Nélida A. Candurra

The entry of Junin virus into Vero cells

SummaryThe 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 polykaryoctye formation. Taken together, these results demonstrate that JV entry occurs through an endocytic mechanism requiring a low pH dependent membrane fusion.

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.

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.

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 μM 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.

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

Summary. 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.

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.

Involvement of cellular proteins in Junin arenavirus entry

Junin arenavirus (JUNV) entry is dependent on clathrin‐mediated pathways and it relies on the integrity of the actin cytoskeleton as well as the dynamics of microtubules. To determine the method of entry used by this human pathogen, we have demonstrated that in Vero cells JUNV is trafficked via the cellular dynamin 2 (dyn2) endocytic pathway and it is dependent on the Eps15 GTPase. In addition, we have shown that the virus travels through Rab5‐mediated early and Rab7‐mediated late endosomes in its pH‐dependent entry. Altogether, this study gives further inside into the endocytic pathway utilized by the arenavirus JUNV

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.

Intermediate filament integrity is required for Junin virus replication.

The role of the cytoskeletal framework in Junin virus (JUNV) replication has already been demonstrated with compounds interfering with the microfilament (MF) and microtubule (MT) networks. In this work, we evaluated the role of intermediate filaments (IF) during JUNV infection. We tested the effect of acrylamide, a compound that selectively disrupts IF, in culture of three different cell types: Vero cells, murine astrocytes and human foreskin fibroblasts. Perturbation of intermediate filaments had an inhibitory effect on JUNV production within a range of acrylamide concentration of 0.5-3mM in a dose-dependent manner, without cell viability modification. Recovery experiments showed that viral production was partially increased when medium containing acrylamide was replaced by normal maintenance medium (MM). The adsorption and internalization steps were not affected by IF disruption. The expression of JUNV proteins was highly reduced in the presence of 2mM acrylamide while immunofluorescence staining of IF showed network disruption with the formation of cytoplasmic aggregates containing vimentin or glial fibrillary acidic protein (GFAP). We conclude that the IF network may play a role in the early step of JUNV multiplication, subsequent to virus entry and that its integrity is a necessary condition for the normal replication of JUNV in neural and fibroblast cells as well as in the Vero cell line.

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.