Filoviruses are highly infectious, and no FDA-approved drug therapy for filovirus infection is available. Most work to find a treatment has involved only a few strains of Ebola virus and testing of relatively small drug libraries or compounds that have shown efficacy against other virus types. Here we report the findings of a high-throughput screening of 319,855 small molecules from the Molecular Libraries Small Molecule Repository library for their activities against Marburg virus and Ebola virus. Nine of the most potent, novel compounds that blocked infection by both viruses were analyzed in detail for their mechanisms of action. The compounds inhibited known key steps in the Ebola virus infection mechanism by blocking either cell surface attachment, macropinocytosis-mediated uptake, or endosomal trafficking. To date, very few specific inhibitors of macropinocytosis have been reported. The 2 novel macropinocytosis inhibitors are more potent inhibitors of Ebola virus infection and less toxic than ethylisopropylamiloride, one commonly accepted macropinocytosis inhibitor. Each compound blocked infection of primary human macrophages, indicating their potential to be developed as new antifiloviral therapies.

A systematic screen of FDA-approved drugs was performed to identify compounds with in vitro antiviral activities against Ebola virus (EBOV). Compounds active (>50% viral inhibition and <30% cellular toxicity) at a single concentration were tested in dose-response assays to quantitate the antiviral activities in replication and viral entry assays as well as cytotoxicity in the Vero cell line used to conduct these assays. On the basis of the approved human dosing, toxicity/tolerability, and pharmacokinetic data, seven of these in vitro hits from different pharmacological classes (chloroquine (CQ), amiodarone, prochlorperazine, benztropine, azithromycin, chlortetracycline, and clomiphene) were evaluated for their in vivo efficacy at a single dose and were administered via either intraperitoneal (ip) or oral route. Initially, azithromycin (100 mg/kg, twice daily, ip), CQ (90 mg/kg, twice daily, ip), and amiodarone (60 mg/kg, twice daily, ip) demonstrated significant increases in survival in the mouse model. After repeat evaluation, only CQ was found to reproducibly give significant efficacy in the mouse model with this dosing regimen. Azithromycin and CQ were also tested in a guinea pig model of EBOV infection over a range of doses, but none of the doses increased survival, and drug-related toxicity was observed at lower doses than in the mouse. These results show the benefits and specific challenges associated with drug repurposing and highlight the need for careful evaluation of approved drugs as rapidly deployable countermeasures against future pandemics.

Ebola virus causes sporadic outbreaks of lethal hemorrhagic fever in humans, but there is no currently approved therapy. Cells take up Ebola virus by macropinocytosis, followed by trafficking through endosomal vesicles. However, few factors controlling endosomal virus movement are known. Here we find that Ebola virus entry into host cells requires the endosomal calcium channels called two-pore channels (TPCs). Disrupting TPC function by gene knockout, small interfering RNAs, or small-molecule inhibitors halted virus trafficking and prevented infection. Tetrandrine, the most potent small molecule that we tested, inhibited infection of human macrophages, the primary target of Ebola virus in vivo, and also showed therapeutic efficacy in mice. Therefore, TPC proteins play a key role in Ebola virus infection and may be effective targets for antiviral therapy.

Background: The rapid development of effective medical countermeasures against potential biological threat agents is vital. Repurposing existing drugs that may have unanticipated activities as potential countermeasures is one way to meet this important goal, since currently approved drugs already have well-established safety and pharmacokinetic profiles in patients, as well as manufacturing and distribution networks. Therefore, approved drugs could rapidly be made available for a new indication in an emergency.Methodology/Principal Findings: A large systematic effort to determine whether existing drugs can be used against high containment bacterial and viral pathogens is described. We assembled and screened 1012 FDA-approved drugs for off-label broad-spectrum efficacy against Bacillus anthracis; Francisella tularensis; Coxiella burnetii; and Ebola, Marburg, and Lassa fever viruses using in vitro cell culture assays. We found a variety of hits against two or more of these biological threat pathogens, which were validated in secondary assays. As expected, antibiotic compounds were highly active against bacterial agents, but we did not identify any non-antibiotic compounds with broad-spectrum antibacterial activity. Lomefloxacin and erythromycin were found to be the most potent compounds in vivo protecting mice against Bacillus anthracis challenge. While multiple virus-specific inhibitors were identified, the most noteworthy antiviral compound identified was chloroquine, which disrupted entry and replication of two or more viruses in vitro and protected mice against Ebola virus challenge in vivo.Conclusions/Significance: The feasibility of repurposing existing drugs to face novel threats is demonstrated and this represents the first effort to apply this approach to high containment bacteria and viruses.

Acid sphingomyelinase (ASMase) converts the lipid sphingomyelin (SM) to phosphocholine and ceramide and has optimum activity at acidic pH. Normally, ASMase is located in lysosomes and endosomes, but membrane damage or the interaction with some bacterial and viral pathogens can trigger its recruitment to the plasma membrane. Rhinovirus and measles viruses each require ASMase activity during early stages of infection. Both sphingomyelin and ceramide are important components of lipid rafts and are potent signaling molecules. Each plays roles in mediating macropinocytosis, which has been shown to be important for ebolavirus (EBOV) infection. Here, we investigated the role of ASMase and its substrate, SM, in EBOV infection. The work was performed at biosafety level 4 with wild-type virus with specificity and mechanistic analysis performed using virus pseudotypes and virus-like particles. We found that virus particles strongly associate with the SM-rich regions of the cell membrane and depletion of SM reduces EBOV infection. ASM-specific drugs and multiple small interfering RNAs strongly inhibit the infection by EBOV and EBOV glycoprotein pseudotyped viruses but not by the pseudotypes bearing the glycoprotein of vesicular stomatitis virus. Interestingly, the binding of virus-like particles to cells is strongly associated with surface-localized ASMase as well as SM-enriched sites. Our work suggests that ASMase activity and SM presence are necessary for efficient infection of cells by EBOV. The inhibition of this pathway may provide new avenues for drug treatment.

ABSTRACT For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2–mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2–mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking.

Arenaviruses and filoviruses are capable of causing hemorrhagic fever syndrome in humans. Limited therapeutic and/or prophylactic options are available for humans suffering from viral hemorrhagic fever. In this report, we demonstrate that pre-treatment of host cells with the kinase inhibitors genistein and tyrphostin AG1478 leads to inhibition of infection or transduction in cells infected with Ebola virus, Marburg virus, and Lassa virus. In all, the results demonstrate that a kinase inhibitor cocktail consisting of genistein and tyrphostin AG1478 is a broad-spectrum antiviral that may be used as a therapeutic or prophylactic against arenavirus and filovirus hemorrhagic fever.

BACKGROUND & AIMS: The hepatitis C virus (HCV) serine protease NS3/4A can cleave mitochondria-associated antiviral signaling protein (MAVS) and block retinoic acid-inducible gene I-mediated interferon (IFN) responses. Although this mechanism is thought to have an important role in HCV-mediated innate immunosuppression, its significance in viral persistence is not clear. METHODS: We generated transgenic mice that express the HCV NS3/4A proteins specifically in the liver and challenged the animals with a recombinant vesicular stomatitis virus, a synthetic HCV genome, IFN alfa, or IFN beta. We evaluated the effects of HCV serine protease on the innate immune responses and their interactions. RESULTS: Expression of HCV NS3/4A resulted in cleavage of intrahepatic MAVS; challenge of transgenic mice with vesicular stomatitis virus or a synthetic HCV genome induced strong, type I IFN-mediated responses that were not significantly lower than those of control mice. Different challenge agents induced production of different ratios of IFN alfa and beta, resulting in different autophagic responses and vesicular trafficking patterns of endoplasmic reticulum-and mitochondria-associated viral proteins. IFN beta promoted degradation of the viral proteins by the autolysosome. Variant isoforms of MAVS were associated with distinct, type I IFN-mediated autophagic responses; these responses have a role in trafficking of viral components to endosomal compartments that contain Toll-like receptor-3. CONCLUSIONS: IFN beta mediates a distinct autophagic mechanism of antiviral host defense. MAVS has an important role in type I IFN-induced autophagic trafficking of viral proteins.

ABSTRACT Axl, a plasma membrane-associated Tyro3/Axl/Mer (TAM) family member, is necessary for optimal Zaire ebolavirus (ZEBOV) glycoprotein (GP)-dependent entry into some permissive cells but not others. To date, the role of Axl in virion entry is unknown. The focus of this study was to characterize entry pathways that are used for ZEBOV uptake in cells that require Axl for optimal transduction and to define the role of Axl in this process. Through the use of biochemical inhibitors, interfering RNA (RNAi), and dominant negative constructs, we demonstrate that ZEBOV-GP-dependent entry into these cells occurs through multiple uptake pathways, including both clathrin-dependent and caveola/lipid raft-mediated endocytosis. Other dynamin-dependent and -independent pathways such as macropinocytosis that mediate high-molecular-weight dextran uptake also stimulated ZEBOV-GP entry into these cells, and inhibitors that are known to block macropinocytosis inhibited both dextran uptake and ZEBOV infection. These findings provided strong evidence for the importance of this pathway in filovirus entry. Reduction of Axl expression by RNAi treatment resulted in decreased ZEBOV entry via macropinocytosis but had no effect on the clathrin-dependent or caveola/lipid raft-mediated endocytic mechanisms. Our findings demonstrate for the first time that Axl enhances macropinocytosis, thereby increasing productive ZEBOV entry.

Expression of gastrin and cholecystokinin 2 (CCK 2 ) receptor splice variants (CCK 2 R and CCK 2i4sv R) are upregulated in human colonic adenomas where they are thought to contribute to tumor growth and progression. To determine the effects of ectopic CCK 2 receptor variant expression on colonic epithelial cell growth in vitro and in vivo , we employed the non‐tumorigenic colonic epithelial cell line, NCM356. Receptor expression was induced using a retroviral expression vector containing cDNAs for either CCK 2i4sv R or CCK 2 R. RT‐PCR and intracellular Ca 2+ ([Ca 2+ ] i ) imaging of RIE/CCK 2 R cells treated with conditioned media (CM) from NCM356 revealed that NCM356 cells express gastrin mRNA and secrete endogenous, biologically active peptide. NCM356 cells expressing either CCK 2 R or CCK 2i4sv R (71 and 81 fmol/mg, respectively) grew faster in vitro , and exhibited an increase in basal levels of phosphorylated ERK (pERK), compared with vector. CCK 2 receptor selective antagonist, YM022, partially inhibited the growth of both receptor‐expressing NCM356 cells, but not the control cells. Inhibitors of mitogen activated protein kinase pathway (MEK/ERK) or protein kinase C (PKC) isozymes partially inhibited the elevated levels of basal pERK and in vitro growth of receptor‐expressing cells. Vector‐NCM356 cells did not form tumors in nude mice, whereas, either CCK 2 receptor‐expressing cells formed large tumors. Autocrine activation CCK 2 receptor variants are sufficient to increase in vitro growth and tumorigenicity of non‐transformed NCM356 colon epithelial cells through a pathway involving PKC and the MEK/ERK axis. These findings support the hypothesis that expression of gastrin and its receptors in human colonic adenomas contributes to tumor growth and progression.

Introduction: Reciprocal interplay bewteen epithelial and stromal cells contributes to the initiation, progression and metastasis of colorectal cancer. The importance of carcinoma-associated fibroblasts (CAFs) in tumor develoment is highlighted by the observation that nontumorigenic prostate epithelial cells can form tumors in nude mice when co-implanted with CAF, but not when co-implanted with fibroblasts from benign prostatic hypertrophy. The precise role of colorectal CAFs in tumor-stroma interactions has been difficult to study, in part because of the difficulty of obtaining primary CAFs in sufficient quantity for experiments. Once isolated, the CAFs can be passaged for a limited number of times before they undergo senescence. Further characterization of human colorectal CAFs would provide the basis for stromal-specific therapeutic targeting in colorectal cancers. The purpose of this study was two-fold: 1) immortalize human CAFs derived from colorectal cancers in order to begin to investigate their influence on human colorectal cancer cells and 2) document gene-specific changes attributable to the immortalization process. Methods: Freshly dissected and dissociated cells were cultured from a resected colon cancer specimen under an IRB-approved protocol. The patient had not received prior chemoradiotherapy. The cells were cultured on collagen I and the purity of the fibroblast culture was verified by immunohistochemistry. Fibroblasts are positive for vimentin and/or α-smooth muscle actin, but negative for cytokeratin. CAFs were infected with lentiviral vectors containing a CMV promotor upstream of SV40 Large T antigen and human telomerase. Total RNA was extracted using RNAqueous (Ambion) from triplicate cultures of transformed (t)CAFs and parental (p)CAFs. The purity and concentration of the RNA samples were determined using Agilent 2100 Bioanalyzer. Samples were hybridized to Affymetrix Gene Chips (HGU133 Plus 2.0). Analyses using the Partek Genomics Suite included routine quality controls, raw data background subtraction and normalization. Differentially expressed candidate genes were identified using the Benjamani-Hochberg multiple hypothesis comparisons (ANOVA, p<0.01 and p<0.005) to decrease the false discovery rate. Ingenuity Pathways analyses were performed to identify differences between the t- and p-CAFs. Results: ANOVA identified 2779 and 648 genes that were significant at the p≤0.01 and 0.005 level, respectively. The major pathways representing the genes differentially expressed bewteen the t- and p-CAFs include: 1) the transcriptional regulator TP53 2) growth factor TGFB1, 3) mitogen-activated protein kinase pathways (p38 and p-extracellularly regulated kinase), and 4) the transcriptional regulator nuclear factor-κB. As expected, one canonical pathway that was altered by the transformation, were genes involved in cell cycle and G1/S checkpoint regulation. Conclusions: The immortalization and subsequent careful characterization of human colorectal CAFs provides an invaluable tool for further study of stromal-epithelial interactions in colorectal carcinogenesis and metastasis. Although genes related to the cell cycle has been significantly altered, future studies are required to verify that the t-CAFs alone are non-tumorigenic in nude mice, similar to the p-CAFs. Additionally, immortalized normal fibroblasts derived from the normal a colonic mucosa will need to be generated as an important control.

ABSTRACT The bacterial pathogen Listeria monocytogenes causes food-borne illnesses culminating in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some mammalian cells through binding of the bacterial surface protein InlB to the host receptor tyrosine kinase Met. Interaction of InlB with the Met receptor elicits host downstream signaling pathways that promote F-actin cytoskeletal changes responsible for pathogen engulfment. Here we show that the mammalian signaling protein ARAP2 plays a critical role in cytoskeletal remodeling and internalization of Listeria. Depletion of ARAP2 through RNA interference (RNAi) caused a marked inhibition of InlB-mediated F-actin rearrangements and bacterial entry. ARAP2 contains multiple functional domains, including a GTPase-activating protein (GAP) domain that antagonizes the GTPase Arf6 and a domain capable of binding the GTPase RhoA. Genetic data indicated roles for both the Arf GAP and RhoA binding domains in Listeria entry. Experiments involving Arf6 RNAi or a constitutively activated allele of Arf6 demonstrated that one of the ways in which ARAP2 promotes bacterial uptake is by restraining the activity of Arf6. Conversely, Rho activity was dispensable for Listeria internalization, suggesting that the RhoA binding domain in ARAP2 acts by engaging a host ligand other than Rho proteins. Collectively, our findings indicate that ARAP2 promotes InlB-mediated entry of Listeria, in part, by antagonizing the host GTPase Arf6.

Zaire ebolavirus (ZEBOV), a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can be applied to development of new therapeutics as well as provide potential insight into the trafficking and entry mechanism of other filoviruses.

While much progress has been made in developing drugs against a few prominent viruses such as HIV, few examples exist for emerging infectious agents. In some cases broad spectrum anti-viral drugs, such as ribavirin, are effective, but for some groups of viruses, these show little efficacy in animal models. Traditional methods focus on screening small molecule libraries to identify drugs that target virus factors, with the intention that side-effects to the host can be minimized. However, this greatly limits potential drug targets and virus genes can rapidly mutate to avoid drug action. Recent advances in siRNA gene targeting technologies have provided a powerful tool to specifically target and suppress the expression of cell genes. Since viruses are completely dependent upon host cell proteins for propagation, siRNA screening promises to reveal novel cell proteins and signaling pathways that may be viable targets for drug therapy regimens. Here we used an siRNA screening approach to identify gene products that play critical roles in Ebola virus infection. By gene cluster analysis, proteins in phosphatidylinositol-3-kinase and calcium/calmodulin kinase related networks were identified as important for Zaire Ebola virus infection and prioritized for further evaluation. Key roles of each were confirmed by testing available drugs specific for members of each pathway. Interestingly, both sets of proteins are also important in cancer and subject to intense investigation. Thus development of new drugs against these cancer targets may also prove useful in combating Ebola virus.

Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNAval promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes.

Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection.

The phosphoinositide-3 kinase (PI3K) pathway regulates diverse cellular activities related to cell growth, migration, survival, and vesicular trafficking. It is known that Ebola virus requires endocytosis to establish an infection. However, the cellular signals that mediate this uptake were unknown for Ebola virus as well as many other viruses. Here, the involvement of PI3K in Ebola virus entry was studied. A novel and critical role of the PI3K signaling pathway was demonstrated in cell entry of Zaire Ebola virus (ZEBOV). Inhibitors of PI3K and Akt significantly reduced infection by ZEBOV at an early step during the replication cycle. Furthermore, phosphorylation of Akt-1 was induced shortly after exposure of cells to radiation-inactivated ZEBOV, indicating that the virus actively induces the PI3K pathway and that replication was not required for this induction. Subsequent use of pseudotyped Ebola virus and/or Ebola virus-like particles, in a novel virus entry assay, provided evidence that activity of PI3K/Akt is required at the virus entry step. Class 1A PI3Ks appear to play a predominant role in regulating ZEBOV entry, and Rac1 is a key downstream effector in this regulatory cascade. Confocal imaging of fluorescently labeled ZEBOV indicated that inhibition of PI3K, Akt, or Rac1 disrupted normal uptake of virus particles into cells and resulted in aberrant accumulation of virus into a cytosolic compartment that was non-permissive for membrane fusion. We conclude that PI3K-mediated signaling plays an important role in regulating vesicular trafficking of ZEBOV necessary for cell entry. Disruption of this signaling leads to inappropriate trafficking within the cell and a block in steps leading to membrane fusion. These findings extend our current understanding of Ebola virus entry mechanism and may help in devising useful new strategies for treatment of Ebola virus infection.