The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50 -60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80 -90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for ␣ 6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted ␤-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface.

Butanol, at sub-growth-inhibitory levels, caused a ca. 20 to 30% increase in fluidity of lipid dispersions from Clostridium acetobutylicum. When grown in the presence of butanol or into stationary phase, C. acetobutylicum synthesized increased levels of saturated acyl chains at the expense of unsaturated chains.

Unsaturated monoglycerides and alcohols of chain lengths of 16 or 18 carbons were found to be extremely potent inactivators of two enveloped viruses, herpes simplex virus type 2 and bacteriophage (p6. The lipid-containing bacteriophage PM2 was also inactivated by some of these amphiphilic molecules. Treatment of herpes simplex virus type 2 with these compounds at concentrations as low as 0.2 ,M reduced virus survival to 50% in 30 min, making these agents the most potent inactivators of herpes simplex viruses discovered that are not cytotoxic to mammalian cells. Detailed characterizations of the effects of unsaturated monoglycerides and alcohols on bacteriophages (p6 and PM2 showed that the inactivated (p6 virion remained nearly intact but that PM2 was almost completely disrupted by the inactivating treatment. Some of the compounds inactivate the viruses even at low temperature (0°C). Excess amounts of diglycerides and phospholipids interfere with the inactivating abilities of some of the unsaturated monoglycerides and alcohols against (p6 and PM2. Our findings suggest that the unsaturated monoglycerides and some of the unsaturated alcohols should be further studied as potential antiviral agents, particularly for application to herpesvirus-infected areas of the skin and accessible epithelium.It was reported 30 years ago that an unsaturated fatty acid (oleic acid, 18:1, A9 cis) inactivates influenza type A virions (16). Very little work on the potential antiviral activity of low concentrations of fatty acids and other hydrocarbon derivatives has been done until recently. We recently reported that oleic acid and palmitoleic acid (16:1, A9 cis) are potent inactivators of the lipid-containing bacteriophage p6 (10) and inhibitors of the entry process of another lipidcontaining bacteriophage, PR4 (8). The common food additive BHT (butylated hydroxytoluene) has been shown to inactivate a variety of lipidcontaining viruses, including herpes simplex virus (HSV), Newcastle disease virus, bacteriophage (6, and bacteriophage PM2 (2,3,14,19). The methyl ester derivative of the polyene antibiotic amphotericin B inactivates vesicular stomatitus virus (6), and some saturated long-chain alcohols inactivate HSV, (p6, and PM2 (15).None of these agents inactivates non-lipid-containing viruses at the low concentrations that cause inactivation of the tested lipid-containing viruses.We are conducting a comprehensive survey of the antiviral effects of fatty acid derivatives. We report here that some unsaturated long-chain alcohols and monoglycerides exhibit extremely potent virucidal effects against HSV and bacteriophages (p6 and PM2. We also report the results of studies on the mechanism(s) of these effects on the two lipid-containing bacterial viruses.MATERIALS AND METHODS Viruses and cells. Bacteriophage (p6 (18) contains a loose envelope structure composed of phospholipid and protein (1, 9, 12), making it structurally similar to the enveloped animal viruses. Bacteriophage PM2 (4) contains an internal lipid bilayer whose structure an...

Butylated hydroxytoluene (BHT) is a potent inactivator of the enveloped bacterial virus 46 at concentrations as low as 3 x 10-5 M. The viral envelope is not removed by BHT treatment, in contrast to the effects of exposure to the detergent Triton X-100. BHT-treated viruses are morphologically indistinguishable from controls but are defective in their ability to attach to the host cell. Temperature at the time of exposure was found to .be a crucial factor in the effectiveness of BHT against gb6. A precipitous drop in the degree of inactivation by 3 x 10-5 M BHT occurred when the temperature was lowered from 20 to 15 C. Calcium ions were found to potentiate the effect of BHT, particularly at lower temperatures where BHT alone was relatively ineffective. Barium and strontium, but not magnesium, were also effective in enhancing the activity of BHT. A structurally related molecule, butylated hydroxyanisole (BHA), was also found to inactivate 46 virus, but higher concentrations were required than with BHT. Both BHT and BHA are commonly used as food additives, have apparent low toxicity to humans and other animals, and are potentially useful as antiviral agents.It was recently reported from our laboratory that butylated hydroxytoluene (BHT) is a potent inactivator of lipid-containing viruses (11). This compound is generally recognized as safe by the Food and Drug Administration (5) and is widely used as an antioxidant in food and cosmetic preparations. The combination of effectiveness against viruses and apparent low toxicity to humans has generated considerable interest in the potential use of BHT as an antiviral agent, particularly for topical application to virus-infected areas of the skin. Consequently, we are characterizing the effects of BHT on lipid-containing viruses in vitro, hoping to improve our understanding of the mechanisms whereby BHT destroys viral infectivity.In a detailed study of the effects of BHT on the bacterial virus PM2, it was found that virus particles are completely disrupted by BHT, with the viral deoxyribonucleic acid being released to the medium as a very slowly sedimentable material (1). Several factors which influence the inactivation of PM2 by BHT, including the initial virus titer, the time of exposure, and the presence of certain "protective" agents including surfactants, bovine serum al- ' Present address: Washington University Medical School, Cancer Center, St. Louis, Mo. 63130. bumin, and bacterial cells, were studied. Potentially important with regard to the use of BHT as an antiviral substance was the observation that its effectiveness is strongly dependent upon the solvent from which it is added to the culture medium.Different viruses have different susceptibilities to BHT, and the most susceptible one studied thus far is the enveloped bacterial virus 46. This virus is bounded by a rather loose membrane structure (3, 13) and has many similarities to several animal viruses. We have now studied in more detail the inactivation of46 by BHT, and the results of this research are de...

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