The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes.

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 alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-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.

Physical map of the Clostridium difficile chromosome.

Clostridium difficile is a causative agent in antibiotically induced diarrhea and pseudomembraneous colitis. The ability of strains of C. difficile to cause disease depends upon the presence of two toxin genes and their corresponding proteins, designated toxin A and toxin B. Previous studies conducted in this laboratory indicated that toxigenic strains of C. difficile possess both toxin genes, whereas non-toxigenic strains do not. Likewise, the studies showed that toxigenic and non-toxigenic strains of C. difficile differ significantly in chromosomal organization by ribotype analysis. Therefore, the chromosomal organization of a reference strain was investigated. Pulsed field gel electrophoresis was utilized to generate a physical map of the chromosome of the toxigenic Clostridium difficile strain ATCC 43594. Restriction digestions of whole chromosomes with the enzymes NruI and SacII generated consistent macrofragment profiles. NruI digestion resulted in 14 discernible bands containing 16 fragments of DNA. SacII digestions resulted in 14 discernible bands containing 15 fragments of DNA. Restriction digestions with both SacII and NruI resulted in 21 discernible bands containing 31 fragments of DNA. Probing of single and double digests with an extensive set of NruI and SacII single- and double-digest bands clarified the location of individual fragments in relation to one another, resulting in a restriction map of the chromosome. PCR-generated probes of five loci of C. difficile were used to map the location of seven genes on the chromosome. Finally, the addition of all fragments from NruI, SacII and NruI/SacII digestions resulted in an approximate chromosome size of 4.4 Mb.

Macrofragment localization of the toxin A and toxin B genes of Clostridium difficile.

We report the physical mapping of the toxin A and B genes to the bacterial chromosome of Clostridium difficile ATCC 43594 by pulsed-field gel electrophoresis. Single and double digestions with restriction endonucleases NruI and SacII allowed localization of the toxin genes to a specific 577-kb fragment and estimation of genome size to be approximately 3.8 megabases. This effort represents the initial step in the construction of a physical map of the whole genome.

Incidence of pulmonary aspiration in intubated patients receiving enteral nutrition through wide- and narrow-bore nasogastric feeding tubes.

A descriptive study was performed to compare the incidence of pulmonary aspiration in 25 critically ill patients who had endotracheal tubes in place and were receiving enteral nutrition through a narrow-bore nasogastric tube (n = 10) or a wide-bore nasogastric tube (n = 15). Results of chi-square analysis of this comparison were not significant, p less than 0.05. Aspiration occurred in one subject. The amount and rate of tube feeding delivered was examined. The number of checks for residual feeding was found to be significantly greater in the wide-bore tube group. A comparison of the assessment of nasogastric tube placement on x-ray examination showed that tube placement was reported on x-ray results with more frequency in the wide-bore group. Questions are raised by these observations regarding the use of narrow-bore tubes in the critically ill population with endotracheal tubes in place.

Ethanol Perturbs Glycosylation and Inhibits Hypersecretion in Trichoderma reesei.

The effects of ethanol and phenylethanol on the growth of and glycoprotein secretion by Trichoderma reesei were studied. Low levels (1.5%, vol/vol) of ethanol perturbed the glycosylation process, as shown by alterations in the isoelectric profile of the secreted proteins and a reduction in the rate of incorporation of mannose into oligosaccharides. In addition to these effects on posttranslational modification, ethanol drastically lowered the protein secretion level of a hypersecretory strain.

Preliminary characterization of bacteriophages infecting the thermophilic actinomycete thermomonospora.

Bacteriophages lysing strains of Thermomonospora alba and T. fusca were isolated, following specific enrichment, from vegetable composts. Four Thermomonospora phages were distinguished by plaque morphology and host range. Electron microscopy of phage particles, termperature inactivation profiles, and electrophoretic analyses of major virion proteins and genomic DNA were used in the comparison and initial characterization of these phages. The four phages studied possessed polyhedral heads and long tails; genomes were linear double-stranded DNA molecules, 35 to 45 kilobases in length, which probably contain cohesive ends. Transfection of Thermomonospora protoplasts with purified genomic DNA from one of the phages was demonstrated.

Genetic Recombination and Transformation in Protoplasts of Thermomonospora fusca.

Protoplasts were produced from the thermophilic actinomycete Thermomonospora fusca and were regenerated to 0.1% of the direct count on regeneration agar. Recombination after protoplast fusion was demonstrated with drug-resistant mutants of T. fusca YX. A single thiostrepton-resistant colony was isolated after transformation of T. fusca YX with the streptomycete vector pIJ702, providing the first evidence for transformation in the genus Thermomonospora and suggesting that some mesophilic streptomycete genes can be expressed in thermophilic actinomycetes. Of 20 thermophilic actinomycete strains isolated from self-heated composts, 3 were found to harbor native plasmid DNA, providing potential sequences for the development of Thermomonospora-Streptomyces shuttle vectors.

Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824.

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.

Photochemical destruction of the virucidal activities of retinoids and unsaturated fatty acids.

Photochemical damage either to retinoids by near-ultraviolet radiation or to unsaturated fatty acids by near-ultraviolet radiation in the presence of a hydrophobic photosensitizer destroys the virucidal activities of the compounds, as determined by studies on the enveloped bacteriophage ø6.

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.

Inhibition of entry of the lipid-containing bacteriophage PR4 by fatty acid derivatives.

Amphiphilic fatty acid derivatives having a 14- to 20-carbon cis-unsaturated alkyl chain were found to be potent inhibitors of the entry process of the lipid-containing bacteriophage PR4.

Enveloped virus inactivation by fatty acid derivatives.

The enveloped bacteriophage phi6 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 phi6. 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.

Inhibitory effect of fatty acids on the entry of the lipid-containing bacteriophage PR4 into Escherichia coli.

Various unsaturated fatty acids (notably palmitoleic acid and oleic acid) interfered with plaque production by the lipid-containing bacteriophage PR4 on lawns of Escherichia coli. Addition of fatty acid to give 50 mug/ml ( approximately 0.2 mM) at the time of infection prevented phage replication. If, however, the fatty acid was added after infection, normal amounts of phage were produced. If the fatty acid was added (to 50 mug/ml) to the host cell culture a long enough time before infection such that the fatty acid concentration in the growth medium at the time of infection was reduced to less, similar5 mug/ml (due to fatty acid incorporation by the host cells), normal phage replication occurred also. Neither palmitoleic acid nor oleic acid prevented PR4 attachment to E. coli. Several types of experiments indicated that it is the entry process of the virus that is inhibited by these fatty acids. Specifically, if the fatty acid was added at the time of infection, the host cells were not killed by the virus and no detectable amounts of viral protein were synthesized. In addition, experiments using purified radioisotope-labeled virions showed directly that entry is inhibited. Mutants of PR4 that did replicate in the presence of oleic acid arose spontaneously at a frequency of 10(-6). Three of these mutants that have been further characterized have protein and phospholipid compositions indistinguishable from those of wild-type PR4.

Inactivation and inhibition of replication of the enveloped bacteriophage phi6 by fatty acids.

The enveloped bacteriophage phi6 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 phi6. Oleic acid (18:1) and palmitoleic acid (16:1) were potent inactivators of the virus. Treatment with either fatty acid at 50 mug/ml at 25 or 0 degrees 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 mug/ml ( approximately 10(-2) mM) reduced the virus titer to <1% of the initial titer within 30 min. Ultracentrifugation analyses of (14)C-amino acid- and (32)P-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 phi6 at 50 mug/ml, but nevertheless did prevent phi6 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 phi6 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 greater, similar10 mug/ml. phi6 could attach to its host cell in the presence of myristic acid at 50 mug/ml. We conclude that the fatty acids that prevent phi6 replication probably do so by interfering with the entry of the viral genome into the host cell.

Structural proteins of a lipid-containing bacteriophage which replicates in Escherichia coli: phage PR4.

PR4 is a lipid-containing bacteriophage which is able to replicate in Escherichia coli. The virus was labeled with either [14C]leucine and [14C]threonine or H235SO4 and then purified by several rounds of sucrose gradient centrifugation. Autoradiographs showed the virus to be composed of six major protein species with molecular weights (1) 68 000, (2) 47 500, (3) 38 500, (4) 35 000, (5) 20 700, (6) 16 500 daltons. Electropherograms showed protein No. 2 to be the major protein, comprising about 43% of the total weight of viral protein.

Effects of temperature and host cell genetic characteristics on the replication of the lipid-containing bacteriophage PR4 in Escherichia coli.

The lipid-containing bacteriophage PR4 is of special intest because it can replicate in various gram-negative bacteria, including Escherichia coli, that carry one of a group of drug resistance plasmids. PR4 grown in E. coli strain PS2R contains about 10% lipid by weight, with the negatively charged phospholipid phosphatidylglycerol being the most abundant lipid in the virion. We now report the following. (i) PR4 attaches to E. coli with an attachment rate constant of Ka approximately 6.2 X 10(-10) ml/min, which is about twice that of the enveloped phage phi6 (to Pseudomonas phaseolicola), but a factor of 5 less than that of phage PM2 (to Pseudomonas BAL-31). (ii) Use of an E. coli glycerol auxotroph indicated that a normal amount of PR4 replication occurs only if glycerol starvation (inhibition of all phospholipid synthesis) begins no earlier than about halfway through the lytic cycle. (iii) Use of an E. coli fatty acid synthesis temperature-sensitive mutant and an E. coli phosphatidylethanolamine synthesis temperature-sensitive mutant indicate that PR4 replication can occur in the absence of either normal fatty acid synthesis or normal phospholipid synthesis if the infection takes place prior to the termination of overall cell growth and the onset of cell death, (iv) Whereas PR4 burst size in nutrient media at 30 degrees C to 42%C is about 40, the burst size at 20 degrees C is less than 3, Temperature-shift experiments show that the temperature late in infection determines the burst size.

Origin of the phospholipids of a lipid-containing virus that replicates in Escherichia coli: bacteriophage PR4.

Bacteriophage PR4 contains lipid and can reproduce in strains of Escherichia coli that carry an appropriate drug-resistance plasmid. Cultivated in either of two E. coli strains, PR4 acquires a lipid region that contains a relatively high level of phosphatidylglycerol and significant amounts of three phospholipids, including phosphatidylserine, which are present in only very low levels in the host cell membranes. To do this, however, PR4 does not significantly alter the relative levels of synthesis of the various E. coli phospholipids after infection. Production of PR4 virions from E. coli cultures labeled with 32PO4 either before or after infection showed that about two-thirds of the viral phospholipid is synthesized after infection. The use of E. coli as the host organism for PR4 should allow a detailed understanding of the assembly process of this lipid-containing virus due to the wealth of biochemical and genetic techniques available.