Antiviral activity of natural and semisynthetic polysaccharides on the early steps of rubella virus infection.

The natural and semisynthetic carbohydrates scleroglucan, locust bean gum, tamarind gum (glyloid) and its three sulphate derivatives (GP4311, GP4327 and GP4324), glycogen and its two sulphate derivatives (GP4427 and GP4435), alginic acid and dextran sulphate, were investigated for their inhibitory effect on rubella virus (RV) infection of Vero cells. The neutral polymer scleroglucan and two highly negatively charged compounds, glyloid sulphate 4324 and dextran sulphate, had the highest inhibitory effect on RV antigen synthesis. The antiviral properties of active molecules appears to be dependent on the shape of the macromolecule and/or on the electric charge, while saccharide units play a minor role. The results indicated that polysaccharides blocked a step in virus replication subsequent to virus attachment, such as internalization and/or uncoating. Confirmation that the inhibitory activity of the compounds was directed at the early steps of RV multiplication, was that none of the polysaccharides had any effect on infection initiated by transfection of cells with RVRNA.

Pathway of rubella virus infectious entry into Vero cells.

The mechanism and the kinetics of rubella virus (RV) penetration into Vero cells were studied. By using pronase or acid treatment to inactivate virus which had adsorbed to cell membrane but had not been internalized, it was found that a period of 7 h was required in order for all of the adsorbed virus to enter the host cells. Lysosomotropic agents (monensin, methylamine, ammonium chloride and chloroquine) were used to study the mechanism by which RV penetrates host cells. Virus replication was inhibited if treatment of cells with these compounds was performed for at least 9 h after infection. However, if extracellular adsorbed virions were eliminated by acid treatment following removal of the lysosomotropic compounds, RV replication was completely inhibited by treatment with these drugs for any time period after adsorption. This indicated that the prolonged period of treatment with these compounds necessary to inhibit virus replication is due to the slow rate of RV internalization. None of the compounds had any effect on infection initiated by transfection of RV RNA, confirming that these drugs were exerting their inhibitory activity at penetration. The inhibition of RV replication by lysosomotropic compounds indicates that RV penetrates host cells by the endosomal pathway.

Inhibition of Sindbis virus replication by cyclopentenone prostaglandins: a cell-mediated event associated with heat-shock protein synthesis.

Cyclopentenone prostaglandins (PGs) have been shown to inhibit the replication of several DNA and RNA viruses. Here we report on the effect of prostaglandin A1 (PGA1) on the multiplication of a positive strand RNA virus, Sindbis virus, in Vero cells under one-step multiplication conditions. PGA1 was found to inhibit Sindbis virus production dose-dependently, and virus yield was reduced by more than 90% at the concentration of 8 micrograms/ml, which was non-toxic to the cells and did not inhibit DNA, RNA or protein synthesis in Vero cells. The cyclopentenone prostaglandin delta 12-PGJ2 was also shown to be a potent inhibitor of Sindbis virus replication. Virus-induced reduction of [3H]uridine uptake by cells was partially prevented by PGA1 treatment, which also caused a 1 h delay in the peak of virus RNA synthesis. SDS-PAGE analysis of [35S]methionine-labeled proteins showed that PGA1 moderately inhibited the synthesis of the viral structural proteins E1, E2 and C, and induced the synthesis of a 72 kDa M(r) protein, identified as a heat-shock protein related to the HSP70 group, in both virus-infected and uninfected cells. Actinomycin D treatment completely prevented PGA1-antiviral activity, indicating that a cellular product is responsible for this action. PGA1-induced HSP70 is a good candidate for this role.

Influence of polyions on the early steps of enterovirus infection.

The influence of electric charged molecules on the early phases of enterovirus infection was studied in order to select antiviral compounds able to prevent viral attachment. The effect of different polyelectrolytes on the multiplication of coxsackie virus B3, echovirus 6 and hepatitis A virus was investigated in susceptible cells by adding the drug before, during or after the viral adsorption period. Among polyanions, the polysaccharides heparin and dextran sulfate inhibited viral infectivity, dextran sulfate being the most effective mainly towards hepatitis A virus infection. DEAE-dextran and protamine sulfate, generally recognized as enhancers of infectivity of naked and enveloped viruses, exhibited an inhibitory effect towards the three picornaviruses tested. Only in the case of hepatitis A did DEAE-dextran slightly improve viral antigen synthesis. The inhibitory effect shown by compounds belonging to positive and negative polyions suggests that the electric charge is not sufficient by itself to explain the antiviral activity of these drugs.

Effect of polyions on the early events of Sindbis virus infection of Vero cells.

To clarify the role of electrostatic interactions in the binding of Sindbis virus (SNV) to cell membrane receptors, we investigated the effect of different polyions on the initial steps of infection of Vero cells by the virus. Several polyanions (mucin, heparin, polygalacturonic acid) and polycations (polylysine, protamine, polybrene) were able to reduce the replication of SNV when present in the viral adsorption period, whereas others (chondroitin sulfate, polymyxin B sulfate, histone) were devoid of any activity. Therefore the electric charge alone is not sufficient to explain the action of compounds. The effects of polyions on receptor binding, on bound virus, and on internalized virus have been examined. All the drugs inhibited SNV infection by affecting its binding to the cellular receptor. The results indicated that heparin and mucin act directly on the virus particle while polycations bind to the cell membrane receptor for the virus, protamine being effective on both targets. Since among polyanions glycosaminoglycans showed a strong inhibiting activity, the involvement of these molecules in the virus surface receptor was assessed by enzyme digestion of cell membrane with heparinase and chondroitin ABC lyase.