Journal of Chemotherapy
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Effect of Biological and Synthetic Polymers on BK Virus Infectivity and Hemagglutination L. Sinibaldi, V. Pietropaolo, P. Goldoni, C. Di Taranto & N. Orsi To cite this article: L. Sinibaldi, V. Pietropaolo, P. Goldoni, C. Di Taranto & N. Orsi (1992) Effect of Biological and Synthetic Polymers on BK Virus Infectivity and Hemagglutination, Journal of Chemotherapy, 4:1, 16-22, DOI: 10.1080/1120009X.1992.11739132 To link to this article: http://dx.doi.org/10.1080/1120009X.1992.11739132
Published online: 15 Jul 2016.
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Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [Monash University Library]
Date: 30 November 2016, At: 22:29
Journal of Chemotherapy
Effect of Biological and Synthetic Polymers on BK Virus Infectivity and Hemagglutination L. SINIBALDI - V. PIETROPAOLO P. GOLDONI- C. DITARANTO N. ORSI
Summary ------------------------------The effect of several biological and synthetic polymers, chosen on the basis of different physical and chemical properties, was investigated on BK virus infectivity and hemagglutination. It was observed that polyanions like mucin, dextran sulfate and heparin depressed the viral binding, whereas polycations hod no significant activity, with the exception of polyL-Iysine, which enhanced it. The effect of the active polymers was studied in different experimental conditions and the results obtained suggested that polyanions may oct directly on the virus particle, whereas the target of polycations could be at the level of cell membranes. However, the effect shown by the active compounds did not appear to be simply related to the electric charge since neutral compounds, such as tamarind gum and locust bean gum, showed a marked inhibitory effect on BK virus binding to the cells. Key words: BK virus, polymers, infectivity, hemagglutination.
Istituto Pas teur - Fondazione Ce nci Bolognetti. Institute of Microbiology, Faculty of Medicine, University of Rome «La Sapienza», Italy. A11thor's address: L. Sinibaldi, Institute of Microbiology, Faculty of Medicine, University of Rome "«La Sapienza». Piazzale Aida Mora, 5 - 00185 Rome, Italy. © Edhioni Riviste Scientifiche - Firenze
Vol. 4 - n. 1 (16-22) - 1992
INTRODUCTION
The first event in viral infection is represented by the binding of viral recognition units to specific cell membrane receptors, a process which involves different forces, such as electrostatic interactions, Van der Waal's forces, hydrogen and hydrophobic bonds. This phase of viral attachment can therefore be highly influenced by all the conditions and molecules capable of modifying the charge distribution on both the ligand and the receptor 1 • Previous investigations have shown that polyelectrolytes have a noticeable effect on the ad sorption of different enveloped viruses 2-8 although naked viruses such as picornavirus have also been considered 7 • 9 • In general it has been noticed that polyanions depress the viral binding 2 • 5 • 7 ' 8 ' 10 , whereas polycations h ave an enhancing effect 3 • 8 • 9 • 1 1 • However the results obtained in all these investigations are not univocal and do not appear to be simply related to the anionic or cationic nature of the compound. Although electrostatic interactions play an essential role in the viral binding to the cell membrane, it has been pointed out 10 that more specific features other than electric charge play a role in the action of these compounds. We have therefore investigated the effect shown by different biological and synthetic polymers on the cellular binding of BK virus, a naked Papovavirus 12 , which is known to be widely spread in human populations 13 • 14 and highly oncogenic in some laboratory animal species 15 • 16 • The compounds tested were chosen on the basis of different physical and chemical properties, such as the electric charge, the molecular weight and the content of sulfate groups, and the results obtained are here reported. ISSN 1120-009X
EFFECT OF BIOLOGICAL AND SYNTH ETIC POLYMERS ON BK VIRUS INFECTIVITY AND HEMAGGLUTINATION
MATERIALS AND METHODS
Cells Vero cells, a monkey kidney cell line, were grown at 37°C in 199 medium (Flow Laboratories) supplemented with 10% inactivated fetal calf serum (FCS), penicillin (100 IU/ml), streptomycin (100 ~-tg/ml) and 2 mM glutamine (growth medium). For cell maintenance the serum concentration was lowered to 2% (maintenance medium).
Virus BK virus (Gardner prototype strain) was propagated in Vero cells grown in 199 medium supplemented with 10% FCS. After adsorption for 1 h at 37°C, the infected cells were incubated in 199 medium with 2% FCS for 21 days at 37°C. The supernatant was then collected, clarified by centrifugation at 3,000 rpm for 30 min to remove cell debris and stored at - 70°C.
Chemicals The activity of the following substances was tested: dextran sulfate sodium salt (MW 5,000 and 500,000), mucin from bovine submaxillary glands, polymyxin B sulfate, histone from calf thymus, heparin sodium salt from porcine intestinal mucosa, poly-L-lysine hydrobromide (MW 10,200, 26,500 and 141,000), DEAE-Dextran and locust bean gum from Sigma Chemical Co. St. Louis, USA; Keltro TF from Kelco, Division of Merk and Co., USA; scleroglucan from Sanofi, Bio-Industries, Paris; alginic acid from Fluka, Switzerland; tamarind gum (or glyloid) supplied by Dainippon Pharmaceutical Co., Osaka and purified in the laboratories of Francesco Angelini Research Institute - Italy; three distinct sulfated tamarind gums (glyloid sulfate) differing in the content of sulfur (i.e . different degree of sulfation), namely: GP4311, S = 0.41%; GP4327, S = 2.2%; GP4324, S = 7.1% were prepared from purified glyloid and kindly supplied by the same laboratories. Stock solutions were made up in PBS (phosphate buffered saline) pH 7 .2, stored at - 20°C and further diluted in cell culture medium before use .
17
Vera cell toxicity assay In order to establish the maximal non-cytotoxic concentrations, two-fold serial dilutions of the drugs in serum-free medium were incubated for 1 h at 37°C with Vera cell monolayers grown in 96-well plates (Falcon). The assays were carried out in duplicate. The drugs were then removed and cells, washed three times with PBS, were overlaid with maintenance medium. After 5 days at 37°C in 5% C0 2, cell morphology, viability (as determined by neutral red uptake in dispersed cells) and yield were examined. Drug dilutions which did not affect any of these parameters in 100% of cells were considered as non-cytotoxic concentrations (Table 1).
Effect of polymers on BK virus infection Cells were grown in microtissue chamber slides (Lab-tek, Miles Laboratory) for 24 h in 5% C02 at 37°C. The monolayers were placed in an ice-water bath, washed with ice cold medium and infected with BK virus suspended in precooled medium at concentrations capable of infecting 50% of the untreated cells. After 1 h at 0°C, the cells were washed with prewarmed medium and incubated for 5 days, in 5% C02 at 3 7 ° C. The percentage of infected cells was determined by indirect immunofluorescence. Different types of experiments were carried out: i) Cells were preincubated at 37°C for 1 h with the drugs which were removed just before virus infection; ii) Drugs were incubated with the cells during the virus attachment step at 0°C for 1 h. They were then removed together with the virus inoculum before the temperature shift to 37°C. iii) Virus was preincubated with the drugs at 26°C for 2 h, then the mixtures were added to the monolayers at 3 7°C for 1 h.
Immunofluorescence Virus-infected cell monolayers were washed with PBS and fixed with absolute acetone for 5 min at - 20°C. After incubation with anti-BK human serum for 1 hat 37°C, cells were washed with PBS and stained with fluorescein isothyo-
18
L. SINIBALDI - V. PIETROPAOLO - P . GOLDONI - C. DI TARANTO - N. ORSI
TABLE 1 -
Toxicity of different polymers. On Vero cells
On red cells
Polymers Highest non-toxic concentration (mg/ml)
Acid polymers (Polyanions) Heparin
2.500
10.000
Mucin
0.018
0.195
Dextran sulfate (MW 5,000)
0.500
0.062
Dextran sulfate (MW 500,000)
0.625
0.625
Polymyxin B sulfate
1.560
0.014
Alginic acid
0.025
0.030
Keltrol
0.025
0.030
Glyloid sulfate 4311
0.025
0.030
Glyloid sulfate 4327
0.025
0.062
Glyloid sulfate 4324
0.025
0.062
Poly-L-Iysine (MW 10,200)
0.010
0.006
Poly-L-lysine (MW 26,500)
0.031
0.0005
Poly-L-lysine (MW 141 ,000)
0.016
0.0002
DEAE-Dextran
0.500
0.0005
Histone
0.625
0.625
Locust bean gum
0.025
0.030
Tamarind gum
0.025
0.030
Scleroglucan
0.025
0.030
Basic polymers (Polycations)
Neutral polymers
cyanate-conjugated anti-human gammaglobulin antibodies (Behring). After 1 h at 37°C, cells were washed with PBS, mounted in buffered glycerol and examined under a Leitz fluorescence microscope . The results were expressed as the percentage of infected cells in treated versus untreated cultures.
Erythrocytes An 8% suspension in PBS of human group 0 Rh + erythrocytes was used .
Erythrocyte toxicity assay The test was carried out by means of a double series of two-fold dilutions of the drugs in HSA (Hepes Saline Albumin) - Buffer pH 7.2 and by adding to each dilution 50 J..l.l of a 0.2% suspension of erythrocytes in HSA. After 24 h of contact at + 4°C the normal sedimentation of red cells or the degree of the eventual hemolysis were checked (Table 1) .
Hemagglutination (HA) and hemagglutination inhibiting (HI) tests These tests were carried out in plastic microplates as previously described 17 •
Control of HA and HI activity of polymers towards BK virus The compounds (at the highest non-cytotoxic concentration) were serially diluted and each dilution was mixed with four hemagglutinating doses of virus. The addition of the erythrocytes to the virus-drug mixture took place immediately.
EHect of polymers on BK virus and on erythrocytes The various substances have been tested directly on both the BK virus and the erythrocytes . In the first case serial dilutions of virus were tested with the highest non-toxic concentration of each compound. After 1 h of contact at room temperature the erythrocytes were added . In order to ascertain whether the various compounds had a direct effect on red cells, the erythrocyte suspension was added to the highest non-toxic concentration of each substance and after 1 h of contact at room temperature the mixture was added to serial viral dilutions.
RESULTS
a) EHect of polymers on BK virus infectivity. This was studied by adding the maximal non-cytotoxic concentration of compounds to the Vera cell monolayers for 1 h prior to infection and incubated at 37°C . The tissue cultures
19
EFFECT OF BIOLOGICAL AND SYNTHETIC POLYMERS ON BK VIRUS INFECTIVITY AND HEMAGGLUTINATION
were then washed and infected with appropriately diluted stock virus . The results obtained in these experiments showed that the pre-exposure of cells to the drugs exerted a slight effect on subsequent BK virus infection as monitored by indirect immunofluorescence (Table 2: b). In parallel, virus infection was synchronized by a temperature shift as described in Materials and Methods in order to verify whether the drug effect took place during the attachment step. In this case the maximal non-cytotoxic concentration of polymers was added to the cells together with the virus during the incubation at 0°C and then removed after 1 h. In this second type of experiment the results obtained showed that among all compounds tested polyanions such as mucin, heparin, dextran sulfate 500,000, alginic acid and glyloid sulfate significantly depressed virus adsorption, whereas among polycations, poly-L-lysine was able to enhance the susceptibility of Vero cells to BK virus adsorption. As to neutral polymers, locust bean gum and tamarind gum showed a strong reducing effect similar to that obtained with the most active polyanions (Table 2: a). An analysis of the data obtained showed that the activity of compounds did not appear exclusively related to the charge of the molecules, since some neutral polymers appeared to be very effective. As to the importance of the molecular weight, it will be seen that the highest effect was obtained with dextran sulfate of 500,000 molecular weight (MW) whereas among the various poly-L-lysines tested the most enhancing action referred to poly-L-lysine of 141,000 MW (Table 2: a, b). Results reported in Table 2 also show that some polyelectrolytes (polymyxin B sulfate, histone and DEAE Dextran) were devoid of any activity in the case of the BK virus we tested. In order to ascertain the site of action, i.e . the target of polymers, experiments were carried out by preincubation of the virus with the drugs at 26°C for 2 h before infection. After this time of contact, the mixtures were added to the cell monolayers at 37°C for 1 h. Virus pretreated with neutral and acid polymers showed, in general, a reduction of infection as monitored by indirect immunofluorescence, whereas preincubation of virus with basic polymers had no effect on subsequent infection (Table 2: c).
TABLE 2 · Effect of polymers on the early steps of BK virus
infection. Polymers maximal non-cytotoxic concentration)
Immunofluorescence (% ) a
b
c
None
50
50
50
Heparin
10
30
10
5
25
10
Dextran sulfate (MW 5,000)
35
40
35
Dextran sulfate (MW 500,000)
5
30
5
Polymyxin B sulfate
50
50
50
Alginic acid
20
30
15
Keltrol
35
40
20
Glyloid sulfate 4311
10
20
25
Glyloid sulfate 4327
25
40
35
Glyloid sulfate 4324
20
30
20
Poly-L-Iysine (MW 10,200)
70
60
50
Poly-L-Iysine (MW 26,500)
80
60
50
Poly-L-Iysine
95
80
50
Mucin
(MW 141,000) DEAE-Dextran
50
50
50
Histone
50
50
50
Locust bean gum Tamarind gum Scleroglucan
10 10 40
30 25 45
15 30 40
a = The polymers were incubated with Vera cells during the virus attachment step at oac for 1 h; then they were removed together with the virus inoculum before the temperature shift to 3 7°C. b = Vera cells were preincubated at 37°C for 1 h with the polymers which were removed just before virus infec tion. c = BK virus was preincubated with the polymers at 26°C for 2 h, then the mixtures were added to cell monolayers at 37 °C for 1 h.
b) Effect of polymers on BK hemagglutination. The effect of compounds on BK hemagglutination was studied by different experimental approaches. The various drugs were tested separately on both the BK virus and the erythrocytes in order to verify whether a reduction or an enhancement of the hemagglutination took place.
L. SINIBALDI - V. PIETROPAOLO - P. GOLDONI - C. DI TARANTO - N. ORSI
20
TABLE 3 - Action of polymers on BK hemagglutination. Polymers (maximal non-cytotoxic concentration) Control Heparin
HA titer a
b
128
128
128
128
96
96
Dextran sulfate (MW 5,000)
128
128
Dextran sulfate (MW 500,000)
56
64
128
128
96
128
128
128
Mucin
Polymyxin B sulfate Alginic acid Keltrol Glyloid sulfate 4311
128
128
Glyloid sulfate 4327
384
256
Glyloid sulfate 4324
256
128
Poly-L-lysine (MW 10,200)
160
288
Poly-L-lysine (MW 26,500)
384
384
Poly-L-lysine (MW 141,000)
768
5 12
DEAE-Dextran
256
512
Histone
128
128
Locust bean gum Tamarind gum Scleroglucan
128 64 96
128 64 64
a = Serial dilutions of BK virus were tested with the highes t non-toxic concentration of each compound. After 1 h of contact at room temperature the erythrocytes were added . b = The erythrocyte suspension was added to the highest non-toxic concentration of each compound and after 1 h of contact at room temperature serial viral dilutions were added to the mixture.
In Table 3 (a) the HA titers obtained after contact of the BK virus with the various substances are reported. It appears that dextran sulfate 500,000 (MW) and, to a lesser extent, mucin, tamarind gum, scleroglucan and alginic acid noticeably reduced the HA activity of BK virus whereas DEAE-Dextran, gly loid sulfate and poly-L-lysine enhanced it.
Among the various preparations of poly-Llysine and glyloid sulfate tested, the most active compounds were respectively poly-L-lysine 141,000 and glyloid sulfate 4327. Table 3 (b) reports the HA titer after contact of all compounds with the erythrocytes. Results showed that mucin, scleroglucan, tamarind gum and dextran sulfate 500,000 MW kept in contact with cells before the addition of virus, reduced the HA activity. In the case of poly-Llysine, DEAE-Dextran and glyloid sulfate, the preliminary contact between the substances and red cells produced an increase in the HA titer. DISCUSSION
Results reported here confirm the role which electrostatic forces play in the early interactions between BK virus and host cells and that polyelectrolytes are able to modify these interactions and consequently viral infectivity. In agreement with results obtained by other Authors, we have noticed that, as also in the case of the BK virus, polyanions depress viral binding, whereas polycations can enhance it; however some of the compounds we tested (DEAE-Dextran and histone among polycations and polymyxin B sulfate among polyanions) are devoid of any effect. However, the effect on viral infectivity is not shown only by polyelectrolytes but also by neutral polymers such as tamarind gum and locust bean gum, which significantly decrease the susceptibility of Vero cells to BK virus infectivity. This interesting observation indicates that in the polymers tested the electric charge of the molecule is not sufficient by itself to explain the effect of these compounds. This hypothesis has already been suggested in preceding research 10 and is clearly demonstrated by our results which show a marked effect also in the use of tamarind gum and locust bean gum, which have a neutral charge . The effect of the active compounds was studied by different types of experiments, i.e., by preincubating the substances with virus or the cells and by the contemporary addition of virus and drugs to the cells at the moment of infection, at 0°C. The greatest effect on virus infection was noticed in this last case. In particular among
EFFECT OF BIOLOGICAL AND SYNTHETIC POLYMERS ON BK VIRUS INFECTIVITY AND HEMAGGLUTINATION
polyanions, mucin, heparin, dextran sulfate 500,000, alginic acid and glyloid sulfate reduce BK virus infectivity very strongly, whereas among polycations, poly-L-lysine (MW. 10,200, 26,500, 141,000) enhance it. Moreover even neutral polymers such as locust bean gum and tamarind gum produce a marked inhibition of BK virus infectivity. The same reducing effect is noticed also in the experiments in which Vera cells are preincubated with acid or neutral polymers before virus infection, but to a lesser degree. An enhancement of viral infectivity is, on the other hand, observed when a polycation such as poly-Llysine is used in the same experimental conditions. This last result is of interest when taking into account the well-known action of polycations on plasma membrane permeability 18 • Finally, in the experiments with BK virus preincubated with polymers, our results show a slight effect on the infection only in the case of virus pretreated with acid or neutral polymers, whereas preincubation of virus with basic polymers has no effect on subsequent infection. The results obtained in these three types of experiments suggest that polyanions act directly on the viral capsid proteins since a low effect is obtained when the compounds are preincubated with cells, before virus addition . A similar action can be argued also for neutral polymers. Another interesting indication of the mechanism of the action of the drugs tested is obtained by using different concentrations of compounds (heparin and dextran sulfate) showing low cell toxicity. In the case of heparin, for example, it is possible to notice that concentrations included in a range between 2.500 mg/ml and 0.025 mgfml did not show a dose-effect ratio, and all the concentrations were practically equally active (data not shown). As to the importance of the molecular weight of compounds on their biological effect on BK virus infectivity, results obtained by the use of two preparations of dextran sulfate (MW 5,000 and 500,000) and three preparations of poly-L-lysine (MW 10,200, 26,500 and 141,000) show that the most active compounds are those with the highest molecular weight. As to the role played by the content of sulfate groups present in the same type of com-
21
pound, experiments carried out with glyloid sulfate 4311, glyloid sulfate 4324 and glyloid sulfate 4 327, do not show a correlation between this parameter and the biological activity on BK virus infection. As to the importance played by the viscosity of the different compounds in their biological effect, it is not possible to obtain any indication since our experiments are carried out with very low concentrations of the compounds in order to avoid any toxic effect on the cells. As to the results obtained by studying the effect of polymers on BK virus hemagglutination, among all compounds tested dextran sulfate 500,000 and, to a lesser extent, mucin, tamarind gum, scleroglucan and alginic acid reduce the HA activity of BK virus whereas DEAE dextran, glyloid sulfate and poly,L-Lysine are able to enhance the susceptibility of erythrocytes to BK virus hemagglutination and consequently the HA titer. All these data concerning the effect of biological and synthetic polymers on BK virus hemagglutination fundamentally confirm the results obtained in the infectivity test, although to a lesser extent. This is probably due to the fact that the hemagglutination by the BK virus is a less sensitive biological system compared to that represented by the infectivity of the same virus. AcKNOWLEDGEMENT - We thank Prof. Rosa rio Nicoletti of the University of Rome «La Sapienza» for rem arks and helpful di scussion and Prof. Giuseppe Barbanti Brodano of the University of Ferrara for having provided the BK virus. The authors would like to express their appreciation to Mrs. Marina Fortunato for skillful technical assistance.
REFERENCES ' Lonberg-Holm K . Attachment of animal viru s to cell, an introduction . In: Receptors and Recognition Series B, Vol. 8, Virus Receptors, Part 2 (Lonberg-Holm K. and Philipson L. eds) London : Chapman and Hall, 1980: 1-20 . 2 Vaheri A. Heparin and heparinoids as virus inhibitors. Acta Pathol Microbial Scand (Suppl) 1964; 171 : 7-97 . 1 Kaplan MM, Wiktor TJ, Maes RF, Campbell JB , Koprowski H . Effect of polyions on the infec tivity of rabies virus in tissue culture: construction of a single-cycle growth curve. J Viral 1964; 1: 145-151. • Toyoshima K, Vogt PK. Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions. Virology 1969 ; 38: 414-426 . 1 Baba M , Pauwels R, Balzarini J , Arnout J , Des myter J, De Clercq E. Mechanism of inhibitory effect of dextran sulphate and heparin on replication of human immunodefi -
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L. SINIBALDI
ciency virus in vitro. Proc Nat! Acad Sci USA 1988; 85: 61326136. 6 Langeland N, Moore LJ, Holmsen H, Haarr L. Interaction of poly lysine with the cellular receptor for herpes simplex virus type 1. J Gen Virol 1988; 69: 1137-1145. ' Mastromarino P, Conti C, Petruzziello R, Lapadula R, Orsi N. Effect of polyions of the early events of Sindbis virus infection of Vero cells. Arch Virol 1991 (in press) . 1 Conti C, Mastromarino P, Riccioli A, Orsi N. Electrostatic interactions in the early events of VSV infection. Res Virol 1991; 142: 17-24. ' Liebhaber K, Takemoto KK. The basis for the size differences in plaques produced by variants of encephalomyocarditis (ECM) virus. Virology 1963; 20: 566-599 . 10 Mastromarino P, Seganti L, Petruzziello R,
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Effect of Biological and Synthetic Polymers on BK Virus Infectivity and Hemagglutination L. Sinibaldi, V. Pietropaolo, P. Goldoni, C. Di Taranto & N. Orsi To cite this article: L. Sinibaldi, V. Pietropaolo, P. Goldoni, C. Di Taranto & N. Orsi (1992) Effect of Biological and Synthetic Polymers on BK Virus Infectivity and Hemagglutination, Journal of Chemotherapy, 4:1, 16-22, DOI: 10.1080/1120009X.1992.11739132 To link to this article: http://dx.doi.org/10.1080/1120009X.1992.11739132
Published online: 15 Jul 2016.
Submit your article to this journal
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [Monash University Library]
Date: 30 November 2016, At: 22:29
Journal of Chemotherapy
Effect of Biological and Synthetic Polymers on BK Virus Infectivity and Hemagglutination L. SINIBALDI - V. PIETROPAOLO P. GOLDONI- C. DITARANTO N. ORSI
Summary ------------------------------The effect of several biological and synthetic polymers, chosen on the basis of different physical and chemical properties, was investigated on BK virus infectivity and hemagglutination. It was observed that polyanions like mucin, dextran sulfate and heparin depressed the viral binding, whereas polycations hod no significant activity, with the exception of polyL-Iysine, which enhanced it. The effect of the active polymers was studied in different experimental conditions and the results obtained suggested that polyanions may oct directly on the virus particle, whereas the target of polycations could be at the level of cell membranes. However, the effect shown by the active compounds did not appear to be simply related to the electric charge since neutral compounds, such as tamarind gum and locust bean gum, showed a marked inhibitory effect on BK virus binding to the cells. Key words: BK virus, polymers, infectivity, hemagglutination.
Istituto Pas teur - Fondazione Ce nci Bolognetti. Institute of Microbiology, Faculty of Medicine, University of Rome «La Sapienza», Italy. A11thor's address: L. Sinibaldi, Institute of Microbiology, Faculty of Medicine, University of Rome "«La Sapienza». Piazzale Aida Mora, 5 - 00185 Rome, Italy. © Edhioni Riviste Scientifiche - Firenze
Vol. 4 - n. 1 (16-22) - 1992
INTRODUCTION
The first event in viral infection is represented by the binding of viral recognition units to specific cell membrane receptors, a process which involves different forces, such as electrostatic interactions, Van der Waal's forces, hydrogen and hydrophobic bonds. This phase of viral attachment can therefore be highly influenced by all the conditions and molecules capable of modifying the charge distribution on both the ligand and the receptor 1 • Previous investigations have shown that polyelectrolytes have a noticeable effect on the ad sorption of different enveloped viruses 2-8 although naked viruses such as picornavirus have also been considered 7 • 9 • In general it has been noticed that polyanions depress the viral binding 2 • 5 • 7 ' 8 ' 10 , whereas polycations h ave an enhancing effect 3 • 8 • 9 • 1 1 • However the results obtained in all these investigations are not univocal and do not appear to be simply related to the anionic or cationic nature of the compound. Although electrostatic interactions play an essential role in the viral binding to the cell membrane, it has been pointed out 10 that more specific features other than electric charge play a role in the action of these compounds. We have therefore investigated the effect shown by different biological and synthetic polymers on the cellular binding of BK virus, a naked Papovavirus 12 , which is known to be widely spread in human populations 13 • 14 and highly oncogenic in some laboratory animal species 15 • 16 • The compounds tested were chosen on the basis of different physical and chemical properties, such as the electric charge, the molecular weight and the content of sulfate groups, and the results obtained are here reported. ISSN 1120-009X
EFFECT OF BIOLOGICAL AND SYNTH ETIC POLYMERS ON BK VIRUS INFECTIVITY AND HEMAGGLUTINATION
MATERIALS AND METHODS
Cells Vero cells, a monkey kidney cell line, were grown at 37°C in 199 medium (Flow Laboratories) supplemented with 10% inactivated fetal calf serum (FCS), penicillin (100 IU/ml), streptomycin (100 ~-tg/ml) and 2 mM glutamine (growth medium). For cell maintenance the serum concentration was lowered to 2% (maintenance medium).
Virus BK virus (Gardner prototype strain) was propagated in Vero cells grown in 199 medium supplemented with 10% FCS. After adsorption for 1 h at 37°C, the infected cells were incubated in 199 medium with 2% FCS for 21 days at 37°C. The supernatant was then collected, clarified by centrifugation at 3,000 rpm for 30 min to remove cell debris and stored at - 70°C.
Chemicals The activity of the following substances was tested: dextran sulfate sodium salt (MW 5,000 and 500,000), mucin from bovine submaxillary glands, polymyxin B sulfate, histone from calf thymus, heparin sodium salt from porcine intestinal mucosa, poly-L-lysine hydrobromide (MW 10,200, 26,500 and 141,000), DEAE-Dextran and locust bean gum from Sigma Chemical Co. St. Louis, USA; Keltro TF from Kelco, Division of Merk and Co., USA; scleroglucan from Sanofi, Bio-Industries, Paris; alginic acid from Fluka, Switzerland; tamarind gum (or glyloid) supplied by Dainippon Pharmaceutical Co., Osaka and purified in the laboratories of Francesco Angelini Research Institute - Italy; three distinct sulfated tamarind gums (glyloid sulfate) differing in the content of sulfur (i.e . different degree of sulfation), namely: GP4311, S = 0.41%; GP4327, S = 2.2%; GP4324, S = 7.1% were prepared from purified glyloid and kindly supplied by the same laboratories. Stock solutions were made up in PBS (phosphate buffered saline) pH 7 .2, stored at - 20°C and further diluted in cell culture medium before use .
17
Vera cell toxicity assay In order to establish the maximal non-cytotoxic concentrations, two-fold serial dilutions of the drugs in serum-free medium were incubated for 1 h at 37°C with Vera cell monolayers grown in 96-well plates (Falcon). The assays were carried out in duplicate. The drugs were then removed and cells, washed three times with PBS, were overlaid with maintenance medium. After 5 days at 37°C in 5% C0 2, cell morphology, viability (as determined by neutral red uptake in dispersed cells) and yield were examined. Drug dilutions which did not affect any of these parameters in 100% of cells were considered as non-cytotoxic concentrations (Table 1).
Effect of polymers on BK virus infection Cells were grown in microtissue chamber slides (Lab-tek, Miles Laboratory) for 24 h in 5% C02 at 37°C. The monolayers were placed in an ice-water bath, washed with ice cold medium and infected with BK virus suspended in precooled medium at concentrations capable of infecting 50% of the untreated cells. After 1 h at 0°C, the cells were washed with prewarmed medium and incubated for 5 days, in 5% C02 at 3 7 ° C. The percentage of infected cells was determined by indirect immunofluorescence. Different types of experiments were carried out: i) Cells were preincubated at 37°C for 1 h with the drugs which were removed just before virus infection; ii) Drugs were incubated with the cells during the virus attachment step at 0°C for 1 h. They were then removed together with the virus inoculum before the temperature shift to 37°C. iii) Virus was preincubated with the drugs at 26°C for 2 h, then the mixtures were added to the monolayers at 3 7°C for 1 h.
Immunofluorescence Virus-infected cell monolayers were washed with PBS and fixed with absolute acetone for 5 min at - 20°C. After incubation with anti-BK human serum for 1 hat 37°C, cells were washed with PBS and stained with fluorescein isothyo-
18
L. SINIBALDI - V. PIETROPAOLO - P . GOLDONI - C. DI TARANTO - N. ORSI
TABLE 1 -
Toxicity of different polymers. On Vero cells
On red cells
Polymers Highest non-toxic concentration (mg/ml)
Acid polymers (Polyanions) Heparin
2.500
10.000
Mucin
0.018
0.195
Dextran sulfate (MW 5,000)
0.500
0.062
Dextran sulfate (MW 500,000)
0.625
0.625
Polymyxin B sulfate
1.560
0.014
Alginic acid
0.025
0.030
Keltrol
0.025
0.030
Glyloid sulfate 4311
0.025
0.030
Glyloid sulfate 4327
0.025
0.062
Glyloid sulfate 4324
0.025
0.062
Poly-L-Iysine (MW 10,200)
0.010
0.006
Poly-L-lysine (MW 26,500)
0.031
0.0005
Poly-L-lysine (MW 141 ,000)
0.016
0.0002
DEAE-Dextran
0.500
0.0005
Histone
0.625
0.625
Locust bean gum
0.025
0.030
Tamarind gum
0.025
0.030
Scleroglucan
0.025
0.030
Basic polymers (Polycations)
Neutral polymers
cyanate-conjugated anti-human gammaglobulin antibodies (Behring). After 1 h at 37°C, cells were washed with PBS, mounted in buffered glycerol and examined under a Leitz fluorescence microscope . The results were expressed as the percentage of infected cells in treated versus untreated cultures.
Erythrocytes An 8% suspension in PBS of human group 0 Rh + erythrocytes was used .
Erythrocyte toxicity assay The test was carried out by means of a double series of two-fold dilutions of the drugs in HSA (Hepes Saline Albumin) - Buffer pH 7.2 and by adding to each dilution 50 J..l.l of a 0.2% suspension of erythrocytes in HSA. After 24 h of contact at + 4°C the normal sedimentation of red cells or the degree of the eventual hemolysis were checked (Table 1) .
Hemagglutination (HA) and hemagglutination inhibiting (HI) tests These tests were carried out in plastic microplates as previously described 17 •
Control of HA and HI activity of polymers towards BK virus The compounds (at the highest non-cytotoxic concentration) were serially diluted and each dilution was mixed with four hemagglutinating doses of virus. The addition of the erythrocytes to the virus-drug mixture took place immediately.
EHect of polymers on BK virus and on erythrocytes The various substances have been tested directly on both the BK virus and the erythrocytes . In the first case serial dilutions of virus were tested with the highest non-toxic concentration of each compound. After 1 h of contact at room temperature the erythrocytes were added . In order to ascertain whether the various compounds had a direct effect on red cells, the erythrocyte suspension was added to the highest non-toxic concentration of each substance and after 1 h of contact at room temperature the mixture was added to serial viral dilutions.
RESULTS
a) EHect of polymers on BK virus infectivity. This was studied by adding the maximal non-cytotoxic concentration of compounds to the Vera cell monolayers for 1 h prior to infection and incubated at 37°C . The tissue cultures
19
EFFECT OF BIOLOGICAL AND SYNTHETIC POLYMERS ON BK VIRUS INFECTIVITY AND HEMAGGLUTINATION
were then washed and infected with appropriately diluted stock virus . The results obtained in these experiments showed that the pre-exposure of cells to the drugs exerted a slight effect on subsequent BK virus infection as monitored by indirect immunofluorescence (Table 2: b). In parallel, virus infection was synchronized by a temperature shift as described in Materials and Methods in order to verify whether the drug effect took place during the attachment step. In this case the maximal non-cytotoxic concentration of polymers was added to the cells together with the virus during the incubation at 0°C and then removed after 1 h. In this second type of experiment the results obtained showed that among all compounds tested polyanions such as mucin, heparin, dextran sulfate 500,000, alginic acid and glyloid sulfate significantly depressed virus adsorption, whereas among polycations, poly-L-lysine was able to enhance the susceptibility of Vero cells to BK virus adsorption. As to neutral polymers, locust bean gum and tamarind gum showed a strong reducing effect similar to that obtained with the most active polyanions (Table 2: a). An analysis of the data obtained showed that the activity of compounds did not appear exclusively related to the charge of the molecules, since some neutral polymers appeared to be very effective. As to the importance of the molecular weight, it will be seen that the highest effect was obtained with dextran sulfate of 500,000 molecular weight (MW) whereas among the various poly-L-lysines tested the most enhancing action referred to poly-L-lysine of 141,000 MW (Table 2: a, b). Results reported in Table 2 also show that some polyelectrolytes (polymyxin B sulfate, histone and DEAE Dextran) were devoid of any activity in the case of the BK virus we tested. In order to ascertain the site of action, i.e . the target of polymers, experiments were carried out by preincubation of the virus with the drugs at 26°C for 2 h before infection. After this time of contact, the mixtures were added to the cell monolayers at 37°C for 1 h. Virus pretreated with neutral and acid polymers showed, in general, a reduction of infection as monitored by indirect immunofluorescence, whereas preincubation of virus with basic polymers had no effect on subsequent infection (Table 2: c).
TABLE 2 · Effect of polymers on the early steps of BK virus
infection. Polymers maximal non-cytotoxic concentration)
Immunofluorescence (% ) a
b
c
None
50
50
50
Heparin
10
30
10
5
25
10
Dextran sulfate (MW 5,000)
35
40
35
Dextran sulfate (MW 500,000)
5
30
5
Polymyxin B sulfate
50
50
50
Alginic acid
20
30
15
Keltrol
35
40
20
Glyloid sulfate 4311
10
20
25
Glyloid sulfate 4327
25
40
35
Glyloid sulfate 4324
20
30
20
Poly-L-Iysine (MW 10,200)
70
60
50
Poly-L-Iysine (MW 26,500)
80
60
50
Poly-L-Iysine
95
80
50
Mucin
(MW 141,000) DEAE-Dextran
50
50
50
Histone
50
50
50
Locust bean gum Tamarind gum Scleroglucan
10 10 40
30 25 45
15 30 40
a = The polymers were incubated with Vera cells during the virus attachment step at oac for 1 h; then they were removed together with the virus inoculum before the temperature shift to 3 7°C. b = Vera cells were preincubated at 37°C for 1 h with the polymers which were removed just before virus infec tion. c = BK virus was preincubated with the polymers at 26°C for 2 h, then the mixtures were added to cell monolayers at 37 °C for 1 h.
b) Effect of polymers on BK hemagglutination. The effect of compounds on BK hemagglutination was studied by different experimental approaches. The various drugs were tested separately on both the BK virus and the erythrocytes in order to verify whether a reduction or an enhancement of the hemagglutination took place.
L. SINIBALDI - V. PIETROPAOLO - P. GOLDONI - C. DI TARANTO - N. ORSI
20
TABLE 3 - Action of polymers on BK hemagglutination. Polymers (maximal non-cytotoxic concentration) Control Heparin
HA titer a
b
128
128
128
128
96
96
Dextran sulfate (MW 5,000)
128
128
Dextran sulfate (MW 500,000)
56
64
128
128
96
128
128
128
Mucin
Polymyxin B sulfate Alginic acid Keltrol Glyloid sulfate 4311
128
128
Glyloid sulfate 4327
384
256
Glyloid sulfate 4324
256
128
Poly-L-lysine (MW 10,200)
160
288
Poly-L-lysine (MW 26,500)
384
384
Poly-L-lysine (MW 141,000)
768
5 12
DEAE-Dextran
256
512
Histone
128
128
Locust bean gum Tamarind gum Scleroglucan
128 64 96
128 64 64
a = Serial dilutions of BK virus were tested with the highes t non-toxic concentration of each compound. After 1 h of contact at room temperature the erythrocytes were added . b = The erythrocyte suspension was added to the highest non-toxic concentration of each compound and after 1 h of contact at room temperature serial viral dilutions were added to the mixture.
In Table 3 (a) the HA titers obtained after contact of the BK virus with the various substances are reported. It appears that dextran sulfate 500,000 (MW) and, to a lesser extent, mucin, tamarind gum, scleroglucan and alginic acid noticeably reduced the HA activity of BK virus whereas DEAE-Dextran, gly loid sulfate and poly-L-lysine enhanced it.
Among the various preparations of poly-Llysine and glyloid sulfate tested, the most active compounds were respectively poly-L-lysine 141,000 and glyloid sulfate 4327. Table 3 (b) reports the HA titer after contact of all compounds with the erythrocytes. Results showed that mucin, scleroglucan, tamarind gum and dextran sulfate 500,000 MW kept in contact with cells before the addition of virus, reduced the HA activity. In the case of poly-Llysine, DEAE-Dextran and glyloid sulfate, the preliminary contact between the substances and red cells produced an increase in the HA titer. DISCUSSION
Results reported here confirm the role which electrostatic forces play in the early interactions between BK virus and host cells and that polyelectrolytes are able to modify these interactions and consequently viral infectivity. In agreement with results obtained by other Authors, we have noticed that, as also in the case of the BK virus, polyanions depress viral binding, whereas polycations can enhance it; however some of the compounds we tested (DEAE-Dextran and histone among polycations and polymyxin B sulfate among polyanions) are devoid of any effect. However, the effect on viral infectivity is not shown only by polyelectrolytes but also by neutral polymers such as tamarind gum and locust bean gum, which significantly decrease the susceptibility of Vero cells to BK virus infectivity. This interesting observation indicates that in the polymers tested the electric charge of the molecule is not sufficient by itself to explain the effect of these compounds. This hypothesis has already been suggested in preceding research 10 and is clearly demonstrated by our results which show a marked effect also in the use of tamarind gum and locust bean gum, which have a neutral charge . The effect of the active compounds was studied by different types of experiments, i.e., by preincubating the substances with virus or the cells and by the contemporary addition of virus and drugs to the cells at the moment of infection, at 0°C. The greatest effect on virus infection was noticed in this last case. In particular among
EFFECT OF BIOLOGICAL AND SYNTHETIC POLYMERS ON BK VIRUS INFECTIVITY AND HEMAGGLUTINATION
polyanions, mucin, heparin, dextran sulfate 500,000, alginic acid and glyloid sulfate reduce BK virus infectivity very strongly, whereas among polycations, poly-L-lysine (MW. 10,200, 26,500, 141,000) enhance it. Moreover even neutral polymers such as locust bean gum and tamarind gum produce a marked inhibition of BK virus infectivity. The same reducing effect is noticed also in the experiments in which Vera cells are preincubated with acid or neutral polymers before virus infection, but to a lesser degree. An enhancement of viral infectivity is, on the other hand, observed when a polycation such as poly-Llysine is used in the same experimental conditions. This last result is of interest when taking into account the well-known action of polycations on plasma membrane permeability 18 • Finally, in the experiments with BK virus preincubated with polymers, our results show a slight effect on the infection only in the case of virus pretreated with acid or neutral polymers, whereas preincubation of virus with basic polymers has no effect on subsequent infection. The results obtained in these three types of experiments suggest that polyanions act directly on the viral capsid proteins since a low effect is obtained when the compounds are preincubated with cells, before virus addition . A similar action can be argued also for neutral polymers. Another interesting indication of the mechanism of the action of the drugs tested is obtained by using different concentrations of compounds (heparin and dextran sulfate) showing low cell toxicity. In the case of heparin, for example, it is possible to notice that concentrations included in a range between 2.500 mg/ml and 0.025 mgfml did not show a dose-effect ratio, and all the concentrations were practically equally active (data not shown). As to the importance of the molecular weight of compounds on their biological effect on BK virus infectivity, results obtained by the use of two preparations of dextran sulfate (MW 5,000 and 500,000) and three preparations of poly-L-lysine (MW 10,200, 26,500 and 141,000) show that the most active compounds are those with the highest molecular weight. As to the role played by the content of sulfate groups present in the same type of com-
21
pound, experiments carried out with glyloid sulfate 4311, glyloid sulfate 4324 and glyloid sulfate 4 327, do not show a correlation between this parameter and the biological activity on BK virus infection. As to the importance played by the viscosity of the different compounds in their biological effect, it is not possible to obtain any indication since our experiments are carried out with very low concentrations of the compounds in order to avoid any toxic effect on the cells. As to the results obtained by studying the effect of polymers on BK virus hemagglutination, among all compounds tested dextran sulfate 500,000 and, to a lesser extent, mucin, tamarind gum, scleroglucan and alginic acid reduce the HA activity of BK virus whereas DEAE dextran, glyloid sulfate and poly,L-Lysine are able to enhance the susceptibility of erythrocytes to BK virus hemagglutination and consequently the HA titer. All these data concerning the effect of biological and synthetic polymers on BK virus hemagglutination fundamentally confirm the results obtained in the infectivity test, although to a lesser extent. This is probably due to the fact that the hemagglutination by the BK virus is a less sensitive biological system compared to that represented by the infectivity of the same virus. AcKNOWLEDGEMENT - We thank Prof. Rosa rio Nicoletti of the University of Rome «La Sapienza» for rem arks and helpful di scussion and Prof. Giuseppe Barbanti Brodano of the University of Ferrara for having provided the BK virus. The authors would like to express their appreciation to Mrs. Marina Fortunato for skillful technical assistance.
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