Inhibition of dengue virus infection by trifluoperazine.

Dengue virus (DENV), a member of the genus Flavivirus, family Flaviviridae, is the most widespread viral pathogen transmitted to humans by mosquitoes. Despite the increased incidence of DENV infection, there are no antiviral drugs available for treatment or prevention. Phenothiazines are heterocyclic compounds with various pharmacological properties that are very adaptable for drug repurposing. In the present report, we analyzed the antiviral activity against DENV and the related Zika virus (ZIKV) of trifluoperazine (TFP), a phenothiazine derivative in clinical use as an antipsychotic and antiemetic agent. TFP exhibited dose-dependent inhibitory activity against the four DENV serotypes and ZIKV in monkey Vero cells at non-cytotoxic concentrations with 50% effective concentration values in the range 1.6-6.4 µM. A similar level of antiviral efficacy was exhibited by TFP against flavivirus infection in the human cell lines A549 and HepG2. Mechanistic studies, performed using time-dependent infectivity assays, real-time RT-PCR, Western blot, and immunofluorescence techniques, indicated that uncoating of the virus during penetration into the cell was the main target for TFP in infected cells, but the compound also exerted a minor effect on a late stage of the virus multiplication cycle. This study demonstrates that TFP, a pharmacologically active phenothiazine, is a selective inhibitor of DENV multiplication in cell culture. Our findings open perspectives for the repositioning of phenothiazines like TFP with a wide spectrum of antiviral efficacy as potential agents for the control of pathogenic flaviviruses.

Inhibitors of the p38 cell signaling pathway as antiviral compounds against Junín virus.

In the present study, we analyzed the modulation of p38 cell signaling by Junín virus (JUNV) and evaluated the antiviral activity of p38 inhibitors against JUNV. While JUNV induced a progressive activation of p38 throughout the infection in Vero cells, a partial downregulation of p38 phosphorylation was observed in HEK293 and HeLa cells. The compounds SB203580 and SB202190, which are selective inhibitors of p38, significantly reduced viral protein expression and viral yield in the cell lines examined, indicating that the p38 signaling pathway might be a promising antiviral target against JUNV infection.

Inhibition of the PI3K/Akt pathway by Ly294002 does not prevent establishment of persistent Junín virus infection in Vero cells.

In previous work, we demonstrated that the arenavirus Junín virus (JUNV) is able to activate Akt by means of the phosphatidylinositol-3-kinase (PI3K) survival pathway during virus entry. This work extends our study, emphasizing the relevance of this pathway in the establishment and maintenance of persistent infection in vitro. During the course of infection, JUNV-infected Vero cells showed a typical cytopathic effect that may be ascribed to apoptotic cell death. Treatment of infected cultures with Ly294002, an inhibitor of the PI3K/Akt pathway, produced an apoptotic response similar to that observed for uninfected cells treated with the drug. This result suggests that virus-induced activation of the PI3K/Akt pathway does not deliver a strong enough anti-apoptotic signal to explain the low proportion of apoptotic cells observed during infection. Also, inhibition of the PI3K/Akt pathway during the acute stage of infection did not prevent the establishment of persistence. Furthermore, treatment of persistently JUNV-infected cells with Ly294002 did not alter viral protein expression. These findings indicate that despite the positive modulation of the PI3/Akt pathway during Junín virus entry, this would not play a critical role in the establishment and maintenance of JUNV persistence in Vero cells.

Raf/MEK/ERK pathway activation is required for Junín virus replication.

In the present work we investigated the importance of the Raf/MEK/ERK signalling pathway in the multiplication of the arenavirus Junín (JUNV) in monkey and human cell cultures. We established that JUNV induces a biphasic activation of ERK and we proved that a specific inhibitor of the ERK pathway, U0126, impairs viral replication. Furthermore, U0126 exerted inhibitory action against the arenaviruses Tacaribe and Pichinde. Moreover, treatment with known ERK activators such as phorbol 12-myristate 13-acetate and serum increased viral yields whereas ERK silencing by small interfering RNAs caused the inhibition of viral multiplication. Therefore, activation of the Raf/MEK/ERK signalling pathway is required to ensure efficient JUNV replication and may constitute a host target for the development of novel effective therapeutic strategies to deal with arenavirus infections.

Differential effect of acute and persistent Junin virus infections on the nucleo-cytoplasmic trafficking and expression of heterogeneous nuclear ribonucleoproteins type A and B.

Heterogeneous nuclear ribonucleoproteins A and B (hnRNPs A/B), cellular RNA-binding proteins that participate in splicing, trafficking, translation and turnover of mRNAs, have been implicated in the life cycles of several cytoplasmic RNA viruses. Here, we demonstrate that silencing of hnRNPs A1 and A2 significantly reduces the replication of the arenavirus Junín virus (JUNV), the aetiological agent of Argentine haemorrhagic fever. While acute JUNV infection did not modify total levels of expression of hnRNPs A/B in comparison with uninfected cells, non-cytopathic persistent infection exhibited low levels of these cell proteins. Furthermore, acutely infected cells showed a cytoplasmic relocalization of overexpressed hnRNP A1, probably related to the involvement of this protein in virus replicative cycle. This cytoplasmic accumulation was also observed in cells expressing viral nucleoprotein (N), and co-immunoprecipitation studies revealed the interaction between hnRNP A1 and N protein. By contrast, a predominantly nuclear distribution of overexpressed hnRNP A1 was found during persistent infection, even in the presence of endogenous or overexpressed N protein, indicating a differential modulation of nucleo-cytoplasmic trafficking in acute and persistent JUNV infections.

Alternative infectious entry pathways for dengue virus serotypes into mammalian cells.

The entry of two dengue virus (DENV) serotypes into Vero cells was analysed using biochemical inhibitors, dominant negative mutants of cellular proteins involved in endocytic pathways, fluorescence microscopy and infectivity determinations. By treatment with dansylcadaverine and chlorpromazine and overexpression of a dominant negative form of the Eps15 protein, a clathrin-mediated endocytosis for productive DENV-1 internalization into Vero cells was demonstrated whereas the infectious entry of DENV-2 in the same cell system was independent of clathrin. Treatment with the inhibitors nystatin and methyl-beta-cyclodextrin, as well as transfection of Vero cells with dominant negative caveolin-1, had no effect on DENV-2 virus infection. It was also shown, by using the K44A mutant and the inhibitor dynasore, that dynamin was required for DENV-2 entry. Consequently, the infectious entry of DENV-2 into Vero cells occurs by a non-classical endocytic pathway independent of clathrin, caveolae and lipid rafts, but dependent on dynamin. By contrast, DENV-2 entry into A549 cells was clathrin-dependent, as previously reported in HeLa, C6/36 and BS-C-1 cells. Our results conclusively show, for the first time, a differential mode of infective entry for DENV-1 and DENV-2 into a common host cell, Vero cells, as well as alternative entry pathways for a given serotype, DENV-2, into different types of cells.

Anti-adenovirus activity of epiandrosterone and dehydroepiandrosterone derivatives.

BACKGROUND: Dehydroepiandrosterone (DHEA) exhibits a wide range of biological functions including antiviral activity. In this work, we present in vitro anti-adenovirus (AdV) activity of seven DHEA and twelve epiandrosterone (EA) analogues. METHODS: The cytotoxic effect of the compounds was determined by the MTT assay and the antiviral activity by a virus yield inhibition assay. The mode of antiviral activity was examined using time-of-addition experiments, adsorption and internalization assays and Western blot analysis. RESULTS: EA, DHEA, and two synthetic derivatives inhibit virus replication with selectivity indices ranging between 42 and 83. Virus adsorption and internalization are not the target of the inhibitory action; meanwhile, AdV protein synthesis was diminished in the presence of DHEA. CONCLUSIONS: DHEA and some synthetic derivatives present antiviral activity similar to cidofovir, which was used as reference drug. These steroidal compounds adversely affect virus protein synthesis and viral mature particle formation.

Dehydroepiandrosterone, epiandrosterone and synthetic derivatives inhibit Junin virus replication in vitro.

In the present paper the in vitro antiviral activity of dehydroepiandrosterone (DHEA), epiandrosterone (EA) and 16 synthetic derivatives against Junin virus (JUNV) replication in Vero cells was studied. DHEA and EA caused a selective inhibition of the replication of JUNV and other members of the Arenaviridae family such as Pichinde virus and Tacaribe virus. The compounds were not virucidal to cell-free JUNV. The impairment of viral replication was not due to an inhibitory effect of the steroids on virus adsorption or internalization. An inhibitory effect of the compounds on JUNV protein synthesis and both intracellular and extracellular virus production was demonstrated. A partial inhibitory action on cell surface expression of JUNV glycoprotein G1 was also detected on DHEA- and EA-treated cultures. Like DHEA and EA, three compounds obtained from EA by chemical synthesis showed selectivity indexes higher than ribavirin, the only antiviral compound that has shown partial efficacy against arenavirus infections.

Role of the ERK1/2 Signaling Pathway in the Replication of Junín and Tacaribe Viruses.

We have previously shown that the infection of cell cultures with the arenaviruses Junín (JUNV), Tacaribe (TCRV), and Pichindé promotes the phosphorylation of mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases 1 and 2 (ERK1/2) and that this activation is required for the achievement of a productive infection. Here we examined the contribution of ERK1/2 in early steps of JUNV and TCRV multiplication. JUNV adsorption, internalization, and uncoating were not affected by treatment of cultured cells with U0126, an inhibitor of the ERK1/2 signaling pathway. In contrast, U0126 caused a marked reduction in viral protein expression and RNA synthesis, while JUNV RNA synthesis was significantly augmented in the presence of an activator of the ERK1/2 pathway. Moreover, U0126 impaired the expression of a reporter gene in a TCRV-based replicon system, confirming the ability of the compound to hinder arenavirus macromolecular synthesis. By using a cell-based assay, we determined that the inhibitor did not affect the translation of a synthetic TCRV-like mRNA. No changes in the phosphorylation pattern of the translation factor eIF2α were found in U0126-treated cells. Our results indicate that U0126 impairs viral RNA synthesis, thereby leading to a subsequent reduction in viral protein expression. Thus, we conclude that ERK1/2 signaling activation is required for an efficient arenavirus RNA synthesis.

Antiviral effect of a synthetic brassinosteroid on the replication of vesicular stomatitis virus in Vero cells.

The antiviral mode of action of the synthetic brassinosteroid (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (6b) against replication of vesicular stomatitis virus (VSV) in Vero cells was investigated. Time-related experiments showed that 6b mainly affects a late event of the virus growth cycle. Virus adsorption, internalisation and early RNA synthesis are not the target of the inhibitory action. Results obtained indicate that the antiviral compound adversely affects virus protein synthesis and viral mature particle formation.

Antiviral activity of natural and synthetic ß-carbolines against dengue virus.

Dengue virus (DENV) is the most prevalent mosquito borne viral pathogen worldwide. In this work we first evaluated the antiviral activity of natural and synthetic ß-carbolines against DENV-2 multiplication in cell cultures. We determined that the natural ß-carboline harmol and a synthetic harmine derivative, 9N-methylharmine, exhibit inhibitory effect on DENV-2 production without virucidal activity. The active compounds were inhibitory of all DENV serotypes, being DENV-2 the more susceptible to their antiviral action. The mode of action of 9N-methylharmine against DENV-2 was further explored. We determined that the derivative neither affects viral adsorption-internalization events nor viral RNA synthesis. The quantification of intracellular and extracellular viral genomes and infectious virus particles indicated that 9N-methylharmine would impair the maturation and release of virus particles to the extracellular medium affecting the spreading of the infection. Furthermore, we also determined that 9N-methylharmine antiviral activity is not related to the ability of the compound to downregulate p38 MAPK phosphorylation.

Antiviral mode of action of a synthetic brassinosteroid against Junin virus replication.

The antiviral mode of action of the synthetic brassinosteroid (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (6b) against Junin virus replication in Vero cells was investigated. Time-related experiments showed that 6b mainly affects an early event of virus growth cycle. Neither adsorption nor internalization of viral particles was the target of the inhibitory action. The analysis of the effect of 6b on viral RNA synthesis demonstrated that the presence of the compound adversely affects virus RNA replication by preventing the synthesis of full length antigenomic RNA. Although 6b was most effective the earlier it was added to the cells after infection with JV, a high level of inhibition of JV yield and fusion activity of newly synthesized viral glycoproteins was still detected when the compound was present during the last hours of infection. Therefore, we cannot rule out an inhibitory action of 6b on later events of JV replicative cycle.

Dengue-3 Virus Entry into Vero Cells: Role of Clathrin-Mediated Endocytosis in the Outcome of Infection.

The endocytic uptake and intracellular trafficking for penetration of DENV-3 strain H-87 into Vero cells was analyzed by using several biochemical inhibitors and dominant negative mutants of cellular proteins. The results presented show that the infective entry of DENV-3 into Vero cells occurs through a non-classical endocytosis pathway dependent on low pH and dynamin, but non-mediated by clathrin. After uptake, DENV-3 transits through early endosomes to reach Rab 7-regulated late endosomes, and according with the half-time for ammonium chloride resistance viral nucleocapsid is released into the cytosol approximately at 12 min post-infection. Furthermore, the influence of the clathrin pathway in DENV-3 infective entry in other mammalian cell lines of human origin, such as A549, HepG2 and U937 cells, was evaluated demonstrating that variable entry pathways are employed depending on the host cell. Results show for the first time the simultaneous coexistence of infective and non -infective routes for DENV entry into the host cell, depending on the usage of clathrin-mediated endocytosis.

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a host factor required for dengue virus and Junín virus multiplication.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are cellular factors involved in the replication of several viruses. In this study we analyzed the expression and intracellular localization of hnRNP A2 and hnRNP K in cell cultures infected with two viruses that cause human hemorrhagic fevers: dengue virus type 2 (DENV-2) and Junín virus (JUNV). We determined that DENV-2 promoted the cytoplasmic translocation of hnRNP K and to a lesser extent of hnRNP A2, meanwhile, JUNV infection induced an increase in hnRNP K cytoplasmic localization whereas hnRNP A2 remained mainly in the nucleus of infected cells. Both hnRNP K and hnRNP A2 were localized predominantly in the nucleus of JUNV persistently-infected cells even after superinfection with JUNV indicating that persistent infection does not alter nucleo-cytoplasmic transport of these hnRNPs. Total levels of hnRNP K expression were unaffected by DENV-2 or JUNV infection. In addition we determined, using small interfering RNAs, that hnRNP K knockout inhibits DENV-2 and JUNV multiplication. Our results indicate that DENV-2 and JUNV induce hnRNP K cytoplasmic translocation to favor viral multiplication.

Enterocin CRL35 inhibits late stages of HSV-1 and HSV-2 replication in vitro.

The replication of herpes simplex virus (HSV) type 1 and 2 in Vero cells is inhibited in the presence of enterocin CRL35 (ECRL), a bacteriocin produced by Enterococcus faecium CRL35. Attempts to resolve the mode of action of ECRL indicate that virus adsorption and penetration are not affected. Instead, a late step of virus multiplication is hindered since the addition of 100 microg/ml of ECRL at 8h post infection still causes a 90% inhibition of virus release. The effect of ECRL on HSV antigen expression was studied by immunofluorescence using a polyclonal serum and a monoclonal antibody against glycoprotein D (gamma protein). These studies indicated that ECRL impeded the second round of infection, apparently as a consequence of the inhibition of glycoprotein D expression. The replication of syncytial mutants of HSV-1 was significantly inhibited at a ECRL concentration of 25 microg/ml. Both the percentage of fused cells and the polykaryocyte size were affected. Studies on the effect of ECRL on viral protein synthesis showed that in the presence of ECRL, HSV late gamma proteins were not synthesized. From these findings, it is concluded that inhibition of HSV spreading by ECRL is due to the prevention of mainly late glycoprotein synthesis.

Antiviral activity of antimicrobial cationic peptides against Junin virus and herpes simplex virus.

The in vitro antiviral activity of antimicrobial cationic peptides: cecropin A, melittin, magainin I and II and indolicidin against the arenavirus Junin virus (JV), and herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) was evaluated. Cecropin A effectively inhibited JV multiplication and failed to affect HSV replication whereas melittin impeded the multiplication of JV and HSV, but was highly toxic for the host cell. Magainins I and II exhibited inhibitory action toward HSV-1 and HSV-2 but were inactive against JV. Only indolicidin showed a direct inactivation effect on cell-free virus stocks. Besides its inhibitory effect on JV replication cecropin A also was active against the arenaviruses Tacaribe and Pichinde, mainly affecting late events of arenavirus multiplication cycle by preventing viral morphogenesis and egress from infected cells.

Antiherpetic mode of action of (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one in vitro.

The replication of herpes simplex virus (HSV) type 1 in Vero cells is inhibited in the presence of (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (6b), a synthetic brassinosteroid derivative. Attempts to disclose the mode of action of 6b indicate that a late step of virus multiplication is affected. In the presence of 6b, HSV late protein synthesis was severely diminished and this inhibitory effect of 6b on HSV antigen expression was confirmed by immunofluorescence assays.

Changes in antiviral susceptibility to entry inhibitors and endocytic uptake of dengue-2 virus serially passaged in Vero or C6/36 cells.

The aim of the present study was to analyze the influence of virus origin, mammalian or mosquito cell-derived, on antiviral susceptibility of DENV-2 to entry inhibitors and the association of this effect with any alteration in the mode of entry into the cell. To this end, ten serial passages of DENV-2 were performed in mosquito C6/36 cells or monkey Vero cells and the antiviral susceptibility of each virus passage to sulfated polysaccharides (SPs), like heparin and carrageenans, was evaluated by a virus plaque reduction assay. After serial passaging in Vero cells, DENV-2 became increasingly resistant to SP inhibition whereas the antiviral susceptibility was not altered in virus propagated in C6/36 cells. The change in antiviral susceptibility was associated to a differential mode of entry into the host cell. The route of endocytic entry for productive Vero cell infection was altered from a non-classical clathrin independent pathway for C6/36-grown virus to a clathrin-mediated endocytosis when the virus was serially propagated in Vero cells. Our results show the impact of the cellular system used for successive propagation of DENV on the initial interaction between the host cell and the virion in the next round of infection and the relevant consequences it might have during the in vitro evaluation of entry inhibitors.

Differential requirements in endocytic trafficking for penetration of dengue virus.

The entry of DENV into the host cell appears to be a very complex process which has been started to be studied in detail. In this report, the route of functional intracellular trafficking after endocytic uptake of dengue virus serotype 1 (DENV-1) strain HW, DENV-2 strain NGC and DENV-2 strain 16681 into Vero cells was studied by using a susceptibility to ammonium chloride assay, dominant negative mutants of several members of the family of cellular Rab GTPases that participate in regulation of transport through endosome vesicles and immunofluorescence colocalization. Together, the results presented demonstrate that in spite of the different internalization route among viral serotypes in Vero cells and regardless of the viral strain, DENV particles are first transported to early endosomes in a Rab5-dependent manner. Then a Rab7-dependent pathway guides DENV-2 16681 to late endosomes, whereas a yet unknown sorting event controls the transport of DENV-2 NGC, and most probably DENV-1 HW, to the perinuclear recycling compartments where fusion membrane would take place releasing nucleocapsid into the cytoplasm. Besides the demonstration of a different intracellular trafficking for two DENV-2 strains that shared the initial clathrin-independent internalization route, these studies proved for the first time the involvement of the slow recycling pathway for DENV-2 productive infection.

Host cell factors as antiviral targets in arenavirus infection.

Among the members of the Arenaviridae family, Lassa virus and Junin virus generate periodic annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of West Africa and Argentina, respectively. Given the human health threat that arenaviruses represent and the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. Since diverse host cell pathways and enzymes are used by RNA viruses to fulfill their replicative cycle, the targeting of a host process has turned an attractive antiviral approach in the last years for many unrelated virus types. This strategy has the additional benefit to reduce the serious challenge for therapy of RNA viruses to escape from drug effects through selection of resistant variants triggered by their high mutation rate. This article focuses on novel strategies to identify inhibitors for arenavirus therapy, analyzing the potential for antiviral developments of diverse host factors essential for virus infection.