Christin Müller

Influenza Virus-Induced Caspase-Dependent Enlargement of Nuclear Pores Promotes Nuclear Export of Viral Ribonucleoprotein Complexes

ABSTRACT 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. IMPORTANCE In 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.

The natural compound silvestrol is a potent inhibitor of Ebola virus replication.

&NA; The DEAD‐box RNA helicase eIF4A, which is part of the heterotrimeric translation initiation complex in eukaryotes, is an important novel drug target in cancer research because its helicase activity is required to unwind extended and highly structured 5′‐UTRs of several proto‐oncogenes. Silvestrol, a natural compound isolated from the plant Aglaia foveolata, is a highly efficient, non‐toxic and specific inhibitor of eIF4A. Importantly, 5′‐capped viral mRNAs often contain structured 5′‐UTRs as well, which may suggest a dependence on eIF4A for their translation by the host protein synthesis machinery. In view of the recent Ebola virus (EBOV) outbreak in West Africa, the identification of potent antiviral compounds is urgently required. Since Ebola mRNAs are 5′‐capped and harbor RNA secondary structures in their extended 5′‐UTRs, we initiated a BSL4 study to analyze silvestrol in EBOV‐infected Huh‐7 cells and in primary human macrophages for its antiviral activity. We observed that silvestrol inhibits EBOV infection at low nanomolar concentrations, as inferred from large reductions of viral titers. This correlated with an almost complete disappearance of EBOV proteins, comparable in effect to the translational shutdown of expression of the proto‐oncoprotein PIM1, a cellular kinase known to be affected by silvestrol. Effective silvestrol concentrations were non‐toxic in the tested cell systems. Thus, silvestrol appears to be a promising first‐line drug for the treatment of acute EBOV and possibly other viral infections. Graphical abstract: Figure. No caption available. HighlightsThe rocaglate silvestrol, a specific eIF4A helicase inhibitor, is a potent antiviral compound to treat Ebola infections.Silvestrol inhibits EBOV in human primary macrophages at low nanomolar concentrations.Silvestrol is not toxic in vitro and in vivo at concentrations of effective antiviral activity.Inhibition of cap‐dependent translation initiation is a potent antiviral strategy.

Broad-spectrum antiviral activity of the eIF4A inhibitor silvestrol against corona- and picornaviruses

Abstract 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. EC50 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 EC50 value of 20 nM silvestrol was determined in MRC‐5 cells. The higher EC50 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. Graphical abstract Figure. No caption available. HighlightsThe eIF4A inhibitor silvestrol is a potent antiviral compound that inhibits the replication of coronaviruses.Silvestrol is also effective against picornaviruses with an eIF4A‐dependent Type 1 IRES element.In primary cells silvestrol has potent antiviral activity and low toxicity.Targeting the host factor eIF4A is a promising broad‐spectrum antiviral strategy.

Inhibition of Cytosolic Phospholipase A2α Impairs an Early Step of Coronavirus Replication in Cell Culture

ABSTRACT 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 A2α (cPLA2α) in coronavirus replication using a low-molecular-weight nonpeptidic inhibitor, pyrrolidine-2 (Py-2). The inhibition of cPLA2α activity, which produces lysophospholipids (LPLs) by cleaving at the sn-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 cPLA2α inhibitor Py-2. Py-2 also displayed antiviral activities against other viruses representing the Coronaviridae and Togaviridae families, while members of the Picornaviridae were not affected. Taken together, the study provides evidence that cPLA2α 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. IMPORTANCE 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 sn-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 cPLA2α inhibitor.

Ribonuclease from Bacillus Acts as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses

Bacillus pumilus ribonuclease (binase) was shown to be a promising antiviral agent in animal models and cell cultures. However, the mode of its antiviral action remains unknown. To assess the binase effect on intracellular viral RNA we have selected single stranded negative- and positive-sense RNA viruses, influenza virus, and rhinovirus, respectively, which annually cause respiratory illnesses and are characterized by high contagious nature, mutation rate, and antigen variability. We have shown that binase exerts an antiviral effect on both viruses at the same concentration, which does not alter the spectrum of A549 cellular proteins and expression of housekeeping genes. The titers of influenza A (H1N1pdm) virus and human rhinovirus serotype 1A were reduced by 40% and 65%, respectively. A preincubation of influenza virus with binase before infection significantly reduced viral titer after single-cycle replication of the virus. Using influenza A virus mini genome system we showed that binase reduced GFP reporter signaling indicating a binase action on the expression of viral mRNA. Binase reduced the level of H1N1pdm viral NP mRNA accumulation in A549 cells by 20%. Since the viral mRNA is a possible target for binase this agent could be potentially applied in the antiviral therapy against both negative- and positive-sense RNA viruses.

Antiviral activity of K22 against members of the order Nidovirales

Abstract Recently, a novel antiviral compound (K22) that inhibits replication of a broad range of animal and human coronaviruses was reported to interfere with viral RNA synthesis by impairing double-membrane vesicle (DMV) formation (Lundin et al., 2014). Here we assessed potential antiviral activities of K22 against a range of viruses representing two (sub)families of the order Nidovirales, the Arteriviridae (porcine reproductive and respiratory syndrome virus [PRRSV], equine arteritis virus [EAV] and simian hemorrhagic fever virus [SHFV]), and the Torovirinae (equine torovirus [EToV] and White Bream virus [WBV]). Possible effects of K22 on nidovirus replication were studied in suitable cell lines. K22 concentrations significantly decreasing infectious titres of the viruses included in this study ranged from 25 to 50 μM. Reduction of double-stranded RNA intermediates of viral replication in nidovirus-infected cells treated with K22 confirmed the anti-viral potential of K22. Collectively, the data show that K22 has antiviral activity against diverse lineages of nidoviruses, suggesting that the inhibitor targets a critical and conserved step during nidovirus replication.

Bacterial ribonuclease binase exerts an intra-cellular anti-viral mode of action targeting viral RNAs in influenza a virus-infected MDCK-II cells

BackgroundInfluenza is a severe contagious disease especially in children, elderly and immunocompromised patients. Beside vaccination, the discovery of new anti-viral agents represents an important strategy to encounter seasonal and pandemic influenza A virus (IAV) strains. The bacterial extra-cellular ribonuclease binase is a well-studied RNase from Bacillus pumilus. Treatment with binase was shown to improve survival of laboratory animals infected with different RNA viruses. Although binase reduced IAV titer in vitro and in vivo, the mode of action (MOA) of binase against IAV at the molecular level has yet not been studied in depth and remains elusive.MethodsTo analyze whether binase impairs virus replication by direct interaction with the viral particle we applied a hemagglutination inhibition assay and monitored the integrity of the viral RNA within the virus particle by RT-PCR. Furthermore, we used Western blot and confocal microscopy analysis to study whether binase can internalize into MDCK-II cells. By primer extension we examined the effect of binase on the integrity of viral RNAs within the cells and using a mini-genome system we explored the effect of binase on the viral expression.ResultsWe show that (i) binase does not to attack IAV particle-protected viral RNA, (ii) internalized binase could be detected within the cytosol of MDCK-II cells and that (iii) binase impairs IAV replication by specifically degrading viral RNA species within the infected MDCK-II cells without obvious effect on cellular mRNAs.ConclusionOur data provide novel evidence suggesting that binase is a potential anti-viral agent with specific intra-cellular MOA.

Verapamil has Antiviral Activities that Target Different Steps of theInfluenza Virus Replication Cycle

Safe and efficacious therapeutic options for infections caused by highly pathogenic influenza viruses (IV) remain limited. Viruses resistant to approved antiviral drugs are increasingly emerging and, in some cases, existing vaccines are insufficient to prevent seasonal epidemics or pandemics caused by these viruses. As a consequence, alternative antiviral strategies that, for example, target cellular factors or mechanisms essential for IV propagation have attracted increasing interest over the past years. Here, we characterized antiviral effects of the calcium channel blocker Verapamil on different steps of the IV replication cycle. Our data suggest that Verapamil (i) efficiently reduces influenza A and B virus titres in cell culture at non-toxic concentrations, (ii) does not influence virus entry, (iii) impairs the replication and transcription activity of the viral polymerase, (iv) decreases viral protein production, (v) reduces virus-induced NF-κB activation and (vi) does not induce the emergence of resistant IV variants, resulting in strongly reduced infectious particle formation.