An antiviral disulfide compound blocks interaction between arenavirus Z protein and cellular promyelocytic leukemia protein.

The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection.

Antiviral activity of the natural alkaloid anisomycin against dengue and Zika viruses.

Flaviviruses constitute a public health concern because of their global burden and the lack of specific antiviral treatment. Here we investigated the antiviral activity of the alkaloid anisomycin against dengue (DENV) and Zika (ZIKV) viruses. At non-cytotoxic concentrations, anisomycin strongly inhibited the replication of reference strains and clinical isolates of all DENV serotypes and Asian and African strains of ZIKV in Vero cells. Anisomycin also prevented DENV and ZIKV multiplication in human cell lines. While initial steps of DENV and ZIKV replicative cycle were unaffected, a high inhibition of viral protein expression was demonstrated after treatment with anisomycin. DENV RNA synthesis was strongly reduced in anisomycin treated cultures, but the compound did not exert a direct inhibitory effect on 2' O-methyltransferase or RNA polymerase activities of DENV NS5 protein. Furthermore, anisomycin-mediated activation of p38 signaling was not related to the antiviral action of the compound. The evaluation of anisomycin efficacy in a mouse model of ZIKV morbidity and mortality revealed that animals treated with a low dose of anisomycin exhibited a significant reduction in viremia levels and died significantly later than the control group. This protective effect was lost at higher doses, though. In conclusion, anisomycin is a potent and selective in vitro inhibitor of DENV and ZIKV that impairs a post-entry step of viral replication; and a low-dose anisomycin treatment may provide some minimal benefit in a mouse model.

The antiviral potency of Fagus sylvatica 4OMe-glucuronoxylan sulfates.

Herpes simplex virus belongs to Herpesviridae family and causes infection of humans from ancient times. 4OMe-glucuronoxylans as the renewable biopolymers can be promising glycomaterials for various applications in pharmacy. Control enzymatic degradation of the native 4OMe-glucuronoxylan (GX1) followed by targeted sulfation procedure afforded a range of 4OMe-glucuronoxylan sulfates differed in the degree of sulfation (10-16%) and molecular mass (21,000-5000g/mol; GXS1>GXS2>GXS3>GXS4). Antiviral activity tests on GXS1-4 against herpes simplex virus (HSV) types 1 and 2 revealed the positive effect of all compounds against strains of herpes virus. Of them, the compounds GXS1 and GXS4 were shown to be the most active for both HSV serotypes. The antiviral activity of GXS1 and GXS4 was similar to those of heparin or dextran sulfate, used as reference compounds. It was found that GXS1 and GXS4 were active as well against Polio and dengue viruses, however, on a smaller scale. The mode of antiviral action of 4OMe-glucuronoxylan sulfates is due to inhibition of the virus binding to the cell receptors.

The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell.

Two homogeneous sulfated polysaccharides obtained from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata, the kappa/iota/nu carrageenan G3d and the dl-galactan hybrid C2S-3, were assayed for their antiviral properties against the four serotypes of dengue virus (DENV) in different host cell types. Both seaweed derivatives were selective inhibitors of DENV-2 multiplication in Vero cells with inhibitory concentration 50% (IC50) values around 1 microg/ml and selectivity indices > 1000. The compounds had a lower antiviral effect against DENV-3 (IC50 values in the range 13.9-14.2 microg/ml), an even lower effect against DENV-4 (IC50 values in the range 29.3 to > 50 microg/ml) and were totally inactive against DENV-1. With respect to the host cell, the polysulfates were inhibitors of DENV-2 and DENV-3 in the human hepatoma HepG2 and foreskin PH cells, with similar antiviral effectiveness as in Vero cells, but were totally inactive in mosquito C6/36 HT cells. Mechanistic studies demonstrated that G3d and C2S-3 were active DENV-2 inhibitors only when added together with the virus or early after infection, and both initial processes of virus adsorption and internalization are the main targets of these compounds. Therefore, the variations in antiviral activity of the polysaccharides depending on the viral serotype and the host cell may be ascribed to differences in the virus-cell interaction leading to virus entry.

Myristic acid analogs are inhibitors of Junin virus replication.

The effects of two myristic acid analogs on Junin virus (JV) replication were investigated. The compounds chosen for the study were DL-2-hydroxymyristic acid (2OHM), an inhibitor of N-myristoyltransferase (NMT), which binds the enzyme and blocks protein myristoylation, and 13-oxamyristic acid (13OM), a competitive inhibitor of NMT which incorporates into the protein instead of myristic acid. Both types of analogs achieved dose-dependent inhibition of viral multiplication at concentrations not affecting cell viability. The 50% inhibitory concentration values determined by a virus-yield inhibition assay for different strains of JV, including a human pathogenic strain, and for the related arenavirus, Tacaribe, were in the range 1.6 to 20.1 microM, with 13OM as the most active compound. From time of addition and removal experiments, it can be concluded that both analogs inhibit a late stage in the JV replicative cycle, and their effect was partially reversible. The cytoplasmic and surface expression of JV glycoproteins was not affected in the presence of the compounds, as revealed by immunofluorescence staining, suggesting that JV glycoprotein myristoylation would not be essential for the intracellular transport of the envelope proteins, but it may have an important role in their interaction with the plasma membrane during virus budding.

Intracellular processing and transport of Junin virus glycoproteins influences virion infectivity.

The influence of glycoprotein processing, cleavage and transport on Junin virus (JV) infectivity was investigated using monensin combined with lectin binding assays. Yields of extracellular virus were more significantly reduced than cell-associated virus, indicating that monensin inhibited the transport of infectious virus to the extracellular space on a late stage of the replicative cycle. Shown by lectin reactivity and immunoprecipitation, the intracellular processing of JV glycoproteins involved first the maturation of GPC oligosaccharides to a complex form and then the precursor cleavage which might occur late in transit through or exit from the Golgi cisternae. Cleavage of GPC to yield the mature GP38 as well as cell surface immunofluorescence were blocked by monensin. Thus, GP38 production together with glycoprotein transport to the cell membrane seemed to be required for the release of infectious virus from JV-infected cells.

Reduced virulence of a Junin virus mutant is associated with restricted multiplication in murine cells.

C167, a mutant derived from the XJC13 strain of Junin virus, is highly attenuated in its pathogenic properties for newborn mice. Whereas 10(2).PFU of XJC13 injected intracerebrally killed 100% of two-day-old mice, the mutant showed no detectable lethality. Survival of mice infected with C167 was associated with a reduced and delayed virus replication in brain and a defective spread of virus from the site of inoculation to the other tissues, including spleen, kidney, thymus, liver, peritoneal cells and serum. As an apparent consequence of the restricted replication of C167 in mice, no detectable interferon induction and low levels of neutralizing antibodies were observed. Analysis of multiplication kinetics of C167 and XJC13 in different cell cultures in vitro has confirmed that the attenuated phenotype of C167 was related to a specific inefficient replication in murine cells. This host-range restriction was due to a combination of adsorption and penetration blockage.

Inhibition of arenavirus multiplication in vitro by phenotiazines.

Trifluoperazine (TFP) and chlorpromazine (CPZ), two pharmacologically active phenotiazine derivatives, were evaluated for their inhibitory activity on the replication of the arenaviruses Junin (JV), the etiological agent of Argentine hemorrhagic fever, Tacaribe virus and Pichinde virus. Both compounds achieved a concentration-dependent inhibition of viral multiplication at concentrations not affecting cell viability. The 50% inhibitory concentration (IC50) values determined by a virus yield inhibition assay for several strains of JV, including a human pathogenic strain, were in the range of 7.7-23.0 microM and the 90% inhibitory concentration (IC90) fluctuated between 16.6 and 35.2 microM. From time of addition and removal experiments, it can be concluded that CPZ inhibited an early stage in the replicative cycle of JV, probably viral entry. TFP also affected JV penetration when present soon after virus adsorption, and also interfered with a later step of viral maturation when added after 7 h of infection. The expression of viral antigens in the cytoplasm of infected cells was highly reduced in the presence of the compounds, as revealed by immunofluorescence staining, whereas no JV proteins were detected at the cell membrane. The distribution pattern of viral proteins was altered in the few cells exhibiting positive fluorescence after treatment with the phenotiazines. The TFP-induced inhibitory effect on JV multiplication was significantly reversed in the presence of 5 microM calmodulin. These data indicate that TFP and CPZ inhibit JV replication in vitro. Our findings suggest that the integrity of the actin microfilaments may be required for optimal arenavirus multiplication.

Mode of inactivation of arenaviruses by disulfide-based compounds.

Several disulfide-based compounds, including intermolecular aromatic disulfides of the type Ph-S-S-Ph and dithianes with the sulfur atoms tethered in a ring structure, have shown effective inhibitory activity against the arenaviruses Junin (JUNV), agent of Argentine hemorrhagic fever, and Tacaribe (TCRV). These compounds showed a strong virucidal effect with inactivating concentration 50% (IC(50)) values in the range 0.6-5.0 microM, and also were effective to reduce virus yields from infected cells. The mode of inactivating action of two active compounds, the aromatic bis disulfide NSC20625 and the dithiane NSC624152, was further studied. Both compounds were able to inactivate arenaviruses after a few minutes of direct contact with virions, in a concentration- and time-dependent manner. The ability of drug-treated virus to perform several steps of the replication cycle was analyzed. The killed virus particles were found to bind and enter to Vero cells with the same efficacy as infectious native virions, but the ability of inactivated virions to synthesize viral proteins in Vero cells was abolished. Thus, treatment of JUNV and TCRV with these compounds destroyed virion infectivity, generating particles which entered the host cell but were unable to complete the viral biosynthetic processes.

Induction of a refractory state to viral infection in mammalian cells by a plant inhibitor isolated from leaves of Melia azedarach L.

A partially purified plant inhibitor (Meliacin) isolated from Melia azedarach L induced in cells a refractory state to virus infection. Meliacin was active in a large variety of continuous and/or primary cell cultures. A state of maximum virus resistance was achieved after 2 h of incubation and was maintained for at least 15 h; later on it declined but it was fully regained after a second pulse of Meliacin. Interferon was not detected in the supernatant of cells treated with Meliacin and a measurable increase in ds-RNA dependent protein kinase activity was not observed in extracts of Meliacin-treated cells. The antiviral state was not transferred by either extracellular fluid or direct cell-to-cell contact. An active cell metabolism was required for Meliacin action, which was partially reversed in the presence of actinomycin D. It appears that Meliacin is not an interferon-like substance, which induces an antiviral state based on a still unexplained mechanism.

Antiviral effects of Melia azedarach L. leaves extracts on Sindbis virus-infected cells.

Partially purified extracts from leaves of Melia azedarach L. (MA) exert a broad range of antiviral effects on DNA and RNA viruses. The effect of MA on different stages of Sindbis virus replicative cycle in BHK cells was investigated. Under one-step growth conditions MA afforded a greater than 90% inhibition in virus yield if added to the cell cultures 2 h before or after infection, and when added 4 h after infection MA still caused a greater than 80% inhibition. Analysis of early events following Sindbis virus infection showed that MA did not affect viral adsorption to or penetration in BHK cell. In contrast, viral RNA and protein synthesis was almost totally inhibited in cells pretreated with MA 2 h before infection, while cellular macromolecular synthesis was similar in MA-treated and untreated cell cultures.

Protective effect of a natural carrageenan on genital herpes simplex virus infection in mice.

In the present study, the protective effect of 1T1, a lambda-carrageenan extracted from the red seaweed Gigartina skottsbergii was evaluated in a murine model of herpes simplex virus type 2 (HSV-2) genital infection. Six to eight-week-old female BALB/c mice were intravaginally inoculated with a lethal dose of HSV-2 (MS strain) and pre- or post-infection treated with different doses of a 10mg/ml solution of 1T1. A single topical administration of 1T1 shortly before infection of BALB/c mice with HSV-2 protected 9 out of 10 mice from HSV-2-induced lesions and mortality, compared with only 10% survival in control mice. In addition, 1T1 produced a total blockade in virus shedding in the vaginal secretions. When 1T1 pre-treatment was reinforced with a second dose 2h after infection, total protection was observed even when the prophylactic administration had taken place at 60min before infection. The irreversible virucidal action of 1T1 against herpes virus seems to be responsible of its protective effect against virus replication and mortality following vaginal HSV-2 infection.

Antiherpetic activity and mode of action of natural carrageenans of diverse structural types.

The lambda-carrageenan 1T1, the kappa/iota-carrageenan 1C1 and the mu/nu-type 1C3, isolated from the red seaweed Gigartina skottsbergii, proved to be potent and selective inhibitors of herpes simplex virus (HSV) types 1 and 2. The antiviral IC50 values determined by virus yield inhibition assay in different cell lines ranged from 0.4 to 3.3 microg/ml, and no cytotoxic effects, measured by trypan blue exclusion on stationary or proliferating cells, tetrazolium salt method or cell protein synthesis, were observed. Time of addition and attachment studies suggested that the main target for antiviral action of the three carrageenans was virus adsorption, whereas no effect on virus internalization, or early or late protein synthesis was detected. However, the lambda-carrageenan 1T1 was still significantly inhibitory when added any time after adsorption. The pretreatment of virions with the carrageenans showed that 1C1 and 1C3 lacked direct inactivating effect at concentrations near the antiviral IC50 but 1T1 exerted virucidal action. The cyclization of 1T1 to afford the derivative 1T1T1 maintained the antiviral activity but eliminated the virucidal properties. Thus, the structure of 1T1 seems to be responsible for its differential behavior from 1C1 and 1C3, probably allowing a more stable binding to HSV, leading to virion inactivation. In contrast, 1C1 and 1C3 fail to bind with high affinity to virus alone, but are able to interfere with the interaction between HSV particles and the cell.

The effects of oligosaccharide trimming inhibitors on glycoprotein expression and infectivity of Junin virus.

The effects of specific inhibitors of glycoprotein trimming reactions on Junin virus (JV) replication were investigated. Bromoconduritol, an inhibitor of glucosidase II, significantly reduced infective virus production (DE50: 1.1 mM) and viral protein expression. Neither 1-deoxynojirimycin, an inhibitor of both glucosidases I and II, nor 1-deoxymannojirimycin and swainsonine, inhibitors of mannosidase I and II, respectively, showed any activity against JV multiplication. These results are the first evidence that the acquisition of a complex form of the envelope glycoprotein oligosaccharide chains is not essential for JV infectivity. The effect of bromoconduritol was reversible and probably due to the formation of an unstable intermediate oligosaccharide structure which may be more sensitive to degradative proteolysis.

Antiviral and virucidal activities against arenaviruses of zinc-finger active compounds.

Fifteen antiretroviral Zn-finger active compounds with diverse chemical structures, including azoic compounds, hydrazide derivatives, disulphide-based reagents and others were screened in vitro against Junin virus (JUNV), the aetiological agent of Argentine haemorrhagic fever, by a virus yield inhibition assay in Vero cells. Cytotoxicity was evaluated simultaneously by the MTT method. Of the compounds, three were totally inactive as antivirals, nine presented moderate anti JUNV-activity and three were truly active with EC50 (effective concentration 50%) values in the range 6.5-9.3 microM and with selectivity indices greater than 10. The most active inhibitors, named NSC20625, 3-7 and 2-71, demonstrated a broad range of action against arenaviruses, including several attenuated and pathogenic strains of JUNV as well as the antigenically related Tacaribe virus (TACV) and Pichinde virus (PICV). The direct treatment of JUNV and TACV virions with the compounds showed two types of behaviour: the aromatic disulphide NSC20625 was a very potent virucidal agent, whereas the other two compounds exhibited moderate or negligible virus-inactivating properties.

Novel DL-galactan hybrids from the red seaweed Gymnogongrus torulosus are potent inhibitors of herpes simplex virus and dengue virus.

A novel series of DL-galactan hybrids extracted from the red seaweed Gymnogongrus torulosus, was evaluated for its in vitro antiviral properties against herpes simplex virus type 2 (HSV-2) and dengue virus 2 (DEN-2). These compounds were very active against both viruses with inhibitory concentration 50% (IC50) values in the range 0.6-16 microg/ml for HSV-2 and 0.19-1.7 microg/ml for DEN-2, respectively, as determined in a virus plaque reduction assay in Vero cells. The DL-galactans lacked of cytotoxic effects, on stationary as well as on actively dividing cells, and anticoagulant properties. Some of the compounds showed a variable level of direct inactivating effect on both virions, with virucidal concentration 50% values exceeding the IC50s obtained by plaque reduction assay. Full inhibitory activity was achieved when the galactans were present during virus adsorption period, suggesting that the mode of action of these compounds is an interference in the binding of the surface envelope glycoprotein with the cell receptor.

Carrageenans from chilean samples of Stenogramme interrupta (Phyllophoraceae): structural analysis and biological activity.

Carrageenans extracted from cystocarpic and tetrasporic Stenogramme interrupta were analysed by chemical and spectroscopic methods. The carrageenan from cystocarpic plants is composed predominantly of 0.5 M KCl-insoluble and 1 M KCl-soluble fractions. The insoluble fraction contained iota-carrageenan as the major component with alpha-carrageenan and pyruvated carrageenan as minor components. The soluble fraction is highly heterogeneous and did not contain the precursors mu- and nu-carrageenans. The polysaccharide from tetrasporic plants is composed of zeta- and lambda-carrageenans, and low sulfated galactans. It is soluble in KCl and partly cyclized by alkaline treatment. The antiviral and anticoagulant properties of the insoluble polysaccharide fraction from cystocarpic S. interrupta and the polysaccharide from tetrasporic S. interrupta are reported the results of which suggest promising antiherpetic activity.

Evaluation of the antiviral activity of natural sulfated polyhydroxysteroids and their synthetic derivatives and analogs.

Disodium 3beta,21-dihydroxypregn-5-en-20-one disulfate (2), sodium 3beta,21-dihydroxypregn-5-en-20-one 3-sulfate (3), sodium 3beta,21-dihydroxypregn-5-en-20-one 21-sulfate (4), and disodium 3beta,6alpha-dihydroxy-5alpha-pregnan-20-one disulfate (6) have been synthesized and completely characterized for the first time from readily available materials. Sulfation was performed using triethylamine-sulfur trioxide complex in dimethylformamide as the sulfating agent. Selective sulfation of 3beta,21-dihydroxypregn-5-en-20-one rendered sodium 3beta,21-dihydroxypregn-5-en-20-one 3-sulfate (3) as the major compound. The synthetic sulfated steroids as well as natural disulfated polyhydroxysteroids (7-9) isolated by us from the antarctic ophiuroid Astrotoma agassizii and the synthetic derivatives disodium 2beta,3alpha,21-trihydroxy-(20R)-cholesta-5,24-diene 3-acetate, 2,21-disulfate (7a) and 2beta,3alpha,21-trihydroxy-(20R)-cholesta-5,24-diene (7b) were comparatively evaluated for their inhibitory effect on the replication of one DNA (HSV-2) and two RNA (PV-3, JV) viruses. In general, steroids with sulfate groups at C-21 and C-2 or C-3 were the most effective in their inhibitory action against HSV-2 and also proved to be active against PV-3 and JV.

The system of sulfated alpha-(1-->3)-linked D-mannans from the red seaweed Nothogenia fastigiata: structures, antiherpetic and anticoagulant properties.

Structural analysis of two xylomannans extracted from Nothogenia fastigiata was carried out. The results are consistent with the general pattern previously reported for other xylo-mannans of the same system, alpha-(1-->3)-linked D-mannans 2- and 6-sulfated and having single stubs of beta-(1-->2)-linked D-xylose, but one of the new samples contains a significant amount of 2,6-disulfated units. Both xylomannans studied are obtained as complexes with a beta-D-(1-->3)-, alpha-L-(1-->4)-galactan and a beta-D-(1-->3)-, beta-D-(1-->4)-'mixed linkage' xylan co-existing in the seaweed, a fact that limits the accuracy of the data determined. The structures of the galactan and the xylan are similar to those previously informed for this seaweed. The antiviral activity against four different herpes simplex viral strains and the anticoagulant properties of all the xylo-mannans of the system are reported.

Antiherpetic and anticoagulant properties of carrageenans from the red seaweed Gigartina skottsbergii and their cyclized derivatives: correlation between structure and biological activity.

The antiviral activity against herpes simplex virus types 1 and 2 of kappa/l-, partially cyclized mu/v-, and lambda-carrageenans isolated from the red seaweed Gigartina skottsbergii and their cyclized derivatives was analyzed. lambda-Carrageenans and the partially cyclized mu/v-carrageenan were the most potent inhibitors of herpes viruses (including acyclovir-resistant variants and clinical isolates), with IC50 values lower than 1 microgram ml-1 against both serotypes and selectivity indices higher than 10(3). kappa/l-Carrageenans were slightly less effective than the other two types with IC50 values in the range 1.6-4.1 micrograms ml-1. Antiherpetic activity was directly correlated to the amount of alpha-D-galactose 2,6-disulfate residues in the natural carrageenans. The cyclization of the alpha-D-galactose 6-sulfate and 2,6-disulfate units into 3,6-anhydro-alpha-D-galactose and 3,6-anhydro-alpha-D-galactose 2-sulfate residues in these polysaccharides, in general, lowers the antiherpetic activity of the derivatives with respect to the natural carrageenans. Some carrageenans showed a very reduced anticoagulant activity only at concentrations that were considerably higher than the IC50, whereas others were totally devoid of anticoagulant properties. Among natural carrageenans, the mu/v-type IC3 shows the best relationship between antiviral efficacy and lack of anticoagulant action, resulting a very promising compound.