Coronaviruses (CoV) and picornaviruses are plus-strand RNA viruses that use 5' cap-dependent and cap-independent strategies, respectively, for viral mRNA translation initiation. Here, we analyzed the effects of the plant compound silvestrol, a specific inhibitor of the DEAD-box RNA helicase eIF4A, on viral translation using a dual luciferase assay and virus-infected primary cells. Silvestrol was recently shown to have potent antiviral activity in Ebola virus-infected human macrophages. We found that silvestrol is also a potent inhibitor of cap-dependent viral mRNA translation in CoV-infected human embryonic lung fibroblast (MRC-5) cells. EC values of 1.3 nM and 3 nM silvestrol were determined for MERS-CoV and HCoV-229E, respectively. For the highly pathogenic MERS-CoV, the potent antiviral activities of silvestrol were also confirmed using peripheral blood mononuclear cells (PBMCs) as a second type of human primary cells. Silvestrol strongly inhibits the expression of CoV structural and nonstructural proteins (N, nsp8) and the formation of viral replication/transcription complexes. Furthermore, potential antiviral effects against human rhinovirus (HRV) A1 and poliovirus type 1 (PV), representing different species in the genus Enterovirus (family Picornaviridae), were investigated. The two viruses employ an internal ribosomal entry site (IRES)-mediated translation initiation mechanism. For PV, which is known to require the activity of eIF4A, an EC value of 20 nM silvestrol was determined in MRC-5 cells. The higher EC value of 100 nM measured for HRV A1 indicates a less critical role of eIF4A activity in HRV A1 IRES-mediated translation initiation. Taken together, the data reveal a broad-spectrum antiviral activity of silvestrol in infected primary cells by inhibiting eIF4A-dependent viral mRNA translation.

Coronavirus replication is associated with intracellular membrane rearrangements in infected cells, resulting in the formation of double-membrane vesicles (DMVs) and other membranous structures that are referred to as replicative organelles (ROs). The latter provide a structural scaffold for viral replication/transcription complexes (RTCs) and help to sequester RTC components from recognition by cellular factors involved in antiviral host responses. There is increasing evidence that plus-strand RNA (+RNA) virus replication, including RO formation and virion morphogenesis, affects cellular lipid metabolism and critically depends on enzymes involved in lipid synthesis and processing. Here, we investigated the role of cytosolic phospholipase Aα (cPLAα) in coronavirus replication using a low-molecular-weight nonpeptidic inhibitor, pyrrolidine-2 (Py-2). The inhibition of cPLAα activity, which produces lysophospholipids (LPLs) by cleaving at the -2 position of phospholipids, had profound effects on viral RNA and protein accumulation in human coronavirus 229E-infected Huh-7 cells. Transmission electron microscopy revealed that DMV formation in infected cells was significantly reduced in the presence of the inhibitor. Furthermore, we found that (i)viral RTCs colocalized with LPL-containing membranes, (ii) cellular LPL concentrations were increased in coronavirus-infected cells, and (iii) this increase was diminished in the presence of the cPLAα inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the and families, while members of the were not affected. Taken together, the study provides evidence that cPLAα activity is critically involved in the replication of various +RNA virus families and may thus represent a candidate target for broad-spectrum antiviral drug development. Examples of highly conserved RNA virus proteins that qualify as drug targets for broad-spectrum antivirals remain scarce, resulting in increased efforts to identify and specifically inhibit cellular functions that are essential for the replication of RNA viruses belonging to different genera and families. The present study supports and extends previous conclusions that enzymes involved in cellular lipid metabolism may be tractable targets for broad-spectrum antivirals. We obtained evidence to show that a cellular phospholipase, cPLA2α, which releases fatty acid from the -2 position of membrane-associated glycerophospholipids, is critically involved in coronavirus replication, most likely by producing lysophospholipids that are required to form the specialized membrane compartments in which viral RNA synthesis takes place. The importance of this enzyme in coronavirus replication and DMV formation is supported by several lines of evidence, including confocal and electron microscopy, viral replication, and lipidomics studies of coronavirus-infected cells treated with a highly specific cPLAα inhibitor.

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Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.

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Rocaglates, a class of natural compounds isolated from plants of the genus Aglaia, are potent inhibitors of translation initiation. They are proposed to form stacking interactions with polypurine sequences in the 5′untranslated region (UTR) of selected mRNAs, thereby clamping the RNA substrate onto eIF4A and causing inhibition of the translation initiation complex. Since virus replication relies on the host translation machinery, it is not surprising that the rocaglate Silvestrol has broad-spectrum antiviral activity. Unfortunately, synthesis of Silvestrol is sophisticated and time-consuming, thus hampering the prospects for further antiviral drug development. Here, we present the less complex structured synthetic rocaglate CR-31-B (−) as a novel compound with potent broad-spectrum antiviral activity in primary cells and in an ex vivo bronchial epithelial cell system. CR-31-B (−) inhibited the replication of corona-, Zika-, Lassa-, Crimean Congo hemorrhagic fever viruses and, to a lesser extent, hepatitis E virus (HEV) at non-cytotoxic low nanomolar concentrations. Since HEV has a polypurine-free 5′-UTR that folds into a stable hairpin structure, we hypothesized that RNA clamping by Silvestrol and its derivatives may also occur in a polypurine-independent but structure-dependent manner. Interestingly, the HEV 5′-UTR conferred sensitivity towards Silvestrol but not to CR-31-B (−). However, if an exposed polypurine stretch was introduced into the HEV 5′-UTR, CR-31-B (−) became an active inhibitor comparable to Silvestrol. Moreover, thermodynamic destabilization of the HEV 5′-UTR led to reduced translational inhibition by Silvestrol, suggesting differences between rocaglates in their mode of action, most probably by engaging Silvestrol's additional dioxane moiety.

Influenza A viruses (IAV) replicate their segmented RNA genome in the nucleus of infected cells and utilize caspase-dependent nucleocytoplasmic export mechanisms to transport newly formed ribonucleoprotein complexes (RNPs) to the site of infectious virion release at the plasma membrane. In this study, we obtained evidence that apoptotic caspase activation in IAV-infected cells is associated with the degradation of the nucleoporin Nup153, an integral subunit of the nuclear pore complex. Transmission electron microscopy studies revealed a distinct enlargement of nuclear pores in IAV-infected cells. Transient expression and subcellular accumulation studies of multimeric marker proteins in virus-infected cells provided additional evidence for increased nuclear pore diameters facilitating the translocation of large protein complexes across the nuclear membrane. Furthermore, caspase 3/7 inhibition data obtained in this study suggest that active, Crm1-dependent IAV RNP export mechanisms are increasingly complemented by passive, caspase-induced export mechanisms at later stages of infection. IMPORTANCEIn contrast to the process seen with most other RNA viruses, influenza virus genome replication occurs in the nucleus (rather than the cytoplasm) of infected cells. Therefore, completion of the viral replication cycle critically depends on intracellular transport mechanisms that ensure the translocation of viral ribonucleoprotein (RNP) complexes across the nuclear membrane. Here, we demonstrate that virus-induced cellular caspase activities cause a widening of nuclear pores, thereby facilitating nucleocytoplasmic translocation processes and, possibly, promoting nuclear export of newly synthesized RNPs. These passive transport mechanisms are suggested to complement Crm1-dependent RNP export mechanisms known to occur at early stages of the replication cycle and may contribute to highly efficient production of infectious virus progeny at late stages of the viral replication cycle. The report provides an intriguing example of how influenza virus exploits cellular structures and regulatory pathways, including intracellular transport mechanisms, to complete its replication cycle and maximize the production of infectious virus progeny.

Cellular sensing of bacterial RNA is increasingly recognized as a determinant of host-pathogen interactions. The intracellular pathogen Listeria monocytogenes induces high levels of type I interferons (alpha/beta interferons [IFN-α/β]) to create a growth-permissive microenvironment during infection. We previously demonstrated that RNAs secreted by L. monocytogenes (comprising the secRNome) are potent inducers of IFN-β. We determined the composition and diversity of the members of the secRNome and found that they are uniquely enriched for noncoding small RNAs (sRNAs). Testing of individual sRNAs for their ability to induce IFN revealed several sRNAs with this property. We examined ril32, an intracellularly expressed sRNA that is highly conserved for the species L. monocytogenes and that was the most potent inducer of IFN-β expression of all the sRNAs tested in this study, in more detail. The rli32-induced IFN-β response is RIG-I (retinoic acid inducible gene I) dependent, and cells primed with rli32 inhibit influenza virus replication. We determined the rli32 motif required for IFN induction. rli32 overproduction promotes intracellular bacterial growth, and a mutant lacking rli32 is restricted for intracellular growth in macrophages. rli32-overproducing bacteria are resistant to H2O2 and exhibit both increased catalase activity and changes in the cell envelope. Comparative transcriptome sequencing (RNA-Seq) analysis indicated that ril32 regulates expression of the lhrC locus, previously shown to be involved in cell envelope stress. Inhibition of IFN-β signaling by ruxolitinib reduced rli32-dependent intracellular bacterial growth, indicating a link between induction of the interferon system and bacterial physiology. rli32 is, to the best of our knowledge, the first secreted individual bacterial sRNA known to trigger the induction of the type I IFN response. IMPORTANCE Interferons are potent and broadly acting cytokines that stimulate cellular responses to nucleic acids of unusual structures or locations. While protective when induced following viral infections, the induction of interferons is detrimental to the host during L. monocytogenes infection. Here, we identify specific sRNAs, secreted by the bacterium, with the capacity to induce type I IFN. Further analysis of the most potent sRNA, rli32, links the ability to induce RIG-I-dependent induction of the type I IFN response to the intracellular growth properties of the bacterium. Our findings emphasize the significance of released RNA for Listeria infection and shed light on a compartmental strategy used by an intracellular pathogen to modulate host responses to its advantage.