Large polyanionic molecules, such as sulfated polysaccharides (including soluble heparin and dextran sulfate), synthetic polyanionic polymers, and negatively charged proteins, have been shown to broadly inhibit several enveloped viruses. We recently reported the antiviral activity of a peptide derived from amino acids 77 to 95 of a potential binding partner of respiratory syncytial virus F protein (RSV F), the GTPase RhoA. A subsequent study with a truncated peptide (amino acids 80 to 94) revealed that optimal antiviral activity required dimerization via intermolecular disulfide bonds. We report here that the net negative charge of this peptide is also a determining factor for its antiviral activity and that it, like other polyanions, inhibits virus attachment. In a flow cytometry-based binding assay, peptide 80-94, heparin, and dextran sulfate inhibited the attachment of virus to cells at 4 degrees C at the same effective concentrations at which they prevent viral infectivity. Interestingly, time-of-addition experiments revealed that peptide 80-94 and soluble heparin were also able to inhibit the infectivity of a virus that had been prebound to cells at 4 degrees C, as had previously been shown for dextran sulfate, suggesting a potential role for postattachment effects of polyanions on RSV entry. Neutralization experiments with recombinant viruses showed that the antiviral activities of peptide 80-94 and dextran sulfate were diminished in the absence of the RSV attachment glycoprotein (G). Taken together, these data indicate that the antiviral activity of RhoA-derived peptides is functionally similar to that of other polyanions, is dependent on RSV G, and does not specifically relate to a protein-protein interaction between F and RhoA.

Previous studies from our laboratory demonstrated that PVC-211 murine leukemia virus (MuLV), a neuropathogenic variant of Friend MuLV (F-MuLV), had undergone genetic changes which allowed it to efficiently infect rat brain capillary endothelial cells (BCEC) in vivo and in vitro. Two amino acid changes from F-MuLV in the putative receptor binding domain (RBD) of the envelope surface protein of PVC-211 MuLV (Glu-116 to Gly and Glu-129 to Lys) were shown to be sufficient for conferring BCEC tropism on PVC-211 MuLV. Recent examination of the unique RBD of PVC-211 MuLV revealed that the substitution of Lys for Glu at position 129 created a new heparin-binding domain that overlapped a heparin-binding domain common to ecotropic MuLVs. In this study we used heparin-Sepharose columns to demonstrate that PVC-211 MuLV, but not F-MuLV, can bind efficiently to heparin and that one or both of the amino acids in the RBD of PVC-211 MuLV that are associated with BCEC tropism are responsible. We further showed that heparin can enhance or inhibit MuLV infection and that the mode of action is dependent on heparin concentration, sulfation of heparin, and the affinity of the virus for heparin. Our results suggest that the amino acid changes that occurred in the envelope surface protein of PVC-211 MuLV may allow the virus to bind strongly to the surface of BCEC via heparin-like molecules, increasing the probability that the virus will bind to its cell surface receptor and efficiently infect these cells.

Glycosaminoglycans (GAGs) on the surface of cultured cells are important in the first step of efficient respiratory syncytial virus (RSV) infection. We evaluated the importance of sulfation, the major biosynthetic modification of GAGs, using an improved recombinant green fluorescent protein-expressing RSV (rgRSV) to assay infection. Pretreatment of HEp-2 cells with 50 mM sodium chlorate, a selective inhibitor of sulfation, for 48 h prior to inoculation reduced the efficiency of rgRSV infection to 40%. Infection of a CHO mutant cell line deficient in N-sulfation was three times less efficient than infection of the parental CHO cell line, indicating that N-sulfation is important. In contrast, infection of a cell line deficient in 2-O-sulfation was as efficient as infection of the parental cell line, indicating that 2-O-sulfation is not required for RSV infection. Incubating RSV with the purified soluble heparin, the prototype GAG, before inoculation had previously been shown to neutralize its infectivity. Here we tested chemically modified heparin chains that lack their N-, C6-O-, or C2-O-sulfate groups. Only heparin chains lacking the N-sulfate group lost the ability to neutralize infection, confirming that N-sulfation, but not C6-O- or C2-O-sulfation, is important for RSV infection. Analysis of heparin fragments identified the 10-saccharide chain as the minimum size that can neutralize RSV infectivity. Taken together, these results show that, while sulfate modification is important for the ability of GAGs to mediate RSV infection, only certain sulfate groups are required. This specificity indicates that the role of cell surface GAGs in RSV infection is not based on a simple charge interaction between the virus and sulfate groups but instead involves a specific GAG structural configuration that includes N-sulfate and a minimum of 10 saccharide subunits. These elements, in addition to iduronic acid demonstrated previously (L. K. Hallak, P. L. Collins, W. Knudson, and M. E. Peeples, Virology 271:264-275, 2000), partially define cell surface molecules important for RSV infection of cultured cells.

Addition of heparin to the virus culture inhibited syncytial plaque formation due to respiratory syncytial virus (RSV). Moreover, pretreatment of the virus with heparinase or an inhibitor of heparin, protamine, greatly reduced virus infectivity. Two anti-heparan sulfate antibodies stained RSV-infected cells, but not noninfected cells, by immunofluorescence. One of the antibodies was capable of neutralizing RSV infection in vitro. These results prove that heparin-like structures identified on RSV play a major role in early stages of infection. The RSV G protein is the attachment protein. Both anti-heparan sulfate antibodies specifically bound to this protein. Enzymatic digestion of polysaccharides in the G protein reduced the binding, which indicates that heparin-like structures are on the G protein. Such oligosaccharides may therefore participate in the attachment of the virus.

A series of sulfonic acid polymers were shown to be potent and selective inhibitors of respiratory syncytial virus (RSV) and influenza A virus. The compounds inhibit the replication of RSV and influenza A virus in HeLa and MDCK cells, at concentrations of 0.16 and 4.0 micrograms/ml, respectively, and are nontoxic to growing cells at concentrations of > 100 micrograms/ml. The mode of antiviral action of the sulfonic acid polymers can be ascribed to inhibition of virus-cell fusion (for influenza A virus) or inhibition of both virus-cell binding and fusion (for RSV). The sulfonic acid prototype PAMPS [poly(2-acrylamido-2-methyl-1-propanesulfonic acid)], when administered intranasally to mice as a single dose of 10 or 50 mg per kg of body weight at the time of infection, completely inhibited influenza A virus replication (in lungs) and virus-associated lung consolidation in immunocompetent mice and completely protected NMRI and SCID (severe combined immune deficiency) mice against influenza A virus-associated mortality. When administered 1 h before or after virus inoculation, no protective effect was observed at a dose of 10 or 100 mg/kg. Sulfonic acid polymers exert selective inhibitory effects on RSV and influenza A virus replication.

A series of novel compounds, 5-alkynyl-1-beta-D-ribofuranosylimidazole-4- carboxamides, have been identified as broad-spectrum antiviral agents. 5-Ethynyl-1-beta-D-ribofuranosylimidazole-4- carboxamide (EICAR), the most potent congener of the group, showed antiviral potency about 10- to 100-fold greater than that of ribavirin. Similar in spectrum to ribavirin, EICAR was particularly active (50% inhibitory concentration, 0.2 to 4 micrograms/ml) against poxviruses (vaccinia virus), togaviruses (Sindbis and Semliki forest viruses), arenaviruses (Junin and Tacaribe viruses), reoviruses (reovirus type 1), orthomyxoviruses (influenza A and B viruses), and paramyxoviruses (parainfluenza virus type 3, measles virus, subacute sclerosing panencephalitis virus, and respiratory syncytial virus). EICAR was also cytostatic for rapidly growing cells (50% inhibitory concentration, 0.2 to 0.9 microgram/ml). EICAR inhibited vaccinia virus tail lesion formation at doses that were not toxic to the host. EICAR is a candidate antiviral drug for the treatment of pox-, toga-, arena-, reo-, orthomyxo, and paramyxovirus infections.

9-(cis-3-Hydroxymethyl-2-methylenecyclobutyl)guanine (3b) and 9-(3-methylene-trans-2-hydroxymethylcyclobutyl)guanine (4b) were prepared from N2-isobutyryl-9-[trans-trans-2,3-bis(hydroxymethyl)cyclobutyl]guanine (2f) or 2,3-bis(hydroxymethyl)-1-cyclobutanol (7b). Carbocyclic oxetanocin analogues (A, 1d; G, 2d) and related compounds including 4b were assayed against a broad variety of viruses. It appeared that the activity of 2d against herpes simplex virus (HSV) and varicella-zoster virus (VZV) at least partially depends on phosphorylation by the virus-induced thymidine kinase (TK). Although 1d and 2d are inhibitory to the replication of human immunodeficiency virus (HIV), they are quite toxic to proliferating human T-lymphocytes.

Six nucleoside analogues, two sulfated polysaccharides, and four protease inhibitors were evaluated in vitro as inhibitors of influenza virus replication. Four guanosine analogues (mizoribine, ribavirin, pyrazofurin, and 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide), the sulfated polysaccharide dextran sulfate (molecular weight 500,000), and two protease inhibitors (camostat mesilate and nafamostat mesilate) were inhibitory to the replication of strains of influenza virus types A and B at concentrations down to 0.3 micrograms/mL. Of these seven compounds, ribavirin, camostat mesilate, and nafamostat mesilate were efficacious in both reducing the virus titer and increasing the survival rate of influenza virus-infected chick embryos. For camostat mesilate, the ED50 (required to improve the survival rate of influenza virus-infected chick embryos by 50%) was 0.80 micrograms/g, and its selectivity index, based on the ratio of the 50% toxic dose (required to reduce the viability of chick embryos by 50%) to ED50, was 280. Camostat mesilate deserves further exploration for its potential in the treatment of influenza virus infection.

A number of adamantaneketoxime ethers and an acetylenic adamantaneketoxime iododerivative have been prepared and tested as potential antifungal agents. They were also examined for their antibacterial and antiviral effects. Most of them proved active against the tested fungi. Their antimicrobial activity was generally low, while they did not exhibit a "specific" antiviral activity against any of the viruses tested.

We examined the inhibitory effect of 20 antiviral compounds, including ribavirin, on the replication of respiratory syncytial virus in HeLa and HEp-2 cell cultures. Of the compounds studied, pyrazofurin and 3-deazaguanine emerged as more potent inhibitors of respiratory syncytial virus than ribavirin. Based on their inhibitory effect on the cytopathogenicity of respiratory syncytial virus in HeLa cells, the average 50% effective dose of pyrazofurin and 3-deazaguanine for eight strains was 0.07 and 1.65 micrograms/ml, respectively; that of ribavirin was 5.82 micrograms/ml. The cytotoxicity of these compounds for HeLa cells was examined by monitoring the incorporation of radiolabeled uridine into cellular RNA. The selectivity indexes of pyrazofurin and 3-deazaguanine exceeded that of ribavirin by 70- and 11-fold, respectively. Pyrazofurin, 3-deazaguanine, and ribavirin inhibited both viral antigen expression and syncytium formation in HeLa cell cultures, as assessed by an indirect immunofluorescence assay. In these assays, pyrazofurin and 3-deazaguanine again proved more potent than ribavirin. 2,5-Diamidinoindole and carbodine were less potent than ribavirin. Various other compounds, i.e., 3-adenin-9-yl-2-hydroxypropanoic acid isobutyl ester, 3-deazauridine, 3'-C-methyluridine, 5'-deoxy-5-fluorouridine, 5-cyanoimidazole-4-carboxamide, and its ribofuranosyl derivative, did not inhibit the cytopathic effect of the Long strain of respiratory syncytial virus at concentrations greater than or equal to 125 micrograms/ml. Tubercidin, 5-chlorotubercidin, xylotubercidin, neplanocin A, thiosemicarbazone R, and 3-methylquercetine were too toxic to HeLa cells for their inhibitory effects on respiratory syncytial virus to be examined.

A variety of antiviral compounds were examined for their inhibitory effect on measles (SSPE) virus plaque formation in VERO cells. The following compounds inhibited SSPE virus (strain Niigata-1) replication at concentrations that were significantly lower than their minimum cytotoxic concentrations: neplanocin A, neplanocin C, carbocyclic 3-deazaadenosine, 9-(trans-2', trans-3'-dihydroxycyclopent-4'-enyl)adenine, 9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)-3-deazaadenine,(RS)-3-adenin-9-yl-2-hydroxypropanoic acid isobutyl ester, carbodine, cyclopentenyl cytosine, 3-deazaguanine, pyrazofurin, ribavirin and 6-azauridine. As the most selective inhibitors of SSPE virus replication emerged pyrazofurin, 3-deazaguanine, 6-azauridine and ribavirin. These compounds were further examined for their relative potency against a number of measles (SSPE) virus strains. Their order of (decreasing) potency was pyrazofurin greater than 6-azauridine approximately 3-deazaguanine greater than ribavirin. Amantadine, inosiplex and glycyrrhizin, that were also included in these assays, did not show appreciable activity against any of the measles (SSPE) virus strains.

Novel neplanocin A analogues modified at the 6'-position, i.e., 6'-deoxy analogues (2, 3, 6, 9, 20), 6'-O-methylneplanocin A (15), and 6'-C-methylneplanocin A's (22a and 22b) have been synthesized and evaluated for their antiviral activity in a wide variety of DNA and RNA virus systems. These compounds showed an activity spectrum that conforms to that of S-adenosylhomocysteine hydrolase inhibitors. They were particularly active against pox-(vaccinia), paramyxo-(parainfluenza, measles, respiratory syncytial), arena-(Junin, Tacaribe), rhabdo-(vesicular stomatitis), reo-, and cytomegalovirus. In order of (increasing) antiviral activity, the compounds ranked as follows: 3 less than 15 approximately 20 less than 6 less than 9 approximately 2 less than 22a. Of the two diastereomeric forms of 22, only 22a was active; 22a surpassed neplanocin A both in antiviral potency and selectivity. Compound 22a appears to be a promising candidate drug for the treatment of pox-, paramyxo-, arena-, rhabdo-, reo-, and cytomegalovirus infections.

A series of four mannose(Man)-, three N-acetylglucosamine (GlcNAc)n-, ten N-acetylgalactosamine/galactose(GalNAc/Gal)-, one 5-acetylneuraminic acid (alpha-2,3-Gal/GalNAc)- and one 5-acetylneuroaminic acid(alpha-2,6-Gal/Gal-NAc)-specific plant agglutinins were evaluated for their antiviral activity in vitro. the mannose-specific lectins from the orchid species Cymbidium hybrid (CA), Epipactis helleborine (EHA) and Listera ovata (LOA) were highly inhibitory to human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in MT-4, and showed a marked anti-human cytomegalovirus (CMV), respiratory syncytial virus (RSV) and influenza A virus activity in HEL, HeLa and MDCK cells, respectively. The 50% effective concentration (EC50) of CA and EHA for HIV ranged from 0.04 to 0.08 micrograms/ml, that is about 3 orders of magnitude below their toxicity threshold (50% inhibitory concentration for MT-4 cell growth: 54 to 60 micrograms/ml). Also, the (GlcNAc)n-specific lectin from Urtica dioica (UDA) was inhibitory to HIV-1-, HIV-2-, CMV-, RSV- and influenza A virus-induced cytopathicity at an EC50 ranging from 0.3 to 9 micrograms/ml. The GalNAc/Gal-, alpha-2,3-Gal/GalNAc- or alpha-2,6-Gal/GalNAc-specific lectins were not inhibitory to HIV or CMV at non-toxic concentrations. CA, EHA and UDA proved to be potent inhibitors of syncytium formation between persistently HIV-1- and HIV-2-infected HUT-78 cells and CD4+ Molt/4 (clone 8) cells (EC50: 0.2-2 micrograms/ml). Unlike dextran sulfate, the plant lectins CA, EHA and UDA did not interfere with HIV-1 adsorption to MT-4 cells and RSV- and influenza A virus adsorption to HeLa and MDCK cells, respectively. They presumably interact at the level of virion fusion with the target cell.

(+/-)-5'-Noraristeromycin (3) has been prepared in three steps beginning with the 2,3-O-isopropylidene derivative of (+/-)-(1 alpha, 2 beta, 3 beta, 4 alpha)-4-amino-1,2,3-cyclopentanetriol (7). Also prepared from the same starting material were the related hypoxanthine (4), guanine (5), and 2,6-diaminopurine (6) analogues. Compounds 3-6 were evaluated for antiviral activity against a large number of viruses with marked activity being observed for 3 towards vaccinia virus, human cytomegalovirus, vesicular stomatitis virus, parainfluenza (type 3) virus, measles virus, respiratory syncytial virus, reovirus (type 1), and the arenaviruses Junin and Tacaribe. None of the compounds showed cytotoxicity to the host cell monolayers used in the antiviral studies. Both 3 and 6 have been found to be inhibitors of S-adenosyl-L-homocysteine hydrolase (AdoHcy hydrolase), which likely accounts for their antiviral activity. Inhibition of AdoHcy hydrolase represents a new approach to human cytomegalovirus drug design that should be pursued. Also, the activity of 3 should be further scrutinized for the treatment of pox-, rhabdo-, paramyxo-, reo-, and arenavirus infections.

Various polyoxometalates proved inhibitory to the replication of a number of enveloped DNA and RNA viruses, i.e., herpesviruses (herpes simplex and cytomegalo), togaviruses (Sindbis), paramyxoviruses (respiratory syncytial), rhabdoviruses (vesicular stomatitis), arenaviruses (Junin and Tacaribe), and retroviruses [human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), simian immunodeficiency virus, and murine sarcoma virus]. The most potent compounds, i.e., JM1590 [K13[Ce(SiW11O39)2]. 26H2O] and JM2766 [K6[BGa(H2O)W11O39]. 15H2O], inhibited HIV-1 and simian immunodeficiency virus at concentrations as low as 0.008-0.8 microM. The polyoxometalates also inhibited giant cell formation in co-cultures of HIV-infected HUT-78 cells and uninfected MOLT-4 cells. Studies designed to unravel the mechanism of action of these compounds revealed that they inhibit the reverse transcriptase activity associated with HIV. The polyoxometalates also proved inhibitory to the binding of HIV-1 virions to the cells. From "time of addition" experiments, whereby the polyoxometalates were added at different times after virus infection, their mechanism of anti-HIV action could be attributed to inhibition of virus-cell binding. There was a good correlation (r = 0.84) between the inhibitory effects of the compounds on HIV-1-induced cytopathicity and their inhibitory effects on syncytium formation and a close correlation (r = 0.902) between their inhibitory effects on syncytium formation and their interaction with gp120, whereas there was no correlation between their anti-HIV-1 activity and their inhibitory effects on HIV-1 reverse transcriptase. In flow cytometric studies, the compounds did not interfere with the binding of OKT4A/Leu-3a monoclonal antibody to the CD4 receptor of uninfected cells, but they inhibited binding of anti-gp120 monoclonal antibody to HIV-1-infected cells. Thus, the binding of the polyoxometalates to the viral envelope glycoprotein gp120 is responsible for their anti-HIV activity.

We studied the antiviral activity of carbohydrate-binding agents (CBAs), including several plant lectins and the non-peptidic small-molecular-weight antibiotic pradimicin A (PRM-A). These agents efficiently prevented hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection of target cells by inhibiting the viral entry. CBAs were also shown to prevent HIV and HCV capture by DC-SIGN-expressing cells. Surprisingly, infection by other enveloped viruses such as herpes simplex viruses, respiratory syncytial virus and parainfluenza-3 virus was not inhibited by these agents pointing to a high degree of specificity. Mannan reversed the antiviral activity of CBAs, confirming their association with viral envelope-associated glycans. In contrast, polyanions such as dextran sulfate-5000 and sulfated polyvinylalcohol inhibited HIV entry but were devoid of any activity against HCV infection, indicating that they act through a different mechanism. CBAs could be considered as prime drug leads for the treatment of chronic viral infections such as HCV by preventing viral entry into target cells. They may represent an attractive new option for therapy of HCV/HIV coinfections. CBAs may also have the potential to prevent HCV/HIV transmission.

The general principle of anti-adhesion therapy is the inhibition of microorganism adhesion to the host cell with the help of a soluble receptor analog. Despite an evident attractiveness of the concept and its long existence, the therapeutics of the 'post-antibiotic era' have not yet appeared. This can be explained by the contradictoriness of requirements for anti-adhesion drugs: to be efficient a drug must be multivalent, i.e. large molecule, but to obtain FDA approval it should be a small molecule. A way to overcome this contradiction is self-assembly of glycopeptides. The carbohydrate part of glycopeptide is responsible for binding with the lectin of microorganisms, whereas a simple peptide part is responsible for an association to the so-called tectomers. Depending on the structure, tectomers are formed either spontaneously or upon promotion of a microorganism. In particular, sialopeptide, which is capable of converting to a tectomer only in the presence of the influenza virus, has been obtained. Thus, the new strategy of anti-adhesion therapy can be formulated as follows:(1) identification of oligosaccharide-receptor for a particular virus (bacteria);(2) optimization of the peptide part;(3) conventional trials. The expected advantages of this strategy are the following:(i) no polymer;(ii) a virion completely covered with a tectomer, i.e. blocking is both complete and irreversible;(iii) rapid and rational lead identification and optimization;(iv) minimum side effects;(v) potential for microorganism resistance to natural receptor is lower than in the case of mimetics.

Several sulfated seaweed polysaccharides show high antiviral activity against enveloped viruses, including important human pathogens such as human immunodeficiency virus, herpes simplex virus, human cytomegalovirus, dengue virus and respiratory syncytial virus. They can be obtained in major amounts and at low costs, have low toxicity and in some cases, lack anticoagulant effects. Even if the systemic applications have many drawbacks, their structure and mode of action indicate potential for topical uses to prevent virus infection. The herpes simplex viruses attach to cells by an interaction between the envelope glycoprotein C and the cell surface heparan sulfate (HS). The virus-cell complex is formed by ionic interactions between the anionic (mainly sulfate) groups in the polysaccharide and basic amino acids of the glycoprotein, and non-ionic ones depending on hydrophobic amino acids interspersed between the basic ones in the glycoprotein-binding zone. Hypothesis are advanced of the corresponding hydrophobic structures in the polysaccharides. The antiviral activity of the sulfated seaweed polysaccharides is based on the formation of formally similar complexes that block the interaction of the viruses with the cells. Correlations are established between different structural parameters and antiviral activity. The minimal, ionic and hydrophobic, structures in the seaweed polysaccharides were hypothesized by comparison of the polysaccharides with the known minimal binding structure in HS/heparin, together with a correlation between those structures of the polysaccharides and their antiviral activity.

Large polyanionic molecules, such as sulfated polysaccharides (including soluble heparin and dextran sulfate), synthetic polyanionic polymers, and negatively charged proteins, have been shown to broadly inhibit several enveloped viruses. We recently reported the antiviral activity of a peptide derived from amino acids 77 to 95 of a potential binding partner of respiratory syncytial virus F protein (RSV F), the GTPase RhoA. A subsequent study with a truncated peptide (amino acids 80 to 94) revealed that optimal antiviral activity required dimerization via intermolecular disulfide bonds. We report here that the net negative charge of this peptide is also a determining factor for its antiviral activity and that it, like other polyanions, inhibits virus attachment. In a flow cytometry-based binding assay, peptide 80-94, heparin, and dextran sulfate inhibited the attachment of virus to cells at 4 degrees C at the same effective concentrations at which they prevent viral infectivity. Interestingly, time-of-addition experiments revealed that peptide 80-94 and soluble heparin were also able to inhibit the infectivity of a virus that had been prebound to cells at 4 degrees C, as had previously been shown for dextran sulfate, suggesting a potential role for postattachment effects of polyanions on RSV entry. Neutralization experiments with recombinant viruses showed that the antiviral activities of peptide 80-94 and dextran sulfate were diminished in the absence of the RSV attachment glycoprotein (G). Taken together, these data indicate that the antiviral activity of RhoA-derived peptides is functionally similar to that of other polyanions, is dependent on RSV G, and does not specifically relate to a protein-protein interaction between F and RhoA.