Compound

The carboxamide-derivatized aromatic disulfide NSC4492 was provided by the National Cancer Institute, Frederick, MD, USA. Stock solutions at a concentration of 100 mM were prepared in dimethylsulfoxide (DMSO). Working solutions of NSC4492 were prepared by dilution of the 100mM stock in the appropriate culture medium.

Cells and viruses

Vero cells were grown as monolayers in Eagle’s minimum essential medium (MEM, Invitrogen-Life Technologies) containing 5 % inactivated bovine serum and 50 µg/ml gentamycin. Maintenance medium (MM) consisted of MEM supplemented with 1.5 % bovine serum. CV1 cells and BSR cells (a BHK-21 clone) were grown in Dulbecco's MEM (D-MEM, Invitrogen-Life Technologies) and Glasgow MEM (G-MEM, Invitrogen - Life Technologies, USA), respectively, supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS) and penicillin (100 U/ml)-streptomycin (100 µg/ml) (Invitrogen-Life Technologies).

All experiments were performed using the attenuated strain IV4454 of JUNV [31]. Virus stocks were prepared in Vero cells and titrated by plaque assay on the same cells.

Virus inactivation

For virus inactivation assays, treatment of JUNV with NSC4492 was carried out by mixing an aliquot of a viral suspension containing approximately 1x10⁶ plaque-forming units (PFU) with the same volume of the appropriate NSC4492 working solution followed by incubation at the temperature and times indicated. As control, an equivalent aliquot of the virus suspension was incubated in parallel with MM under the same conditions. Then, samples were chilled, further diluted with MM and used to determine the remaining viral titer by plaque assay. In a similar inactivation assay, we have previously determined that the incubation of virus with MM containing DMSO 1:5000-1:100000 (dilutions of solvent corresponding to the working solutions of NSC4492) did not affect JUNV infectivity. Incubation times required to produce 50, 90 and 99 % reduction in virus titer, T-50, T-90 and T-99, respectively were calculated from the inactivation kinetics curve.

Virus adsorption and internalization

For evaluation of adsorption, Vero cells were infected with JUNV suspensions previously treated or not with NSC4492, at a multiplicity of infection (m.o.i.) of 1 PFU/cell. The indicated values of m.o.i. always refer to starting PFU activity previous to inactivation. After 5 and 60 min of adsorption at 4°C, cells were extensively washed with cold PBS and total RNA was extracted by using RNeasy Mini Kit (Qiagen) according to the manufacturer´s instructions. To monitor cell-bound viral RNA, cDNA was generated from purified RNA by using murine reverse transcriptase M-MLV (Invitrogen-Life Technologies) and random primers. This cDNA was amplified by real time PCR using SYBRGreen (Roche) detection and specific primers for the Z gene. Actin mRNA was amplified with the corresponding gene specific primers. The primer sequences and reaction conditions for real time RT-PCR were previously reported [32]. Average viral RNA Ct values were normalized to the average Ct values of actin and ΔΔCt based fold-change calculations were set relative to untreated-virus infected cells using Bio-Rad iQ5 2.1 software.

For quantification of internalized virus, after adsorption as above cells were further incubated in MM at 37°C for 1 h. Then, culture media were discarded; cells were washed with PBS and treated with a solution of 1 mg/ml proteinase K (Invitrogen-Life Technologies) for 45 min at 4°C. Proteinase K was inactivated with PBS- 0.2% bovine serum albumin (BSA) containing 2 mM phenylmethylsulfonyl fluoride, detached cells were transferred into a tube and washed twice with PBS-0.2 % BSA by low-speed centrifugation. Total RNA was extracted from pelleted cells and internalized viral RNA was evaluated by real time RT- PCR, as above.

Virus uncoating

Vero cells grown in coverslips were infected with NSC4492-treated or untreated JUNV at a m.o.i of 10 PFU/cell. After 1 h of adsorption at 4°C, cells were washed and incubated at 37°C in MM with the addition -or not- of concanamycin A at a final concentration of 50 nM. At the indicated times, cells were fixed in 4% paraformaldehyde for 10 min at 37 °C, then incubated with 20 mM NH₄Cl for 10 min at 37 °C and permeabilized with PBS 0.5 % Triton X-100 for 15 min at room temperature. Cytoplasmic NP immunofluorescence staining was carried out with the monoclonal antibody (mAb) SA02-BG12 [33], followed by Alexa Fluor 488-goat anti-mouse IgG (Invitrogen-Life Technologies) as secondary antibody. After a final washing with PBS, cells were mounted in a glycerol solution containing 1,4-diazabicyclo[2, 2, 2]octane (DABCO) and visualized under confocal fluorescence microscope.

Virus macromolecular synthesis

To analyze virus RNA synthesis, Vero cells were infected with NSC4492-treated or untreated JUNV at a m.o.i of 1 PFU/cell. At 1, 2.5, 5, 7 and 12 h p.i, total RNA was extracted by using RNeasy Mini Kit (Qiagen) and employed for cDNA synthesis by using M-MLV (Invitrogen-Life Technologies, USA) and a genomic sense JUNV GPC-specific primer. The cDNA was further amplified using GPC-specific primers by real time PCR as above. Average viral RNA Ct values were normalized to the average Ct values of actin and ΔΔCt based fold-change calculations for untreated and treated- virus infected cells were set relative to the value of untreated-virus infected cells at 1 h p.i., defined as 1, using Bio-Rad iQ5 2.1 software.

To determine viral protein expression, Vero cells grown in coverslips were infected with NSC4492-treated or untreated JUNV (m.o.i. 1 PFU/cell). At 16 h p.i., cells were fixed and processed for cytoplasmic and membrane immunofluorescence. For NP cytoplasmic staining, cells were fixed in methanol for 10 min at -20°C and then incubated with the mAb SA02-BG12, followed by fluorescein isothiocyanate (FITC)-goat anti-mouse IgG (Sigma Aldrich Co). For membrane staining, cells were fixed in 4% paraformaldehyde for 10 min at 37 °C and then incubated with 20 mM NH₄Cl for 10 min at 37 °C. Then, cells were incubated with mAb QC03-BF11 for GPC/GP1 [33], followed by the FITC-labeled IgG as secondary antibody. In both cases, after a final washing with PBS, cells were stained with Evans Blue and mounted in DABCO.

VLP purification and Western blotting

Approximately 4 x 10⁵ BSR cells grown in a 12-well dish were transfected with (amounts per well) 1 µg of plasmid pJUNV Z-HA, expressing an HA-tagged version of JUNV Z (JUNV Z-HA) along with 1 µg of plasmid pCMV-T7pol, which expresses the bacteriophage T7 RNA polymerase [34]. At 4 h post-transfection supernatants were removed, cells were washed twice with PBS, and supplemented with G-MEM 2% FBS containing or not NSC4492 at a final concentration of 25 µM. Control cultures were supplemented with medium plus the corresponding volume of DMSO. Following incubation at 37°C for 48 h, culture supernatants were harvested and cell monolayers were lysed in non-reducing SDS-PAGE sample buffer (Invitrogen Life-Techcnologies). VLPs were purified from the cell culture supernatants by ultracentrifugation through 20% (wt/vol) sucrose cushions at 34,000 rpm for 2 h at 4°C in a Beckman SW 50.1 rotor. Purified VLPs, resuspended in nonreducing sample buffer, and cellular lysates were resolved by SDS-PAGE in gels containing 12% polyacrylamide and then transferred to nitrocellulose membranes. Blots were probed with a rabbit anti-HA polyclonal antibody (Santa Cruz Biotechnology) for 2 h at 37°C followed by incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch) according to the supplier’s specifications. Detection was achieved by enhanced chemiluminescence, using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific). Quantification of protein bands were carried out by densitometry using ImageJ software [35]. To normalize the amount of Z to the amount of actin in cell extracts, blots were stripped and then reprobed with an anti-actin primary antibody (Sigma-Aldrich Co), followed by enhanced chemiluminescence and quantification by densitometry, as above.

JUNV inactivating activity of NCS4492: Time and temperature dependence

Previously, we performed a screening of a panel of aromatic disulfides and found that the carboxamide-derivatized NSC4492 (Figure 1A) is a very potent virucidal agent against two closely related NW arenaviruses, TCRV and JUNV, with inactivating concentration 50% (IC₅₀) values in the range of 0.2-0.7 µM [30]. To further characterize the inactivating properties of NSC4492, the temperature dependence of its biological effect was analyzed by incubation of JUNV in the presence of increasing concentrations of NSC4492 at 4, 25 or 37°C (Figure 1B). The results showed a very weak inactivating effect at 4°C, with about 40% remaining infectivity at concentrations of the compound as high as 20 µM. The inactivating effect was drastically enhanced when virus treatment was carried out at 25°C, being maximal upon incubation at 37°C (Figure 1B), indicating that the interaction between NSC4492 and virions is temperature dependent. Next, the kinetics of JUNV inactivation by NSC4492 was evaluated using the optimal temperature and compound concentration established from Figure 1B, 37°C and 10 µM. A very significant loss of infectivity was observed as early as 10-15 min after addition of the compound to the virus suspension since the value of T-50 obtained from data shown in Figure 1C was 10.5 ± 1.8 min. The virucidal effectiveness of the disulfide was further evidenced by the strong inactivation produced after 30 min of incubation, with more than 99 % reduction of viral infectivity and values of T-90 and T-99 of 23.0 ± 3.9 and 29.4 ± 0.3 min, respectively. Finally, complete inactivation of JUNV with more than 4 log reduction in virus titer was observed after 45 min of treatment. These results lead us to adopt incubation conditions assessing potent virus inactivation (10µM NSC4492 for 90 min at 37°C) for the following experiments.

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Figure 1. Time and temperature dependence of NSC4492 inactivating activity on JUNV.

(A) Chemical structure of NSC4492. (B) Suspensions containing 1 x 10⁶ PFU of JUNV were incubated with increasing concentrations of NSC4492 at 4°C (■), 25°C (●) or 37°C (▲) for 90 min. Then, the remaining infectivity was titrated by plaque assay in Vero cells. (C) Suspensions containing 1 x 10⁶ PFU of JUNV were incubated with 10 µM NSC4492 at 37°C. At the indicated times, the remaining infectious virus was determined as in (B). Each value represents the mean of triplicate assays ± standard deviation (SD).

https://doi.org/10.1371/journal.pone.0081251.g001

Mode of inactivation of JUNV virions by NSC4492

As a first approach to elucidate the mechanism of JUNV inactivation by the disulfide NSC4492, we analyzed the effect of the compound on virus entry. To this end, the binding ability of NSC4492-inactivated virions to the cell membrane was first evaluated. Vero cells were inoculated with JUNV previously treated or not with NSC4492 and the amount of cell-bound JUNV RNA that remained after extensive washing of the cell monolayers was quantified by real time RT- PCR. As shown in Figure 2A, treatment with NSC4492 did not alter the level of cell-associated viral RNA detected at the beginning (0 min) of the adsorption step. Moreover, similar levels of viral RNA were observed at 60 min post- inoculation with either treated or untreated virions, suggesting that the receptor binding capacity of inactivated JUNV is comparable to that of control infectious particles.

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Figure 2. Entry of JUNV particles treated with NSC4492.

(A) Binding. JUNV treated -or not- with 10 µM NSC4492 for 90 min at 37°C was adsorbed at 4°C to Vero cells. Immediately after inoculation (time 0) or 60 min later, the inoculum was removed, total RNA was extracted and the amount of cell-bound viral RNA was determined by real-time RT-PCR using JUNV Z gene specific primers and cellular actin amplification for normalization. (B) Internalization. Vero cells were inoculated with JUNV inactivated or not with NSC4492 as in (A). After adsorption for 1 h at 4°C followed by removal of the inoculum, cells were shifted to 37°C. At the indicated times, non internalized virus was removed by treatment with proteinase K, total RNA was extracted and real-time RT-PCR was performed as in (A) to determine the relative amount of internalized viral RNA. Results in (A) and (B) are expressed as fold difference of viral RNA level in cells infected with NSC4492-treated JUNV as compared to the corresponding untreated JUNV, set as 1. The values are averages of duplicate independent experiments ± SD. (C) Uncoating. Vero cells grown in coverslips were mock-infected or infected at 4°C with JUNV suspensions previously treated or not with NSC4492. Unadsorbed virus was removed and cells were supplemented with MM containing or not concanamycin A and incubation proceeded at 37°C for 5 or 60 min. Then, cells were processed to detect NP by IF staining. Representative cells of all fields in each sample are shown in the figure. Magnification: 600X plus digital zoom 2.5X.

https://doi.org/10.1371/journal.pone.0081251.g002

Next, a virus internalization assay was employed to analyze the effect of NSC4492 on virion uptake. Briefly, JUNV previously treated -or not- with NSC4492 was adsorbed to Vero cells for 1 h. Following incubation at 37°C to allow virus entry, cell monolayers were treated with proteinase K to remove adsorbed but not internalized virus, and intracellular JUNV RNA was quantified by real time RT- PCR. The results showed levels of viral RNA in cell monolayers inoculated with NSC4492-treated JUNV that were comparable to those in untreated JUNV-infected cells (Figure 2B). These results clearly demonstrated that NSC4492 did not impair the uptake of JUNV into the host cells.

To further assess the effect of NSC4492 on virus entry, we examined the ability of treated virus to exit the endosomal compartment and release the viral ribonucleoprotein into the cytoplasm. Cells were inoculated with untreated or NSC4492-treated virus and the presence of virions into cytoplasmic endosomal vesicles was detected by IF staining of NP. As control, untreated virus-infected cells were incubated with concanamycin A, a specific inhibitor of vacuolar-type ATPase activity that has been reported to raise endosomal pH of enveloped virus-exposed cells thus preventing membrane fusion [36,37]. As expected, cells infected with untreated virus showed a decreased amount of NP after 60 min of incubation at 37°C, as compared with that observed immediately after adsorption (Figure 2C), indicating that virion uncoating occurred and nucleocapsids were released from cellular endosomes after fusion. Cultures infected with NSC4492-treated virions revealed a pattern of NP immunofluorescence that was comparable to that observed for control untreated virus at 0 and 60 min of infection. In contrast, blockage of virus uncoating was visualized in cultures infected with control virus and treated with concanamycin A: a pattern of bright intracellular NP staining was observed at both times post-infection, with a strong accumulation of fluorescence in the perinuclear zone after 60 min of internalization (Figure 2C). When cells infected with NSC4492-inactivated JUNV were treated with concanamycin A, a similar pattern of NP fluorescence retention in the perinuclear region was detected (Figure 2C), indicative that the uncoating of inactivated virions is dependent on endosomal acidification, similar to untreated virus and in accordance with the penetration mechanism reported for JUNV [38,39]. Altogether, these results suggested that virion uncoating from the endosomal compartment was not impaired by treatment of JUNV with NSC4492.

After penetration into the host cell, synthesis of viral RNA and proteins is the subsequent step in virus multiplication cycle. First, to analyze the possible targeting of NSC4492 on viral RNA synthesis, the levels of JUNV RNA in cells infected with treated or untreated virions were comparatively quantified during 12 h of infection, the time required to complete the multiplication cycle of JUNV [40], by using real time RT- PCR. The amounts of viral RNA in cells infected with untreated or treated virions were calculated in comparison to the content of viral RNA in control infected cells at 1 h p.i., defined as 1. As seen in Figure 3A, the time course of JUNV RNA synthesis in control infected cells was in accordance with previous studies [32,41] with increasing levels of intracellular RNA from earlier to later times. At 1 h p.i., the content of viral RNA in cells infected with inactivated virions was similar to that in control infected cells, confirming that entry and uncoating are not affected and that the initial amount of RNA delivered into the cells through infection was similar. By contrast, the relative contents of viral RNA in cells infected with NSC4492-inactivated JUNV decreased with time, indicating that no new RNA molecules were synthesized whereas initially internalized viral RNA was degraded. The maximal difference between cells infected with control and treated virions was observed at 12 h p.i., when the peak in RNA synthesis was detected for untreated virions (Figure 3A). These results suggested that NSC4492 impairs the ability of virions to direct viral RNA synthesis.

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Figure 3. Analysis of viral macromolecule synthesis in cells infected with NSC4492-inactivated JUNV.

(A) RNA synthesis. Vero cells were infected with JUNV previously treated or not with NSC4492. Total cellular RNA was extracted at the indicated h p.i. and viral RNA was quantified by real-time RT-PCR using primers specific for JUNV GPC. Results are expressed as fold difference of viral RNA level in cells exposed to untreated or NSC4492-treated JUNV as compared to viral RNA level at 1 h p.i. in cells infected with untreated JUNV, reference point defined as 1. The values are averages of duplicate independent experiments ± SD. (B) Protein expression. Vero cells were mock-infected or were inoculated with untreated or NSC4492-inactivated JUNV. At 16 h p.i., either cytoplasmic or membrane IF staining was performed to detect NP or GP1/GPC proteins. Magnification: 400X.

https://doi.org/10.1371/journal.pone.0081251.g003

To further corroborate these results, the level of viral protein expression in infected cells was analyzed by indirect immunofluorescence. Both NP and GPC/GP1, the major structural arenavirus proteins, were clearly detected after 16 h of infection with untreated virus (Figure 3B). In contrast, no viral proteins could be observed either in the cytoplasm or the surface of cells infected with NSC4492-treated JUNV (Figure 3B). Altogether, these results demonstrated that treatment with NSC4492 completely abolished the ability of JUNV particles to drive the biosynthesis of viral macromolecules within the host cell.

Effect of NSC4492 on particle budding

After RNA and protein synthesis, virus assembly and budding represent the final step of JUNV multiplication cycle. It is known that arenavirus Z protein drives the cell surface budding of arenavirus particles in infected cells and is able to direct self-assembly and budding of VLPs, in the absence of any other viral protein [8,9,12,13,42,43]. To evaluate if NSC4492 affects these late processes, we analyzed the effect of the compound on the ability of Z protein to drive VLP formation by employing a well-established VLP assay previously reported [12]. Briefly, mammalian cells were transfected with the plasmids pJUNV Z-HA and pCMV-T7pol to express only JUNV Z-HA followed by incubation of cells with or without NSC4492 for 48 h. Then, VLPs were purified from cell supernatants, aliquots of both cell lysates and purified VLPs were resolved in SDS-polyacrylamide gels and Z protein was detected by immunoblotting (Figure 4). Reprobing of blots with an anti-actin antibody was performed to control for gel loading. Evaluation of the cell lysates revealed comparable levels of Z protein normalized to cellular actin in the presence or absence of the compound, both under reducing or non-reducing conditions (lanes 1 to 4), indicating that NSC4492 did not substantially affect Z expression in transfected cells. With respect to VLP formation, the level of Z self-budding, calculated as the ratio between Z protein detected in VLPs to total Z protein (VLPs plus lysates), was not diminished by NSC4492 (lanes 5 to 8). When samples were analyzed under reducing conditions, similar levels of Z as a monomer were detected in VLPs formed either in the presence or absence of the compound (lanes 5 and 6). By contrast, analysis under non-reducing conditions clearly showed an altered pattern of Z protein electrophoretic migration in VLPs released from NSC4492-treated cells compared to control samples, with decreased amount of Z monomer and increased amount of higher molecular weight multimers (lanes 7 and 8). Overall, these results suggested that NSC4492 altered the capacity of Z protein to self-aggregate into oligomers, although this alteration did not impact on the ability of Z to drive VLP formation.

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Figure 4. Effect of NSC4492 on Z-VLP production.

BSR cells were transfected with plasmids pJUNV Z-HA and pCMV-T7pol, to express JUNV Z-HA protein. After 4 h, transfection medium was replaced with fresh medium with (+) or without (-) 25 µM NCS4492 and incubation proceeded for 48 h. Aliquots of both cell extracts and VLPs purified from cell supernatants were boiled in SDS-PAGE sample buffer after addition (+) or not (-) of 0.1% dithiothreitol (DTT) and then analyzed by Western-blotting, using an anti-HA antibody. Protein bands were quantified by densitometry and the amount of Z in cell lysates was normalized to the amount of actin. Self-budding (%) corresponds to the ratio between Z protein detected in VLPs to total (VLPs plus lysates) Z protein. Bands consistent with monomeric, dimeric, and multimeric forms of Z are indicated.

https://doi.org/10.1371/journal.pone.0081251.g004