Jeffrey A. Sands

Extreme Sensitivity of Enveloped Viruses, Including Herpes Simplex, to Long-Chain Unsaturated Monoglycerides and Alcohols

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 φ6. 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 μM 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 φ6 and PM2 showed that the inactivated φ6 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 φ6 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.

Effects of tunicamycin on secretion and enzymatic activities of cellulase fromTrichoderma reesei

SummaryThe effects of tunicamycin, an inhibi-tor of N-asparagine linked glycosylation, on the synthesis, secretion, and activities of the cellulases produced byTrichoderma reesei wild type QM6a and hypersecrefing mutant RL-P37 were studied. Neither the level of secreted cellutase nor the total amount of secreted protein was affected by the drug at a concentration (5 μg/ml) that slightly in-hibited growth. SDS-polyacrylamide gel electro-phoretic mobilities of proteins secreted during growth in tunicamycin were similar to those of proteins from control cultures that had their N-linked oligosaccharides removed by endoglycosi-dase H. Isoelectric focusing patterns of secreted proteins were also altered by growth in the pres-ence of tunicamycin. All of the bands stained with Schiff’s reagent, indicating that the secreted cellu-lases contained O-linked oligosaccharides in ad-dition to N-linked sugars. Endoglucanase activity in culture broths from tunicamycin grown mycelia was more thermolabile and protease-sensitive than the same activity from control cultures. Thus, N-asparagine linked oligosaccharides do not appear to be necessary forT. reesei cellulase secretion or activity, but do seem to contribute to the stability of the enzymes. The role of O-finked oligosaccharides is being investigated.

Inactivation of the Enveloped Bacteriophage ø6 by Butylated Hydroxytoluene and Butylated Hydroxyanisole

Butylated hydroxytoluene (BHT) is a potent inactivator of the enveloped bacterial virus ø6 at concentrations as low as 3 × 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 ø6. A precipitous drop in the degree of inactivation by 3 × 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 ø6 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.

Enveloped Virus Inactivation by Fatty Acid Derivatives

The enveloped bacteriophage φ6 has been shown to be a valuable model system for the preliminary screening of compounds that might be expected to inactivate herpes simplex virus and other enveloped mammalian viruses. A variety of fatty acid derivatives that form fluid micelles in aqueous media have been found to be potent inactivators of φ6. The chemical nature of the polar head group, the length of the alkyl chain(s), and the extent and geometry of unsaturation are all important parameters in determining the antiviral effectiveness of this class of compounds.

Inactivation and Inhibition of Replication of the Enveloped Bacteriophage φ6 by Fatty Acids

The enveloped bacteriophage φ6 has been shown to be an interesting model system for the study of chemical agents that might have specific antiviral effects against lipid-containing mammalian viruses. In this report, we describe two types of antiviral activity exhibited by several fatty acids against bacteriophage φ6. Oleic acid (18:1) and palmitoleic acid (16:1) were potent inactivators of the virus. Treatment with either fatty acid at 50 μg/ml at 25 or 0°C for 30 min reduced the virus titer to about 0.1% of the initial titer. Oleic acid at a concentration as low as 3 μg/ml (∼10−2 mM) reduced the virus titer to <1% of the initial titer within 30 min. Ultracentrifugation analyses of 14C-amino acid- and 32P-labeled virus treated with oleic acid indicated that the virion is largely disassembled by the treatment. Myristic acid (14:0) and palmitic acid (16:0) did not inactivate φ6 at 50 μg/ml, but nevertheless did prevent φ6 plaque production. Single-step virus growth experiments in which fatty acid was added at various times before or after infection indicated that it was an early stage of the φ6 replication cycle that was inhibited by the presence of myristic acid and that the inhibition occurred only if the myristic acid concentration in the extracellular growth medium was ≳10 μg/ml. φ6 could attach to its host cell in the presence of myristic acid at 50 μg/ml. We conclude that the fatty acids that prevent φ6 replication probably do so by interfering with the entry of the viral genome into the host cell.

The phospholipid composition of bacteriophage φ6

Summary The enveloped bacteriophage φ6 appears to be similar in several ways to the enveloped mammalian viruses. 32 P-labelled stocks of φ6 and its host cell were produced and then subjected to phospholipid extraction and analyzation by thin layer chromatography. The results indicate that φ6 and its host cell contain three major phospholipids: cardiolipin, phosphatidylethanolamine, and phosphatidylglycerol. φ6 contains a higher relative amount of phosphatidylglycerol (57%) than does its host cell (29%), suggesting that φ6 does not acquire its membrane directly from an unaltered cellular membrane.

Virucidal activity of retinal.

Herpes simplex virus type 2 and simian virus 40 were rapidly inactivated by retinal at micromolar concentrations. Other fat-soluble vitamins, particularly vitamin A derivatives, were also active against herpes simplex virus type 2 and several lipid-containing bacteriophages.

Different temperature profiles of enzyme secretion by two common strains of Trichoderma reesei.

SummaryWe have investigated the effects of high and low temperature on the synthesis and secretion of cellulases and other enzymes by two common and readily available strains ofTrichodermareesei. While some effects were similar in both strains QM9414 and RUT-C30 (a reduction in cellulase production but stimulation of xylanase production at high temperature, and alterations in expression of the cellulase complex at low temperature), some specific differences between the strains were determined, most significantly an enhanced specific secretion rate (secretion/growth) at low growth temperature for QM9414.

Detection of acrylic acid formation in Megasphaera elsdenii in the presence of 3-butynoic acid

SummaryThe formation of acrylic acid from lactic acid in the anaerobic rumen bacterium Megasphaera elsdenii was detected in the presence of 3-butynoic acid. While the major end products of lactic acid fermentation in the absence of the inhibitor were propionate, acetate, valerate, and butyrate, the presence of 3-butynoic acid led to the production of propionate, acetate, acrylate, and butyrate. An improvement in the chemical synthesis and purification of 3-butynoic acid was developed.

Electrophoretic karyotype ofTrichoderma reesei

SummaryPulsed field electrophoresis was used to separate the intact chromosomes of the fungi imperfectiTrichoderma reesei for which no previous karyotype has been available.T. reesei was shown to have six chromosomes with molecular weights ranging from 3.3 to 11.9 megabasepairs (Mbp). The genome size was estimated to be 38 Mbp, indicating thatTrichoderma has a genome larger thanAspergillus nidulans (31 Mbp) but smaller thanNeurospora crassa (47 Mbp).