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.

Effect of fatty acids on arenavirus replication: inhibition of virus production by lauric acid.

To study the functional involvement of cellular membrane properties on arenavirus infection, saturated fatty acids of variable chain length (C10-C18) were evaluated for their inhibitory activity against the multiplication of Junin virus (JUNV). The most active inhibitor was lauric acid (C12), which reduced virus yields of several attenuated and pathogenic strains of JUNV in a dose dependent manner, without affecting cell viability. Fatty acids with shorter or longer chain length had a reduced or negligible anti-JUNV activity. Lauric acid did not inactivate virion infectivity neither interacted with the cell to induce a state refractory to virus infection. From mechanistic studies, it can be concluded that lauric acid inhibited a late maturation stage in the replicative cycle of JUNV. Viral protein synthesis was not affected by the compound, but the expression of glycoproteins in the plasma membrane was diminished. A direct correlation between the inhibition of JUNV production and the stimulation of triacylglycerol cell content was demonstrated, and both lauric-acid induced effects were dependent on the continued presence of the fatty acid. Thus, the decreased insertion of viral glycoproteins into the plasma membrane, apparently due to the increased incorporation of triacylglycerols, seems to cause an inhibition of JUNV maturation and release.

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.

Homologous and heterologous glycoproteins induce protection against Junin virus challenge in guinea pigs.

Tacaribe virus (TACV) is an arenavirus that is genetically and antigenically closely related to Junin virus (JUNV), the aetiological agent of Argentine haemorrhagic fever (AHF). It is well established that TACV protects experimental animals fully against an otherwise lethal challenge with JUNV. To gain information on the nature of the antigens involved in cross-protection, recombinant vaccinia viruses were constructed that express the glycoprotein precursor (VV-GTac) or the nucleocapsid protein (VV-N) of TACV. TACV proteins expressed by vaccinia virus were indistinguishable from authentic virus proteins by gel electrophoresis. Guinea pigs inoculated with VV-GTac or VV-N elicited antibodies that immunoprecipitated authentic TACV proteins. Antibodies generated by VV-GTac neutralized TACV infectivity. Levels of antibodies after priming and boosting with recombinant vaccinia virus were comparable to those elicited in TACV infection. To evaluate the ability of recombinant vaccinia virus to protect against experimental AHF, guinea pigs were challenged with lethal doses of JUNV. Fifty per cent of the animals immunized with VV-GTac survived, whereas all animals inoculated with VV-N or vaccinia virus died. Having established that the heterologous glycoprotein protects against JUNV challenge, a recombinant vaccinia virus was constructed that expresses JUNV glycoprotein precursor (VV-GJun). The size and reactivity to monoclonal antibodies of the vaccinia virus-expressed and authentic JUNV glycoproteins were indistinguishable. Seventy-two per cent of the animals inoculated with two doses of VV-GJun survived lethal JUNV challenge. Protection with either VV-GJun or VV-GTac occurred in the presence of low or undetectable levels of neutralizing antibodies to JUNV.

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.

[Inhibition of Junin virus multiplication by caffeine]. / Acción inhibitoria de la cafeína sobre la multiplicación del virus junín.

The inhibitory activity of caffeine, a pharmacologically active compound, on Junin virus (JV) multiplication in Vero cells was evaluated by a virus yield inhibition assay. The compound achieved a dose-dependent inhibition of virus production at concentrations not affecting cell viability. The 50% inhibitory concentration (IC50) and the 90% inhibitory concentration (IC90) were 9.0 mM and 10.8 mM, respectively. From time of addition experiments, it can be concluded that caffeine inhibited an early stage in the replicative cycle of JV occuring before 4 h of infection. Extracellular and cell-associated virus yields were reduced to the same extent. The addition of caffeine after several cycles of infection for a very short treatment period did not significantly affect the formation of JV infectious particles. The expression of viral proteins in the cytoplasm and the membrane of infected cells was highly reduced in the presence of caffeine, as revealed by immunofluorescence staining, confirming that caffeine predominantly exerted its inhibitory action early in the infection of Vero cells with JV.

Acción inhibitoria de la cafeína sobre la multiplicación del virus junín / Inhibition of Junin virus multiplication by caffeine

The inhibitory activity of caffeine, a pharmacologically active compound, on Junin virus (JV) multiplication in Vero cells was evaluated by a virus yield inhibition assay. The compound achieved a dose-dependent inhibition of virus production at concentrations not affecting cell viability. The 50 inhibitory concentration (IC50) and the 90 inhibitory concentration (IC90) were 9.0 mM and 10.8 mM, respectively. From time of addition experiments, it can be concluded that caffeine inhibited an early stage in the replicative cycle of JV occuring before 4 h of infection. Extracellular and cell-associated virus yields were reduced to the same extent. The addition of caffeine after several cycles of infection for a very short treatment period did not significantly affect the formation of JV infectious particles. The expression of viral proteins in the cytoplasm and the membrane of infected cells was highly reduced in the presence of caffeine, as revealed by immunofluorescence staining, confirming that caffeine predominantly exerted its inhibitory action early in the infection of Vero cells with JV.

Acción inhibitoria de la cafeína sobre la multiplicación del virus junín / Inhibition of Junin virus multiplication by caffeine

The inhibitory activity of caffeine, a pharmacologically active compound, on Junin virus (JV) multiplication in Vero cells was evaluated by a virus yield inhibition assay. The compound achieved a dose-dependent inhibition of virus production at concentrations not affecting cell viability. The 50 inhibitory concentration (IC50) and the 90 inhibitory concentration (IC90) were 9.0 mM and 10.8 mM, respectively. From time of addition experiments, it can be concluded that caffeine inhibited an early stage in the replicative cycle of JV occuring before 4 h of infection. Extracellular and cell-associated virus yields were reduced to the same extent. The addition of caffeine after several cycles of infection for a very short treatment period did not significantly affect the formation of JV infectious particles. The expression of viral proteins in the cytoplasm and the membrane of infected cells was highly reduced in the presence of caffeine, as revealed by immunofluorescence staining, confirming that caffeine predominantly exerted its inhibitory action early in the infection of Vero cells with JV.(AU)

Involvement of the cytoskeleton in Junin virus multiplication.

The role of the cellular cytoskeleton in Junin virus (JV) infection was explored in two ways. Firstly, the action of inhibitors that affect individual cytoskeletal systems (microtubules or microfilaments) selectively was analysed. It was found that perturbations of microtubule or microfilament networks caused by colchicine, nocodazole, nifedipine, EGTA or DMSO strongly affected virion production and viral protein expression at non-cytotoxic concentrations. Secondly, the extent of association of viral proteins and infectious virus particles with the cytoskeletal fraction of monkey Vero cells was determined by using three non-ionic detergents, Triton X-100 (TX-100), NP-40 and octyl glucoside (OG). The cytoskeleton retained nearly 70% of the external JV envelope glycoprotein GP38 and about 40% of the JV nucleoprotein NP, according to TX-100 and OG insolubility results. Furthermore, 1% of the total cell-bound infectivity was detected in the detergent-insoluble fraction, suggesting that cytoskeletal components are involved in the initiation of the assembly and budding processes of JV particles at the plasma membrane.

Acción inhibitoria de la cafeína sobre la multiplicación del virus junín. / [Inhibition of Junin virus multiplication by caffeine]

The inhibitory activity of caffeine, a pharmacologically active compound, on Junin virus (JV) multiplication in Vero cells was evaluated by a virus yield inhibition assay. The compound achieved a dose-dependent inhibition of virus production at concentrations not affecting cell viability. The 50

inhibitory concentration (IC50) and the 90

inhibitory concentration (IC90) were 9.0 mM and 10.8 mM, respectively. From time of addition experiments, it can be concluded that caffeine inhibited an early stage in the replicative cycle of JV occuring before 4 h of infection. Extracellular and cell-associated virus yields were reduced to the same extent. The addition of caffeine after several cycles of infection for a very short treatment period did not significantly affect the formation of JV infectious particles. The expression of viral proteins in the cytoplasm and the membrane of infected cells was highly reduced in the presence of caffeine, as revealed by immunofluorescence staining, confirming that caffeine predominantly exerted its inhibitory action early in the infection of Vero cells with JV.

Effect of inhibitors of the intracellular exocytic pathway on glycoprotein processing and maturation of Junin virus.

The effect of glycoprotein processing and transport on Junin virus (JV) maturation was studied by using brefeldin A (BFA) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), two inhibitors affecting different steps of the intracellular exocytic pathway, combined with low temperature incubation. Both compounds inhibited the multiplication of JV, strain IV4454, in Vero cells in a dose dependent manner. The addition of the compounds after several cycles of infection for a very short treatment period produced an immediate arrest on the formation of JV infectious particles, due to their effect on a late event in JV infective cycle. Extracellular and cell-associated virus yields were reduced to the same extent, suggesting that the assembly of virus particles was the blocked stage. In infected cells treated with CCCP and BFA an altered subcellular distribution of partially processed viral glycoproteins was observed, with an accumulation of JV glycoproteins at the endoplasmic reticulum and a blockade of their transport to the site of envelopment in the plasma membrane. Concomitantly, the cleavage of the precursor GPC to the mature glycoprotein GP38 was strongly inhibited when the exocytic pathway was affected. Thus, results obtained with BFA allow to conclude that the proteolytic processing of GPC probably occurs at the trans-Golgi network and this cleavage together with the glycoprotein presence at the cell surface is required for maturation of infectious JV particles.

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.

Influencia del tratamiento enzimático sobre la interacción virus Junín-células Vero / Influence of enzyme treatment on the interaction of Junin virus with Vero cells

Se investigó la naturaleza bioquímica de las estructuras presentes en la superficie de células Vero, implicadas en la interacción con el virus Junín (cepa XJ C13) y una mutante de rango de huésped denominada Cl 67. Los tratamientos enzimáticos realizados a las células antes de la infección indican que las primeras moléculas que interaccionan con el virus Junín serían proteínas o grupos proteícos con aminoácidos básicos o aromáticos expuestos, existiendo diferencias en la afinidad hacia las cepas estudiadas

Influencia del tratamiento enzimático sobre la interacción virus Junín-células Vero / Influence of enzyme treatment on the interaction of Junin virus with Vero cells

Se investigó la naturaleza bioquímica de las estructuras presentes en la superficie de células Vero, implicadas en la interacción con el virus Junín (cepa XJ C13) y una mutante de rango de huésped denominada Cl 67. Los tratamientos enzimáticos realizados a las células antes de la infección indican que las primeras moléculas que interaccionan con el virus Junín serían proteínas o grupos proteícos con aminoácidos básicos o aromáticos expuestos, existiendo diferencias en la afinidad hacia las cepas estudiadas (AU)

[Influence] of enzymatic treatment on Junin virus--Vero cells interaction]. / Influencia del tratamiento enzimático sobre la interacción virus Junín-células Vero.

The chemical nature of cellular structures involved in the attachment of Junin virus (wild type XJC13 and host range mutant Cl 67) to Vero cells was investigated. Enzyme treatment of cells before virus infection indicated that whereas lipids are not directly involved in virus attachment, cellular proteins play a significant role in early interaction with JV. Moreover aromatic residues, leucine and basic amino acid seem to actively participate in this interaction with different affinity for the assayed strains.

Influencia del tratamiento enzimático sobre la interacción virus Junín-células Vero. / [Influence] of enzymatic treatment on Junin virus--Vero cells interaction]

The chemical nature of cellular structures involved in the attachment of Junin virus (wild type XJC13 and host range mutant Cl 67) to Vero cells was investigated. Enzyme treatment of cells before virus infection indicated that whereas lipids are not directly involved in virus attachment, cellular proteins play a significant role in early interaction with JV. Moreover aromatic residues, leucine and basic amino acid seem to actively participate in this interaction with different affinity for the assayed strains.

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.

The entry of Junin virus into Vero cells.

The entry mechanism of Junin virus (JV) into Vero cells was studied analyzing the effect of lysosomotropic compounds and acid pH on JV infection. Ammonium chloride, amantadine, chlorpheniramine and procaine inhibited JV production. The action of ammonium chloride was exerted at early times of infection. Virus internalization was inhibited and viral protein expression was not detected. When the extracellular medium was buffered at low pH, the ammonium chloride induced block on JV infection was overcome. Furthermore, JV was able to induce fusion of infected cells at pH 5.5 leading to polykaryocyte formation. Taken together, these results demonstrate that JV entry occurs through an endocytic mechanism requiring a low pH dependent membrane fusion.

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.

A comparison of Junin virus strains: growth characteristics, cytopathogenicity and viral polypeptides.

The growth characteristics, cytopathogenicity and viral polypeptides of the virulent strain XJ of Junin virus (JV), its attenuated derivative XJC13 and another naturally attenuated JV strain, IV4454, were comparatively studied. IV4454 and XJC13 viruses showed the highest and lowest cytopathology for Vero cells, respectively, as measured by plaque morphology, cell viability and inhibition of host cell protein synthesis. The kinetics and electrophoretic patterns of viral polypeptides in infected cell extracts were very similar among the three strains, whereas differences were detected in the surface glycoprotein GP38 by peptide mapping after limited proteolysis.