The decreasing of adenovirus type 1 resistance against interferon by methisazone in vitro

The mechanism of N-methyl-izatin-thiocarbazone (methisazone, Mel ВТ) antiviral activity has been studied on Ad 1-infected HEp2 and HeLa cells. Mel ВТ did not induce interferon and did not directly inhibit viral and cell translation. The adenoviral infection was not affected by recombinant human interferon a2 (rIFN). Mel ВТ showed antiviral effect in AdI-infected HEp2 or HeLa cells when rIFN had added to HeLa cells or in the period of interferon induction during virus infection (in HEp2 cells). In the presence of this compound, the El A transcription was unchanged in infected cells as compared to untreated control, while early transcription was decreased, the beginning of viral replication being retarded. Futhermore, the VAI RNA synthesis was also greatly suppressed. These effects were independent on interferon treatment and disappeared when MeIВТ had been added during the late phase of virus growth cycle. Actually, MeIВТ can induce the delay of VAI RNA transcription promoting interferon antiviral effect


The mechanism of N-methyl-izatin-thiocarbazone (methisazone, Mel ВТ) antiviral activity has been studied on Ad 1-infected HEp2 and HeLa cells. Mel ВТ did not induce interferon and did not directly inhibit viral and cell translation. The adenoviral infection was not affected by recombinant human interferon a 2 (rIFN). Mel ВТ showed antiviral effect in AdI-infected HEp2 or HeLa cells when rIFN had added to HeLa cells or in the period of interferon induction during virus infection (in HEp2 cells). In the presence of this compound, the El A transcription was unchanged in infected cells as compared to untreated control, while early transcription was decreased, the beginning of viral replication
being retarded.Futhermore, the VAI RNA synthesis was also greatly suppressed.

These effects were independent on interferon treatment and disappeared when MeIВТ had been added during the late phase of virus growth cycle. Actually, MeIВТ can induce the delay of VAI RNA transcription promoting interferon antiviral effect
Introduction.Methisazone (N-methyl-isatin-/i-thiosemicarbazone, N-MeIBT, MelBT) is a known antiviral drug studied earlier [1 ].Izatizon is an original liquid form of MelBT [2].Both izatizon and MelBT possess a large spectrum of antiviral action, but izatizon is more effective (20-100 times higher than MelBT) and demonstrates no toxic effects in therapeutical doses.Izatizon has been shown as an antiviral agent against poxviruses, herpesviruses, enteroviruses, influenza viruses A and B, etc After optimal schemes of drug application, 100 % curability was reported.So, the antiviral action of both MelBT and izatizon in vitro is identical [2].
The methisazone directed mechanism of cell defence is not known.Earlier published data demonstrate that MelBT reduces the late post-replicative protein synthesis in virus-infected cells [3][4][5][6].In the presence of this compound, a disruption of polysomes in vaccinia virus-infected cells has been also observed [7 ].However, MelBT induced no effect on in vitro translation [8 ], It inhibited different DNA-and RNA-viruses [9].The antiviral effect has been observed by treating the adenovirus-infected cells with MelBT during the early phase of infection [10].MelBT also showed a slight suppressive action on RNAsynthesis [8].Early studies have not detected the target of methisazone action in virus-infected cells.The activity of MelBT against vaccinia virus was disappeared when cell transcription had been blocked by actinomycin D [4].So, MelBT cannot provide antiviral action without cell transcription.Moreover, whereas the existence of MelBT-dependent and MelBT-resistant mutants of vaccinia virus has been also reported [11], it is likely some viral gene/protein are methisazone targets, the latter being cooperated with a cellular gene (or genes).
Here we demonstrate that MelBT is capable to reduce a viral defence against interferon antiviral action.Since MelBT does not inhibit the adenoviral infection without interferon, interferon is a cellular compound necessary for methisazone directed cell defence mechanism.
Materials and Methods.MelBT was synthesized as described previously (see [2]).To test its action, the compound was dissolved in dimethyl sulfoxide (or in mixture DMSO:PEG400, 1:3, v/v ), stock concentration being 10-100 mM; it was added to culture medium after virus absorption at different final concentrations (0.001-50 mM).The final concentration of each solvent was not higher than 0.5 %.No toxic effects of solvents were observed up to 2 % concentration.
Cells (HeLa and HEp2) were grown in the minimal essential Eagle medium supplemented with 10 % fetal calf serum, 1.2 mM glutamine and 0.2 % sodium bicarbonate, and a mixture of penicillin.streptomycin(up to 100 units/ml:40 mg/1).The cells were infected with human adenoviruses -Ad HI 3H10 (Adl) and Ad H2 (Ad2) with a moiety of infection 5 PFU per cell.After incubation at 37 °С, the Ad-infected cells were microscopically tested, washed by PBS, frozen at -70 °С and kept until futher studies.
The viral protein immuno assay.
The antiserum against Ad6 was obtained by intramuscular immunization of rabbits.The purification of the hexon Adl was provided by hydrophobic and ion exchange chromatography on the columns with buthyl-toyopearl («Toyosoda») and DEAE-Trisacryl («Pharmacia»).The purified hexon Adl was used for the performance of calibration curve.

3).
On the second stage, the Alul 548 bp fragment was extracted and cloned in the SmaI site of pUC19 {pVA\54%).On the third stage, the plasmid pVA224 was obtained (nt 10255 / Alul site/ to 10479 /Xbal site/) by removing XbaI-fragment from pVAI548.
To test ElA transcription, the synthetic oligonucleotide probe was synthesized (nt 831 to 853 of Ad2).

Hybridization procedures.
RNA preparations from infected cells were extracted as described [13].VAI RNA was purified according to O'Malley et al. [14].The DNA probes were labelled by nick-translation Promega kit with a-32 P-dCTP to a specific radioactivity of (1-5)-10 s cpm/mg of DNA.Electrophoresis of the denatured RNA samples in 2 % agarose gels containing 2 % formaldehyde, blot-transfer to Hybond N membrane, DNA-DNA and DNA-RNA hybridization to the 32 P-labelled DNA probes were performed as described previously [15].Filters were dissected and radioactivity of hybridized probes was detected according to Cerencov program.
Results.The cytotoxicity of MelBT was usually observed with drug concentration above 40 mM.The lower concentrations were inhibiting for the viral protein synthesis in a dose-dependent manner as expected from earlier results [10].The sensitivity of Adl to MelBT in vitro action was determined by immunoassay method.Our data concerning drugs have been shown no differences between MelBT and izatizon in vitro action.The decreasing of viral hexon synthesis was observed in МеІВТ-treated Adl-infected HEp2 cells (Fig. 1, я).The recombinant human interferon a 2 (rIFN) did not damage hexon synthesis was observed at the 5th h.Later the level of interferon increased, and the second maximum of interferon appeared at the 20-21th h p. i.After 40 h, the third maximum was also revealed.Existence of more than one maximum of interferon induction shows the possibility of virus reinfection beyond each virus growth cycle (16-18 h in our experiments).The infective process developed completely on the third day.Almost all the cells were infected and aggregated.On the contrary, Adl induced tenfold lower interferon titer in HeLa cells as compared to HEp2 ones (Fig. 2, b).The same results have been also obtained with HeLa S3 cells and Vero cells (data not shown).The drop of interferon expression correlates to the absence of MelBT antiviral action in virus-infected 77 synthesis, however MelBT antiviral effect increased in the presence of rIFN.On the other hand, the level of hexon accumulation did not decrease in МеІВТ-treated Adl-infected HeLa cells as compared to untreated culture (Fig. l, b).The rIFN did not also inhibit the adenoviral infection in HeLa cells.However, in the presence of both MelBT and rIFN, the antiviral effect appeared again (Fig. 1, b).Hexon accumulation in adenovirus-infected cells was sharply inhibited when the addition of MelBT (+ rIFN) is delayed until 8 h post infection (p.L).Increasing amounts of viral hexon appeared when addition of the drug is delayed beyond this time (not shown).Since adenoviruses are not sensitive to interferon antiviral effects, it is likely the antiviral activity of MelBT is due to activation of both early viral functions and interferon expression.
In the absence of MelBT, Adl stimulated interferon expression in HEp2 cells beginning from the 2nd h p. i. (Fig. 2, a HeLa ceils, and virus hexon synthesis was greatly reduced by simultaneously adding of rIFN and MelBT (see Fig. 1, b and table).
MelBT influenced on the induction of interferon in Adl-infected HEp2 cells (Fig. 2).The interferon expression being low until 8th h p. i. increased sharply after 8-9 h p. L and became further depressed after 12-14 h p. i.Another

Fig 4. The accumulation of DBF-(a), El A-(b), and VAI RNA-(c) transcripts during virus growth cycle in Adl-infected HEp2 cells. The extracts of Adl-infected MelBT (2.5 mM)-treated and untreated HEp2 cells (10° cells in each variant) were hybridized with nick-translated DNA; a-DNA-RNA dot probed with pDBP0.3;
b -DNA-RNA dot-hybridization with synthetic oligonucleotide probe (ElА); с -to test VAI RNA, the nick-translated probe pVA224 was used; 1 -Control; 2 -MelBT growth cycle has revealed that drug retards the beginning of viral DNA synthesis in Adl-infected cells as compared to untreated cells (Fig. 3, b).This effect was obtained when MelBT has been added until 8th h after infection, and disappeared when MelBT has been added later.So, MelBT cannot directly inhibit viral replication.Since the viral DNA-polymerase mRNA is transcribed from the early region of adenoviral genome [16], it is likely this effect of compound follows the delayed early transcription.maxima of interferon synthesis were slight.The appearence of virus cytopathic effect was delayed.Since MelBT (as well as izatizon) does not induce any type of interferon in vitro and in vivo [2], and possesses no direct action on translation [8] we conclude that this compound might act on the early viral transcription resulting to be the modification of interferon expression in virus-infected cells.
The possible mechanism of MelBT action makes predictions retarding the beginning ng of early viral transcription.Accordingly, we measured amounts of total viral RNA and viral DNA in extracts of with and without MelBT-treated infected or uninfected cells (HeLa and HEp2) by dot-hybridization method.In control cells no hybridization signal has been detected.After infection the amounts of viral RNA greatly increased especially from the beginning of the late phase of virus growth cycle (Fig. 3, a).After MelBT treatment the accumulation of early viral RNA was 7-10 fold reduced (Fig. 3, a) as compared to untreated Adl-infected cells up to the 8th h p. i., and increased beyond this time.As found previously on the model of vaccinia virus-infected cells [8], the 50 per cent inhibition of transcription has been also observed during the late phase of virus growth in MelBT-treated culture.Moreover, the time of half-life of RNA transcripts was identical in MelBT-treated and untreated cells [8 ].The delay of viral transcription appeared if MelBT had been added until 8 h p. i. and disappeared when MelBT had been added later.
Earlier it has been shown that MelBT does not inhibit vaccinia virus replication [5].The equal amounts of adenoviral DNA were observed in MelBT-treated cells and in untreated ones only on the late infection phase (18 h p. i.).However, the analysis of Adl DNA accumulation during the virus

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In addition, the same effect of MelBT was shown by testing DBP-gene transcription in Adl-infected cells.Hence, the accumulation of DBP-transcripts was reduced in МеІВТ-treated cells as compared to untreated culture during 8-10 h p. i. (Fig. 4, a).
To test whether MelBT treatment influenced on the transcription of early Adl 1A region (ElA), the amounts of E1A RNA were measured in Adl-infected cells during virus growth using dot-hybridization with polynucleotide kinasephosphorylated oligonucleotide probe.Our experimental data showed no influence of compound on El A transcription (Fig. 4, b).
Since MelBT depresses early transcription and viral replication in Adlinfected HEp2, HeLa, and HeLa S3 cells with and without interferon, it proves MelBT functions to be interferon-independent.
Adenoviruses are relatively resistant to interferon challenge [14], The loss of interferon antiviral activity is due to Pl/eIF2 kinase inhibition by viral VAI RNA.In the presence of interferon and small quantities of dsRNA, the kinase can phosphorylate initiation factor eIF2 inducing polysomes disruption and blocking translation [14].Earlier, the VAI RNA synthesis by RNA-polymerase III has been shown after viral replication beginning [16].Since the viral DNA-synthesis was retarded in МеІВТ-treated cells, we have proposed VAI RNA-gene transcription to decrease.To test the level of VAI RNA synthesis, the dot-hybridization has been used.Our data have been demonstrated VAI RNA accumulation to be retarded and reduced in МеІВТ-treated Adl-infected HEp2 cells as compared to untreated Adl-infected cells (Fig. 4, c).These data w r ere completely confirmed by Northern analysis of VAI RNA (Fig. 5, a).The loss of VAI RNA was not observed when the addition of drug has been provided on the late phase of virus growth cycle (Fig. 5, b).So, it is evident the antiviral agent does not directly suppress VAI RNA synthesis and RNA-polymerase III activity.The rIFN has also shown no effect on VAI RNA transcription (Fig. 5,  b).Moreover, the drop of VAI RNA transcription was appeared in MelBTtreated Adl-infected HeLa cells (Fig. 5, c) when the adenoviral infection was МеІВТ-resistant, and the Adl induced the low titer of interferon.Discussion.Our experimental data show that the main action of MelBT is related with the delay of both early adenoviral transcription and adenoviral replication.Since MelBT does not inhibit the viral protein synthesis without interferon and interferon does not inhibit viral infection without the drug, it is evident that MelBT can suppress the viral defence against interferon action.Actually, interferon is the necessary cell compound participating in the MelBT directed mechanism.How common is such a mechanism restoring interferon action?According to [14], the virus protection against interferon is dependent on VAI RNA which can inhibit interferon activated Pl/eIF2 kinase.The virus defence against interferon action is developed when large amount of VAI RNA appear during the late phase of virus growth cycle.Since the level of VAI RNA synthesis is sharply reduced in MelBT-treated Adl-infected cells, a high concentration of interferon is capable to increase the initiation of factor eIF2 phosphorylation balloned by translation blocking.Moreover, the rate of translation was greatly stimulated in cell-free extracts from MelBT+interferontreated Adl-infected cells when the purified eIF2 was added (data not shown).
The mode of MelBT action described here may be appropriate for another DNA-and RNA-viruses.In addition, we can suggest about the correlation between the interferon-inducing ability of viruses and the antiviral effect of this compound.Moreover, some other МеІВТ-susceptible viruses [9] possess an analogous protective mechanism against interferon action [17].Besides, methisazone does not significantly prevent the production of viral antigen by cells already chronically infected with HIV-1.However, in the presence of rIFN, the compound (nontoxic concentrations) can reduce the yield of infective virus in this culture by 10-100 fold.
The viral target of MelBT is still unknown.Since the MeiBT-resistant mutants of vaccinia virus has been obtained earlier [11], it is probable the existence of one virus gene/protein-target of MelBT.Our experiment showed the delay of early transcription in MelBT-treated Adl-infected cells, but the transcription of early region Al was not significantly changed.A protein encoded in El A region promotes the rapid onset of viral transcription.This phosphoprotein predicted from the sequence of its mRNA is to be 289 amino acids long (289 pp) and required to facilitate early transcription [18].Moreover, it contains a zinc-binding region [19].On the other hand, MelBT is known as an effective chelating agent [20]

Fig. 1 .
Fig. 1.The level of hexon synthesis in Adl-infected HEp2 cells (a) and HeLa cells (b) during virus growth cycle.MelBT (2.5 mM) and rIFN (1000 U/ml) were supplemented to the culture medium at 1 h p. i. Hexon assay were performed in the extracts of infected cells (10 ъ cells in each variant) frozen at -70 °С.Values represent the mean of 3 determinations.J -Control; 2 -MelBT; 3 -MelBT + rIFN; 4 -rIFN plasmid constructed by cloning of EcoRl B-fragment (nt 21338 to 25633) from total Ad2 DNA in the EcoRl site of plasmid pUC19.Then the 336 bp DBP-fragment was cloned in the BamHI site of pUC19 (pDBPO.3).To clone VAI RNA gene, the Hindlll B-fragment (nt 6231 to 11555) Ad2 DNA was excised and cloned in the HindUl site of pUC19 (pAdH5.

Fig. 5 .
Fig. 5. MelBT effect on VAI RNA synthesis.The RNA of Adl-infected cells (l(f cells in each variant) was extracted at the 16th h p. i. and run in electrophoresis.VAI RNA analysis was provided by Northern blot probed with pVA224 a -VAI RNA of Adl-infected HEp2 cells.10 ng purified VAI RNA (line 1) was presented.MelBT (2.5 mM) was added (line 2) or not (line 3) in the culture medium at the 1st h p. i.The low molecular RNA of Adl-infected HEp2 cells (the 1st h p.i. ) is also presented (line 4); b -50 ng of purified VAI RNA was added (line /).VAI RNA of Adl-infected untreated HEp2 cells (line 2), and VAI RNA of Adl-infected HEp2 cells when MelBT (2.5 mM) and rIFN (1000 U/ml) were simultaneously added at the 8th h (line 3) and at the 1st h (line 4) p. i; с -VAI RNA analysis in Adl-infected HeLa cells.50 ng purified VAI RNA was run (line 7).MelBT (2.5 mM) was added (line 2) or not (line 3) at the 1st h p. i.