Anti-viral activity of red microalgal polysaccharides against retroviruses

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.


Background
Retroviruses [1][2][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 [6].
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][8][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][11][12][13]. In general, polysaccharides exhibiting antiviral potential are highly sulfated [10,[14][15][16][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][19][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][24][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 [28], exhibits antiviral activity against various members of the herpes family of viruses [29]. 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.

Characterization of cell transformation by MuSV-124 and MuSV/MuLV
NIH/3T3 cells grown in plastic dishes in RPMI medium with 2% NBCS appear as flat cells (Fig 1a); these cells are completely unable to grow in agar. When these cells were infected with an appropriate dilution of MuSV-124, tiny foci of transformed cells, with a highly refractile spindle shape, growing randomly in a criss-cross fashion, could be detected by microscopic observation within five days of infection (Fig. 1b). Later, these foci gradually increased in size and compactness until they became visible to the naked eye on day 12 to 14 after infection with MuSV-124. The number of foci remained unchanged in these cultures during the entire culture period. When the cells were infected with high titer of MuSV-124 (1 ffu/cell), most of the cells were transformed two-three days after infection. In MuSV/MuLV-infected cultures, the number of foci increased continuously, and at any time foci of various sizes (tiny to large) could be detected. Moreover, if these cultures were maintained for a sufficiently long time, all the cells eventually became transformed. Examination of the culture medium for the presence of viral reverse transcriptase revealed that MuSV/MuLV infection yielded virus-producing cells, whereas MuSV-124 infection resulted in the formation of transformed cells not producing virus. It is therefore likely that the increasing number of foci in the productively infected cells resulted from multiple secondary infections by the virus progenies, the smaller foci being formed by these infections. It was found that the later the infection, the smaller the foci. Both MuSV-124-and MuSV/MuLV-transformed cells could grow efficiently in agar.

Antiretroviral effect of red microalgal polysaccharides
The polysaccharide extracted from Porphyridium sp. was more effective in inhibiting retrovirus replication and cell transformation by MuSV than the polysaccharides obtained from P. aerugineum or Rhodella reticulata ( Table 1). The concentration of Porphyridium sp. polysaccharide required for 50% protection against the formation of foci of transformed cells by MuSV or for a 50% reduction in MuLV production (as evaluated in terms of reverse transcriptase activity) was one or two orders of magnitude lower than that needed when P. aerugineum or R. reticulata polysaccharide was applied. We therefore focused our research on the anti-retroviral effects of Porphyridium sp. polysaccharide.   Porphyridium sp. polysaccharide was also superior to other polysaccharides, such as carrageenan and dextran sulfate 500,000, in preventing the transformation of NIH/3T3 cells. Although the anti-transforming activity of Porphyridium sp. polysaccharide did not seem to be much higher than that of carrageenan or dextran sulfate at a concentration of 10 µg/ml (Fig. 2), at the higher concentrations of Porphyridium sp. polysaccharide was not toxic to the cells whereas the other two biopolymers were extremely toxic to the cells (Fig. 3). Porphyridium sp. polysaccharide had no effect on the proliferation of NIH/3T3 cells, even up to a concentration of 500 µg/ml (results not shown). However, at a concentration of 1,000 µg/ml, Porphyridium sp. polysaccharide caused the cells to stop growing three days after the beginning of the treatment (Fig. 3).
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
The algal polysaccharide significantly inhibited malignant transformation of NIH/3T3 cells by MuSV or MuSV/ MuLV. As can be seen from

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 Ta

Effect of time of addition of Porphyridium sp. polysaccharide on release of progeny virus from MuSV/MuLV-infected cells
The algal polysaccharide probably exerted its effect by preventing secondary infections, which were the major source of foci scored in these cultures under our experimental conditions. To determine whether the effect of the polysaccharide was due to the arrest of virus release from the primary infected cells or merely from blocking the establishment of secondary infections, the polysaccharide was added at various times before and after infection, and the release of progeny virus was followed by assaying viral reverse transcriptase activity in aliquots taken from the culture medium at different times post infection. The results presented in Fig. 4 showed that the infection cycle is completed within 20-24 h after inoculation, this being the time at which the appearance of the first progeny could be detected. The polysaccharide had a significant inhibitory effect on the release of virus progeny even when it was added 48 h after infection. Therefore, the significant prevention of formation of malignant foci by the polysaccharide at this late time was most likely due to its action against the subsequent secondary infection cycle. These results are in agreement with our suggestion that in MuSV/ MuLV-infected cultures, part of the inhibitory effect of the polysaccharide against cell transformation could be a result of inhibiting secondary viral infections.

Effect of time of removal of Porphyridium sp. polysaccharide on cell transformation
To determine whether it is necessary for Porphyridium sp. polysaccharide to remain in the culture during the whole time until scoring of foci, cells were treated with the polysaccharide 2 h before infection and polysaccharide was removed at various times after infection. Foci were scored 12 days after infection. As can be seen from Table  4, about 75 and 65% of the transforming capacity of MuSV-124 and MuSV/MuLV, respectively, were recovered when the polysaccharide was removed at the time of infection. When Porphyridium sp. polysaccharide was removed 72 h post-infection, only 55% and 20% of transforming capacity of MuSV-124 and MuSV/MuLV, respectively, were recovered.
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 [31].
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.

Conclusions
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

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 [33]. 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-cm 2 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 [34].