Anti-viral activity of red microalgal polysaccharides against retroviruses
© Talyshinsky et al; licensee BioMed Central Ltd. 2002
Received: 24 January 2002
Accepted: 5 July 2002
Published: 5 July 2002
Red microalgal polysaccharides significantly inhibited the production of retroviruses (murine leukemia virus- MuLV) and cell transformation by murine sarcoma virus(MuSV-124) in cell culture. The most effective inhibitory effect of these polysaccharides against both cell transformation and virus production was obtained when the polysaccharide was added 2 h before or at the time of infection. Although, addition of the polysaccharide post-infection significantly reduced the number of transformed cells, but its effect was less marked than that obtained when the polysaccharide was added before or at the time of infection.The finding that the inhibition of cell transformation by MuSV-124 was reversible after removal of the polysaccharide suggested that microalgal polysaccharides inhibited a late step after provirus integration into the host genome. In conclusion, our findings could support the possibility that the polysaccharide may affect early steps in the virus replication cycle, such as virus absorption into the host cells, in addition to its effect on a late step after provirus integration.
KeywordsRed microalgae polysaccharides malignant cell transformation retroviruses antiviral activity
Retroviruses [1–3] – viruses that contain reverse transcriptase, an RNA-directed DNA polymerase [4, 5] – have been implicated in various types of human and animal leukemia's and other tumors. Although there are many compounds that exhibit potent anti-viral, and possibly anti-tumor, activity in cell culture and in experimental animals, only very few synthetic compounds and one natural product – alpha interferon – have so far been approved for treatment of viral infections in man. Alpha interferon has been approved for treatment of hairy cell leukemia, of Karposi's sarcoma and of genital warts caused by papiloma virus .
A class of natural products with low mammalian toxicity that are currently regarded as having antitumor activity are polysaccharides of biological origin, e.g., polysaccharides from yeasts, algae, bacteria, higher plants and fungi [7–9]. Of interest in this context are polysaccharides produced by some species of red algae; these compounds have shown promising activity against a variety of animal viruses [10–13]. In general, polysaccharides exhibiting antiviral potential are highly sulfated [10, 14–17]. Dextran sulfate and polysaccharides from marine algae, for example, have been found to be potent in vitro inhibitors of HIV types 1 and 2 [15, 18–20]. They inhibit HIV-1-induced cytopathogenicity and HIV-1 antigen expression [13, 18, 21, 22]. These sulfated polysaccharides also inhibit the activity of purified reverse transcriptase and RNase H, which are essential for retrovirus replication [18, 20]. Some previous studies showed that algal polysaccharides exert their inhibitory action at a very early stage (adsorption, fusion or penetration), in the viral infection cycle [9, 18, 20, 23–25], whereas others showed that these polysaccharides did not interfere with virus attachment or penetration, but they did prevent viral protein synthesis [11, 26, 27].
Our previous research has shown potent antiviral activity of a highly sulfated polysaccharide extracted from red microalga. The polysaccharide, which consists mainly of xylose, glucose and galactose , exhibits antiviral activity against various members of the herpes family of viruses . In the current study, the activity of this red microalgal polysaccharide against the replication and the transforming ability of the retroviruses, Moloney murine sarcoma virus (MuSV) and Moloney murine leukemia virus (MuLV), was studied.
Results and Discussion
Characterization of cell transformation by MuSV-124 and MuSV/MuLV
Antiretroviral effect of red microalgal polysaccharides
Inhibiting effect of red microalgal polysaccharides on formation of foci by MuSV-124 and on virus production, as measured by reverse transcriptase (RT) activity
ffu50 protection (μg/ml)
RT50 reduction (μg/ml)
Microscopy showed that there were there were no changes in cell morphology in the presence of Porphyridium sp. polysaccharide, even at a concentration of 1000 μg/ml.
Effect of dosage of Porphyridium sp. polysaccharide on cell transformation
Effect of dosage of Porphyridium sp. polysaccharide on NIH/3T3 cell transformation by MuSV-124 and MuSV/MuLV
Porphyridium sp. polysaccharide (μg/ml)
Number of foci1
200 ± 10
120 ± 8
150 ± 10
90 ± 7
110 ± 8
65 ± 5
15 ± 6
10 ± 3
Effect of time of addition of Porphyridium sp. polysaccharide on cell transformation
The algal polysaccharide (100 μg/ml) was added to NIH/3T3 cells at various times before and after infection with either MuSV-124 or MuSV/MuLV. As can be seen from Table 3, the polysaccharide fully inhibited formation of foci in MuSV-124- and MuSV/MuLV-infected cultures if it was added before or at the time of infection. If the polysaccharide was added post-infection, it was less effective. In MuSV/MuLV-infected cultures the polysaccharide was still significantly inhibitory to the formation of foci when added 48 h after infection. The protective effect of the polysaccharide in these cultures was lost only if it was added 72 h after infection, whereas in MuSV-124-infected cultures the effectiveness of the polysaccharide was lost when addition was as early as 48 h after infection.
Effect of time of addition of Porphyridium sp. polysacharide on NIH/3T3 cell transformation
Timing of Porphyridiumsp. addition of polysaccharide
Number of foci1
24 h before infection
2 h before infection
0 h at infection
2 h after infection
15 ± 4
6 ± 2
24 h after infection
50 ± 7
14 ± 4
48 h after infection
73 ± 6
30 ± 5
72 h after infection
75 ± 7
64 ± 6
Effect of time of addition of Porphyridium sp. polysaccharide on release of progeny virus from MuSV/MuLV-infected cells
Effect of time of removal of Porphyridium sp. polysaccharide on cell transformation
Focus formation after removal of Porphyridium sp. polysaccharide
Porphyridium sp. polysaccharide
Number of foci1
100 ± 6
70 ± 5
0 h after infection
75 ± 7
65 ± 4
24 h after infection
64 ± 6
50 ± 5
72 h after infection
20 ± 4
As a consequence of the different character of the infection by these two virus stocks, their focus-forming capacity responded quite differently to the timing of polysaccharide addition and removal. In the case of MuSV/MuLV infected cultures, focus formation was significantly inhibited even when the polysaccharide was added or removed at longer times after infection compared to MuSV-124 infected cultures. These findings indicate that the continuous presence of thepolysaccharide in the culture medium after infection with the virus was essential for full prevention of malignant transformation over the tested period (about two weeks). When the treatment with the polysaccharide was terminated immediately post-infection (Table 4), there was a significant recovery in the appearance of malignant transformed cells for all tested concentrations of the polysaccharide. This reversibility strongly suggests that the polysaccharide, partially at least, exerted its inhibitory effect on a certain event occurring after proviral integration. In addition, this reversibility could not be explained only by the possibility of preventing viral reinfections by the polysaccharide because in the case of MuSV-124 infections there are no reifections [30, 31]. The inhibitory effect does not seem to be mediated by interferon or by an interferon-like antiviral state, since interferon has been found to inhibit certain events occurring before proviral integration .
Our results do not rule out the possibility that at least part of the inhibitory effect of the polysaccharide was due to blocking some of the viral receptors, thus interfering with the penetration of the virus into the cells. This possibility was supported by our results showing that treatment of the cells with the polysaccharide post-infection caused a significant inhibition of cell transformation, but that this inhibition was less impressive than that obtained when treatment with the polysaccharide was started before or at the time of infection (Table 3). This possibility is also in agreement with various previous studies [9, 17, 18, 22, 24, 32] that suggested that sulfated polysaccharides prevent early steps in the viral life cycle. In addition, some of our data not presented here showed that the inhibitory effect of Porphyridium sp. polysaccharide on cell malignant transformation by MuSV was not a result of a direct interaction between the polysaccharide and the virus particles. In contrast, our previous data (29) showed a strong interaction between Herpes simplex virus (HSV 1 and HSV-2) particles and Porphyridium sp. polysaccharide. This contradiction could be due to differences in viral envelope composition. Herpes viruses envelope is positively charged, whereas retroviruses are negatively charged. Therefore, the sulfate groups of the polysaccharide could easily interact with positively charged viruses.
The present data show that the red microalgal polysaccharides profoundly inhibited retroviral malignant cell transformation and retrovirus replication. Most effective inhibitory activity of these polysaccarides on cell transformation was obtained when the cells were treated with polysaccharide before or at the time of infection. These results support the possibility that at least part of the inhibitory effect of the polysaccharide was due to blocking some of the viral receptors, thus interfering with the penetration of the virus into the cells. On the other hand, the reversibility of this inhibitory activity strongly suggests that the polysaccharide exerted its inhibitory effect also on a certain event occurring after proviral integration. Thus, it appears that Porphyridium sp. polysaccharide has a pleiotropic mode of action during the infection cycle of MuSV. The exact steps (or step) during the viral replication cycle that are affected by Porphyridium sp. polysaccharide remain to be elucidated.
Materials and Methods
Cells and viruses
NIH/3T3 cells (mouse fibroblast cells) were grown at 37°C in RPMI medium supplemented with 10% new born calf serum (NBCS) and the antibiotics penicillin, streptomycin and neomycin. Clone 124 of TB cells chronically releasing Moloney murine sarcoma virus (MuSV-124) (31) was used to prepare a virus stock that contained an approximately 30-fold excess of MuSV particles over Moloney murine leukemia virus (MuLV) particles. MuLV and MuSV used in this research were grown on NIH/3T3 cells. The virus concentration was determined by counting the number of foci (ffu-focus-forming units) in the case of MuSV and by the reverse transcriptase assay in the case of MuLV.
Preparation and purification of microalgal polysaccharide
Polysaccharides produced from three species of red microalga; Porphyridium sp., P. aerugineum and Rhodella reticulata, were used in this study. The polysaccharides were collected and purified as previously described . Briefly, these polysaccharides are produced and secreted into the growth medium by the appropriate red microalgae. The medium was collected, cells were removed by centrifugation and the supernatant containing the polysaccharides was dialyzed and lyophilized.
Cell infection and determination of viral infection
A monolayer of NIH/3T3 cells was grown in 9-cm2 tissue culture plates and treated with 0.8 μg/ml of polybrene (a cationic polymer required for neutralizing the negative charge of the cell membrane) for 24 h before infection with the virus. Free polybrene was then removed, and the cells were incubated at 37°C for 2 h with the infecting virus (MuSV-124) at various concentrations in RPMI medium containing 2% of NBCS. The unabsorbed virus particles were removed, fresh medium containing 2% NBCS was added, and the monolayers were incubated at 37°C. After 2–3 days, the cell cultures were examined for the appearance of malignant transformed cells. The amount of malignant transformed cells was expressed as the percentage of transformed cells in the inspection field or as the number of foci in the infected culture 10 days after infection.
Reverse transcriptase assay
Viral reverse transcriptase activity was assayed as previously described .
This research was supported by Ma'OF (established by the Kahanoff Foundation).
We thank Ms Marion Milner for typing. and editorial review of the manuscript.
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