a Department of Virology and b Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194; c Department of Biochemistry, Shiga University of Medical Science, Seta, Ohtsu 520-2192; and d Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
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Abstract |
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Introduction |
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For more than a decade, we have screened and characterized antiviral chemicals obtained primarily from natural resources, including various algae and higher plants.47 Recently, we evaluated approximately 2250 samples from acetone extracts of cultured microorganisms for their anti-HSV-1 activity. One of them, extracted from a Streptomyces sp. strain, FV60, showed relatively strong activity in the screening assays. (1R,2R)-1-(5'-Methylfur-3'-yl)propane-1,2,3-triol (MFPT) was present in this extract. MFPT is a stable furan derivative easily transformed from sphydrofuran, a secondary metabolite produced by actinomycetes and initially isolated by Umezawa & Usui.8 In this paper, we report in vitro assessments of anti-HSV-1 activity.
The synthetic antiherpetic agents acyclovir,9 foscarnet,10 5'-iodo-2'-deoxyuridine (IdU)11 and trifluridine12 all inhibit virus replication by acting on viral DNA synthesis. A few natural products have been shown to affect the functions of HSV glycoproteins. Melittin, a 26 amino acid peptide contained in honey bee venom, is an inhibitor of Na+,K+-ATPase13 and inhibits syncytium formation if the mutation responsible maps to glycoprotein K (gK).14 A mutation in gK has been found in heparin-resistant virus.15 So far, there are no reports of agents that interact selectively with gC synthesis.
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Materials and methods |
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Streptomyces sp. strain FV60 was fermented in K medium (2.5% starch, 1.5% soy bean meal, 0.2% Ebios and 0.4% CaCO3, pH 6.2) at 27°C for 5 days. The culture broth of this strain (70 L) was centrifuged to isolate the mycelium. The mycelium was extracted with acetone and the extract was concentrated under reduced pressure to give a brown solid residue. The methanol-soluble part (58.4 g) of this residue was subjected to silica gel column chromatography (7.4 cm x 36.0 cm), with sequential elution with a CHCl3/methanol solvent system (with CHCl3, then with 1%, 2%, 5%, 10%, 20%, 50% and 80% methanol in CHCl3, and finally with methanol). The eluates were separated into six fractions on the basis of their thin layer chromatography (TLC) patterns: fraction 1 eluted with CHCl3; fraction 2 with 110% methanol in CHCl3; fraction 3 with 1020% methanol in CHCl3; fraction 4 with 2050% methanol in CHCl3; fraction 5 with 5080% methanol in CHCl3; and fraction 6 with methanol. The yields were as follows: fraction 1, 80 mg; fraction 2, 12.1 g; fraction 3, 8.1 g; fraction 4, 7.5 g; fraction 5, 5.5 g; and fraction 6, 14.1 g. Fraction 3 (a brown oil) was dissolved in CHCl3 and loaded on a silica gel column (3.5 cm x 46.0 cm). The column was eluted with a CHCl3/ methanol solvent system to give five fractions: fraction 3-1 eluted with 02% methanol in CHCl3; 3-2 with 25% methanol in CHCl3; 3-3 with 5% methanol in CHCl3; 3-4 with 10% methanol in CHCl3; and 3-5 with methanol. The yields were: fraction 3-1, 22 mg; fraction 3-2, 115 mg; fraction 3-3, 900 mg; fraction 3-4, 4.3 g; and fraction 3-5, 2.6 g. Fraction 3-4 showed a main spot of Rf 0.44 (CHCl3: methanol = 9:1) (200 mg) and was further purified by column chromatography on Toyopearl HW40F (1.5 cm x 63.0 cm; TOSOH Inc., Tokyo, Japan). The fractions showing a single spot on TLC were pooled and concentrated under vacuum to yield MFPT (117 mg) as a colourless oil. The total yield from the methanol-soluble fraction was 6.51%.
Cell and virus
Vero cells were grown in Eagle's minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS). HSV-1 (HF strain)16 was propagated on Vero cells. After three cycles of freezethawing and centrifugation (3000 rpm, 10 min), the supernatant was stored as an infectious preparation at 80°C.
Cytotoxicity
For the cytotoxicity assay, actively dividing subconfluent Vero cells were cultured for 72 h at 37°C in the presence of increasing amounts of MFPT. Viable cell yield was determined by the trypan blue exclusion test. Inhibition data were plotted as doseresponse curves, from which the 50% cell growth inhibitory concentration (CC50) was obtained.
Antiviral activity
Vero cell monolayers were infected with HSV-1 at 0.1 plaque-forming units (pfu) per cell at room temperature. Compound was added immediately after virus infection (0 h). After 1 h, the monolayers were washed three times with phosphate-buffered saline (PBS) and incubated in maintenance medium (MEM plus 2% FBS) at 37°C. Virus yields were determined by plaque assay at the 1 day incubation point. Antiviral activity is expressed as the 50% virus replication inhibitory concentration (IC50), the lowest concentration of MFPT that reduced plaque numbers by 50% in the treated cultures compared with untreated ones. In time-of-addition experiments, cells were infected with the virus at 10 pfu/cell, and MFPT was added at the indicated time.
Virucidal activity
HSV-1 (1 x 106 pfu) and MFPT were mixed and incubated for 0, 1, 2, 3 and 6 h at 37°C. The infectivity remaining in the mixtures was determined by plaque assay.
Assay for virus adsorption
The effect of MFPT on the binding process of HSV-1 was determined by means of an infectious centre assay.17 Briefly, Vero cell suspension was prepared to a final density of 4 x 106 cells/mL and cooled at 4°C for at least 2 h. HSV-1 (1 pfu/cell) and different concentrations of drug were cooled to 4°C and then added to the Vero cell suspension. The mixtures were incubated for 1 h at 4°C to prevent the virus from penetrating the cells. After the adsorption period, unbound virus and free drug were removed by washing three times with ice-cold PBS. The cell pellets were diluted serially with cold PBS and added to Vero cell monolayers, which were overlaid with maintenance medium containing 0.5% methylcellulose for plaque assay.
Assay of rate of virus penetration
Virus penetration into host cells was measured by the method reported by Huang & Wagner18 and modified by Highlander et al.19 Vero cell monolayers were cooled at 4°C for 2 h and incubated with HSV-1 at 4°C. After 1 h of adsorption, the inoculum was removed by washing three times with cold PBS, and fresh maintenance medium containing different concentrations of drug was added. Virus penetration was permitted by shifting the temperature to 37°C. At the indicated time, free virus was inactivated by treatment with citrate buffer (pH 3.0) for 1 min. The monolayers were overlaid with 0.5% methylcellulose and incubated for 1 day for plaque assay.
Assay of virus release into the medium
Vero cell monolayers were infected with HSV-1 at 0.1 pfu/ cell. After 1 h of adsorption, they were washed three times with PBS and incubated at 37°C in maintenance medium. After 30 min incubation, the cells were treated with citrate buffer (pH 3.0) to inactivate the free virus, and cultured in fresh maintenance medium containing different concentrations of drug. Twenty-four hours post-infection (p.i.), medium was harvested in to tubes. The infected cell monolayers were washed five times with cold PBS and subjected to three cycles of freezethawing. Virus yields in the medium and cells were separately determined by plaque assay.
SDSPAGE analysis of radiolabelled proteins
To analyse the effect of MFPT on protein synthesis in host cells, Vero cells in 24-well plates were radiolabelled with 1 µCi Tran35S-label (43.47 TBq/mmol; ICN Biomedicals, Inc., Costa Mesa, CA, USA) per well for 4 h in methionine-free MEM (Flow Laboratories, Irvine, UK) containing MFPT. In the infection experiments, Vero cells were infected with HSV-1 at 10 pfu/cell, washed with PBS and replenished with methionine-free MEM. To label immediate early () proteins of HSV-1, Vero cells were incubated in the presence of cycloheximide 50 mg/L from 2 h before (2 h) to 2 h after infection, infected with virus, starting at time 0, for 1 h, and then radiolabelled for 1 h with Tran35S-label in the presence of actinomycin D 10 mg/L. The drug was added from 0 to 2 h p.i., from 2 to 3 h p.i., or from 0 to 3 h p.i. To label both
and ß (early) proteins, Vero cells were treated with phosphonoacetic acid (PAA) 300 mg/L from 2 h before infection to 4 h p.i., and incubated from 0 to 4 h p.i. with the medium supplemented with drug and Tran35S-label. To label late (
) proteins, HSV-1-infected cells were incubated with MFPT for 8 h and radiolabelled with Tran35S-label from 4 h to 8 h p.i. Radiolabelled cells were harvested and extracted with cell lysis buffer (0.05 M TrisHCl pH 7.0, 0.15 M NaCl, 1% SDS, 1% Triton X-100). An aliquot of the cell lysates was treated with rabbit anti-HSV-1 serum4 and protein ASepharose 4 Fast Flow (Pharmacia Biotech AB, Uppsala, Sweden) overnight at 4°C with rocking. The immunoprecipitates and cell lysates were analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE) (8% polyacrylamide). After electrophoresis, the gels were soaked in 1 M sodium salicylate for 30 min, dried and exposed to X-ray films.20
Western blot analysis
Vero cells were infected with HSV-1 at 1 pfu/cell. After 24 h of incubation in the absence or presence of MFPT (10 or 50 µM), whole-cell extracts were prepared in cell lysis buffer. Cell extracts were subjected to 7.5% SDSPAGE and the resulting gels were blotted on nitrocellulose filters in 25 mM Tris, 192 mM glycine and 20% methanol. Blotted proteins were immunoreacted with rabbit antibodies prepared against synthesized 15-mer oligopeptides of HSV-1 gB (amino acids 6579), gC (amino acids 113127) and gD (amino acids 329343) coupled to thyroglobulin. Bands were revealed using a peroxidase-conjugated goat antirabbit IgG (heavy and light chains) (Cappel, West Chester, PA, USA) and 4-chloro-1-naphthol.
Examination of plaque morphology
Vero cell monolayers were infected with HSV-1 at a very low multiplicity (0.001 pfu/cell) and incubated in the absence or presence of 5 µM MFPT for 24 h. Plaque-forming cells were fixed and stained with Giemsa's solution.
Selection of MFPT-resistant virus by passage in MFPT
HSV-1 resistant to MFPT were selected by serial passage in the presence of MFPT. Confluent Vero cell monolayers were infected with the HF strain of HSV-1 at 0.1 pfu/cell, and incubated at 37°C in the presence of 10 µM MFPT. When the cytopathic effect was maximal, cultures were harvested and the titre was determined. Two serial passages were made at 0.1 pfu/cell in 50 µM MFPT, and then two serial passages in 200 µM MFPT. The progeny viruses were plaque purified and their sensitivities (IC50) were determined by plaque reduction assays, with increasing concentrations of MFPT in the overlying medium containing 0.5% methylcellulose.
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Results |
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The structure of MFPT is depicted in Figure 1. For the cytotoxicity assay, uninfected Vero cells were cultured for 72 h in the medium containing 12000 µM of MFPT (Figure 2
). At 2000 µM, MFPT caused marked cell lysis. The CC50 of the compound was 814 ± 65 µM. When Vero cells were infected with HSV-1, a dose-dependent inhibition of virus replication was observed in MFPT-treated cell cultures, and its IC50 was 1.16 ± 0.26 µM (Figure 2
). The resulting in vitro therapeutic index, calculated by dividing the CC50 by the IC50, was 700.
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To investigate the mechanism of antiviral action of MFPT, the drug-sensitive phases were determined by time-of-addition experiments. In these experiments, Vero cells were infected with HSV-1 at a high multiplicity of infection, 10 pfu/cell. As shown in Table I, MFPT potently inhibited viral replication in all treatments; its efficacy was lower in cultures only treated with drug for the first hour after infection. These results indicated that MFPT might interfere with very early to late stages of HSV-1 replication.
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In order to evaluate the stages of virus replication affected by MFPT, its inhibitory effect on virus adsorption was studied. The compound moderately interfered with viral attachment to host cell membranes at concentrations of 0.520 µM, but dose dependence was not clear: at 0.5, 2, 5 and 20 µM, the virus attachment was 82 ± 7.0%, 82 ± 5.0%, 58 ± 5.5% and 57 ± 4.0% of that in untreated controls. The kinetics of penetration were determined by inactivating the bound but unpenetrated viruses with a low pH citric acid buffer at various times after temperature shift from 4°C to 37°C (Table II). Penetration of HSV-1 into the cells was inhibited in a dose-dependent manner in the presence of 220 µM MFPT. At
5 µM, however, the number of plaques detected decreased with increasing time of treatment with MFPT: the plaque numbers at 6 h p.i. were 67%, 58% and 8% of those at 0.5 h p.i. at 5, 10 and 20 µM, respectively.
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Effects of MFPT on protein synthesis
MFPT did not inhibit protein synthesis of uninfected Vero cells at concentrations 50 µM (Figure 3a
, lanes a). When Vero cells were infected with HSV-1 and MFPT was added from 0 to 4 h p.i. in the presence of phosphonoacetic acid (PAA), an inhibitor of
protein synthesis that acts by inhibiting viral DNA polymerase and therefore viral DNA replication required for
gene expression,21 MFPT slightly inhibited the amount of virus-specific protein synthesis [Figure 3a
(lanes b) and b (lanes a)]. In infected cells treated with MFPT from 4 to 8 h p.i., no marked reduction in viral protein synthesis was detected [Figure 3a
(lanes c) and b (lanes b)]. However, protein(s) with a molecular weight of about 120 kDa were more mobile in cells that had been treated with 10 µM MFPT than in untreated cells. At 50 µM MFPT, this abnormality in molecular weight was detected more clearly. The effect of MFPT on HSV-1
proteins was unclear from the results obtained in Figure 3
. HSV
proteins accumulate in cells after treatment with actinomycin D following the infection of cells in the presence of cycloheximide.4 As shown in Figure 4
, the expression of
proteins was not markedly suppressed when infected cells were treated with 250 µM MFPT during transcription of viral DNA to mRNA (from 2 to 2 h p.i.), translation (23 h p.i.) or throughout both transcription and translation (from 2 to 3 h p.i.).
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After 24 h of treatment with 10 or 50 µM MFPT (Figure 5), no obvious changes in the synthesis of gB and gD were observed by immunoblotting: the total amounts and mobilities of these proteins appeared to remain equal in treated and untreated cells. The cell extracts yielded two species of gC with apparent molecular weights of 84 and 116 kDa. These species correspond to the precursor (pgC) and mature (gC) forms of gC, respectively.22 No marked change in pgC profile was induced by MFPT, but MFPT treatment induced a dose-dependent increase in the mobility of gC, indicating a decrease in the molecular weight of the glycoprotein.
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To confirm that MFPT inhibits virus release from infected cells, both cell-associated and extracellular viruses were separately titred by plaque assay. HSV-infected cells were treated with MFPT for 24 h as described in Materials and methods. Under these conditions, a dose-dependent reduction in the yield of cell-associated virus was observed in the range of 0.220 µM MFPT (Table III). Further reduction in extracellular viruses was found over the concentrations tested. Even at 0.2 µM, where MFPT did not decrease the yield of cell-associated viruses, there was approximately 20% repression of virus release into the medium.
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The HF strain of HSV-1 used in this study forms syncytial plaques as shown in Figure 6(a). When this strain was propagated in the presence of 5 µM MFPT, plaques with rounded cells were formed and no syncytium-forming plaques were observed (Figure 6b
). Plaques of MFPT-treated virus determined 24 h after infection were much smaller than those of untreated virus. At MFPT concentrations <2 µM, plaques with the morphology of both syncytium and rounded cells were formed, the ratio of rounded cells increasing with the drug concentration.
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After five passages of HSV-1 strain HF in increasing concentrations of MFPT, mutants that did not show the normal (syncytial) plaque morphology but showed rounding of cells were selected. Maximal cytopathic effect developed more quickly with the passage of progeny. Table IV shows the sensitivities of wild-type virus and two mutants to MFPT as well as their plaque morphology. The IC50s for MFPT-resistant strains 1 and 2 were 170 ± 18 and 147 ± 31 µM, respectively, compared with 1.6 ± 0.70 µM for the HF wild type. These mutants had similar sensitivities to MFPT, and were 92- to 106-fold more resistant than the parent virus.
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Discussion |
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As shown in Table III, a reduction of infectious cell-associated viruses was observed in cultures treated with 5 or 20 µM MFPT, with more significant reduction in release of viruses from the cells. Since conversion of high-mannose glycans into complex-type ones in Golgi apparatus appears to be required for the egress of the virus from HSV-infected cells,2831 inhibition of the maturation of virus glycoproteins would explain the decrease in released virus as observed in cell cultures treated with MFPT. When cells were treated with 10 µM MFPT at late stages of replication (48 h p.i.), virus-specific proteins were synthesized at levels almost equal to those in untreated controls (Figure 3
). At the highest concentration tested, 50 µM, the synthesis of some proteins was selectively inhibited while synthesis of other proteins was not markedly affected. At later stages of virus replication, viral glycoproteins are mainly expressed, and are involved in viral attachment and penetration, egress of progeny and cellcell fusion.32 Thus, specific detection of the glycoproteins would be expected to provide clues to the antiviral target of MFPT. When three glycoproteins (gB, gC and gD) were analysed by immunoblotting, only gC showed a markedly abnormal electrophoretic pattern: the levels of expression of pgC and gC were not affected in the presence of effective concentrations of MFPT, although a limitation of gC maturation was assumed from the detection of gC with reduced molecular weight. While gC enhances viral entry,33 it appears to inhibit the fusion of some cell cultures.24,34 At present, it is unknown whether MFPT affects the synthesis of gC. To elucidate the mechanism of action of the compound, it is important to obtain and characterize resistant mutants. In this study, it was shown that mutants that had abnormal plaque morphology (rounded cells) rendered the virus MFPT resistant. Since the abnormal plaque morphology of the mutants was stable in drug-free conditions, MFPT should have a selective effect against HSV-1 replication. In order to identify the virus-specific molecule(s) with which MFPT interacts, we are analysing the genetics of resistance to MFPT.
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Notes |
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References |
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Received 13 October 1999; returned 21 December 1999; revised 25 February 2000; accepted 10 April 2000