitis, hepatitis and pneumonia to explore viral life machinery and virus-host interactions. In viral replication, non-structural protein 5 (Nsp5), also termed main protease (Mpro), plays a dominant role in processing coronavirus-encoded polyproteins and is thus recognized as an ideal target of anti-coronavirus agents. However, no structure of the MHV-A59 Mpro has been reported, and molecular exploration of the catalysis mechanism remains hindered. Here, we solved the crystal structure of the MHV-A59 Mpro complexed with a Michael acceptor-based inhibitor, N3. Structural analysis revealed that the Cß of the vinyl group of N3 covalently bound to C145 of the catalytic dyad of Mpro, which irreversibly inactivated cysteine protease activity. The lactam ring of the P1 side chain and the isobutyl group of the P2 side chain, which mimic the conserved residues at the same positions of the substrate, fit well into the S1 and S2 pockets. Through a comparative study with Mpro of other coronaviruses, we observed that the substrate-recognition pocket and enzyme inhibitory mechanism is highly conservative. Altogether, our study provided structural features of MHV-A59 Mpro and indicated that a Michael acceptor inhibitor is an ideal scaffold for antiviral drugs.

Murine Hepatitis Virus nsp14 Exoribonuclease Activity Is Required for Resistance to Innate Immunity.

Coronaviruses (CoVs) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode several innate immune antagonists that counteract the host innate immune response to facilitate efficient viral replication. CoV nonstructural protein 14 (nsp14) encodes 3'-to-5' exoribonuclease activity (ExoN), which performs a proofreading function and is required for high-fidelity replication. Outside of the order Nidovirales, arenaviruses are the only RNA viruses that encode an ExoN, which functions to degrade double-stranded RNA (dsRNA) replication intermediates. In this study, we tested the hypothesis that CoV ExoN also functions to antagonize the innate immune response. We demonstrate that viruses lacking ExoN activity [ExoN(-)] are sensitive to cellular pretreatment with interferon beta (IFN-ß) in a dose-dependent manner. In addition, ExoN(-) virus replication was attenuated in wild-type bone marrow-derived macrophages (BMMs) and partially restored in interferon alpha/beta receptor-deficient (IFNAR-/-) BMMs. ExoN(-) virus replication did not result in IFN-ß gene expression, and in the presence of an IFN-ß-mediated antiviral state, ExoN(-) viral RNA levels were not substantially reduced relative to those of untreated samples. However, ExoN(-) virus generated from IFN-ß-pretreated cells had reduced specific infectivity and decreased relative fitness, suggesting that ExoN(-) virus generated during an antiviral state is less viable to establish a subsequent infection. Overall, our data suggest murine hepatitis virus (MHV) ExoN activity is required for resistance to the innate immune response, and antiviral mechanisms affecting the viral RNA sequence and/or an RNA modification act on viruses lacking ExoN activity.IMPORTANCE CoVs encode multiple antagonists that prevent or disrupt an efficient innate immune response. Additionally, no specific antiviral therapies or vaccines currently exist for human CoV infections. Therefore, the study of CoV innate immune antagonists is essential for understanding how CoVs overcome host defenses and to maximize potential therapeutic interventions. Here, we sought to determine the contributions of nsp14 ExoN activity in the induction of and resistance to the innate immune response. We show that viruses lacking nsp14 ExoN activity are more sensitive than wild-type MHV to restriction by exogenous IFN-ß and that viruses produced in the presence of an antiviral state are less capable of establishing a subsequent viral infection. Our results support the hypothesis that murine hepatitis virus ExoN activity is required for resistance to the innate immune response.

Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens.

UNLABELLED: Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(-)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex. IMPORTANCE: RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity. Responsibility for replication fidelity in RNA viruses has been attributed to the RNA-dependent RNA polymerases, with mutations in RdRps for multiple RNA viruses shown to alter fidelity and attenuate virus replication and virulence. Coronaviruses (CoVs) are the only known RNA viruses to encode a proofreading exonuclease (nsp14-ExoN), as well as other replicase proteins involved in regulation of fidelity. This report shows that the CoV RdRp (nsp12) likely functions in replication fidelity; that residue determinants of CoV RdRp nucleotide selectivity map to similar structural regions of other, unrelated RNA viral polymerases; and that for CoVs, the proofreading activity of the nsp14-ExoN is epistatic to the function of the RdRp in fidelity.

The NLRP3 Inflammasome and IL-1ß Accelerate Immunologically Mediated Pathology in Experimental Viral Fulminant Hepatitis.

Viral fulminant hepatitis (FH) is a severe disease with high mortality resulting from excessive inflammation in the infected liver. Clinical interventions have been inefficient due to the lack of knowledge for inflammatory pathogenesis in the virus-infected liver. We show that wild-type mice infected with murine hepatitis virus strain-3 (MHV-3), a model for viral FH, manifest with severe disease and high mortality in association with a significant elevation in IL-1ß expression in the serum and liver. Whereas, the viral infection in IL-1ß receptor-I deficient (IL-1R1-/-) or IL-1R antagonist (IL-1Ra) treated mice, show reductions in virus replication, disease progress and mortality. IL-1R1 deficiency appears to debilitate the virus-induced fibrinogen-like protein-2 (FGL2) production in macrophages and CD45+Gr-1high neutrophil infiltration in the liver. The quick release of reactive oxygen species (ROS) by the infected macrophages suggests a plausible viral initiation of NLRP3 inflammasome activation. Further experiments show that mice deficient of p47phox, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit that controls acute ROS production, present with reductions in NLRP3 inflammasome activation and subsequent IL-1ß secretion during viral infection, which appears to be responsible for acquiring resilience to viral FH. Moreover, viral infected animals in deficiencies of NLRP3 and Caspase-1, two essential components of the inflammasome complex, also have reduced IL-1ß induction along with ameliorated hepatitis. Our results demonstrate that the ROS/NLRP3/IL-1ß axis institutes an essential signaling pathway, which is over activated and directly causes the severe liver disease during viral infection, which sheds light on development of efficient treatments for human viral FH and other severe inflammatory diseases.

Serotonin Receptor Agonist 5-Nonyloxytryptamine Alters the Kinetics of Reovirus Cell Entry.

UNLABELLED: Mammalian orthoreoviruses (reoviruses) are nonenveloped double-stranded RNA viruses that infect most mammalian species, including humans. Reovirus binds to cell surface glycans, junctional adhesion molecule A (JAM-A), and the Nogo-1 receptor (depending on the cell type) and enters cells by receptor-mediated endocytosis. Within the endocytic compartment, reovirus undergoes stepwise disassembly, which is followed by release of the transcriptionally active viral core into the cytoplasm. In a small-molecule screen to identify host mediators of reovirus infection, we found that treatment of cells with 5-nonyloxytryptamine (5-NT), a prototype serotonin receptor agonist, diminished reovirus cytotoxicity. 5-NT also blocked reovirus infection. In contrast, treatment of cells with methiothepin mesylate, a serotonin antagonist, enhanced infection by reovirus. 5-NT did not alter cell surface expression of JAM-A or attachment of reovirus to cells. However, 5-NT altered the distribution of early endosomes with a concomitant impairment of reovirus transit to late endosomes and a delay in reovirus disassembly. Consistent with an inhibition of viral disassembly, 5-NT treatment did not alter infection by in vitro-generated infectious subvirion particles, which bind to JAM-A but bypass a requirement for proteolytic uncoating in endosomes to infect cells. We also found that treatment of cells with 5-NT decreased the infectivity of alphavirus chikungunya virus and coronavirus mouse hepatitis virus. These data suggest that serotonin receptor signaling influences cellular activities that regulate entry of diverse virus families and provides a new, potentially broad-spectrum target for antiviral drug development. IMPORTANCE: Identification of well-characterized small molecules that modulate viral infection can accelerate development of antiviral therapeutics while also providing new tools to increase our understanding of the cellular processes that underlie virus-mediated cell injury. We conducted a small-molecule screen to identify compounds capable of inhibiting cytotoxicity caused by reovirus, a prototype double-stranded RNA virus. We found that 5-nonyloxytryptamine (5-NT) impairs reovirus infection by altering viral transport during cell entry. Remarkably, 5-NT also inhibits infection by an alphavirus and a coronavirus. The antiviral properties of 5-NT suggest that serotonin receptor signaling is an important regulator of infection by diverse virus families and illuminate a potential new drug target.

A screen of the NIH Clinical Collection small molecule library identifies potential anti-coronavirus drugs.

With the recent emergence of Middle East Respiratory Syndrome coronavirus in humans and the outbreak of devastating porcine epidemic diarrhea coronavirus in swine, therapeutic intervention is urgently needed. However, anti-coronavirus drugs currently are not available. In an effort to assist rapid development of anti-coronavirus drugs, here we screened the NIH Clinical Collection in cell culture using a luciferase reporter-expressing recombinant murine coronavirus. Of the 727 compounds screened, 84 were found to have a significant anti-coronavirus effect. Further experiments revealed that 51 compounds blocked virus entry while 19 others inhibited viral replication. Additional validation studies with the top 3 inhibitors (hexachlorophene, nitazoxanide and homoharringtonine) demonstrated robust anti-coronavirus activities (a reduction of 6 to 8log10 in virus titer) with an IC50 ranging from 11nM to 1.2µM. Furthermore, homoharringtonine and hexachlorophene exhibited broad antiviral activity against diverse species of human and animal coronaviruses. Since the NIH Clinical Collection consists of compounds that have already been through clinical trials, these small molecule inhibitors have a great potential for rapid development as anti-coronavirus drugs.