Negative Potentials Across Biological Membranes Promote Fusion by Class II and Class III Viral Proteins

Voltage was investigated as a factor in the fusion of virions. Virions, pseudotyped with a class II, SFV E1 or VEEV E, or a class III protein, VSV G, were prepared with GFP within the core and a fluorescent lipid. This allowed both hemifusion and fusion to be monitored. Voltage clamping the target cell showed that fusion is promoted by a negative potential and hindered by a positive potential. Hemifusion occurred independent of polarity. Lipid dye movement, in the absence of content mixing, ceased before complete transfer for positive potentials, indicating that reversion of hemifused membranes into two distinct membranes is responsible for voltage dependence and inhibition of fusion. Content mixing quickly followed lipid dye transfer for a negative potential, providing a direct demonstration that hemifusion induced by class II and class III viral proteins is a functional intermediate of fusion. In the hemifused state, virions that fused exhibited slower lipid transfer than did nonfusing virions. All viruses with class II or III fusion proteins may utilize voltage to achieve infection.     

Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell

Cells expressing the low pH-triggered class II viral fusion protein E1 of Semliki Forest virus (SFV) were fused to target cells. Fusion was monitored by electrical capacitance and aqueous dye measurements. Electrical voltage-clamp measurements showed that SFV E1-induced cell-cell fusion occurred quickly after acidification for a trans-negative potential across the target membrane (i.e., negative potential inside the target cell) but that a trans-positive potential eliminated all fusion. Use of an ionophore to control potentials for a large population of cells confirmed the dependence of fusion on voltage polarity. In contrast, fusion induced by the class I fusion proteins of human immunodeficiency virus, avian sarcoma leukosis virus, and influenza virus was independent of the voltage polarity across the target cell. Initial pore size and pore growth were also independent of voltage polarity for the class I proteins. An intermediate of SFV E1-induced fusion was created by transient acidification at low temperature. Membranes were hemifused at this intermediate state, and raising the temperature at neutral pH allowed full fusion to occur. Capacitance measurements showed that maintaining a trans-positive potential definitely blocked fusion at steps following the creation of the hemifusion intermediate and may have inhibited fusion at prior steps. It is proposed that the trans-negative voltage across the endosomal membrane facilitates fusion after low-pH-induced conformational changes of SFV E1 have occurred.     

Transglycosylation of benzo[h]quinazolines

This was the first study to apply cyclodextrin glucanotransferases (CGTase, EC 2.4.1.19) produced by mesophilic, thermophilic, alkaliphilic, and halophilic bacilli as well as pullulanase, β-amylase, β-galactosidase, and β-fructofuranosidase for transglycosylation of benzo[h]quinazolines. The combination of CGTase produced by Bacillus stearothermophilus ST-88 and γ-cyclodextrin (CD) used as a donor of glucosyl residues was the most efficient. The derivatives obtained are water-soluble. The synthesized products have been purified by various chromatographic methodsa and their fine structures have been determined.     

Effect of Mechanical Vibrations on the lonMutant of Escherichia coliK-12

It was found that, depending on their frequency, mechanical vibrations (MVs) can either stimulate (4 Hz) or inhibit (50 Hz) the growth and the division of the lonmutant of Escherichia coliK-12. Similar effects were observed when the MV-treated nutrient medium was inoculated with untreated mutant cells. MVs enhanced the motility of mutant cells and the fragmentation of filament cells always present in the populations of lonmutants.     

The Effect of Acute Unilateral Denervation Injury on Purinergic Signaling in the Cholinergic Synapse

Biophysics - Previously, we found a change in the efficiency of the modulatory action of ATP under the influence of some non-physiological factors at neuromuscular synapses in rodents. This study...     

The Six-Helix Bundle of Human Immunodeficiency Virus Env Controls Pore Formation and Enlargement and Is Initiated at Residues Proximal to the Hairpin Turn

Residues that create the grooves of the human immunodeficiency virus type 1 (HIV-1) Env triple-stranded coiled coil (HR1) and the residues that pack into the grooves (HR2) to complete the formation of the six-helix bundle (6HB) were mutated. The extent and kinetics of fusion as well as pore enlargement were measured for each mutant. Mutations near the hairpin turns of each monomer of the 6HB were more important than those far from the turn, for both HR1 and HR2. This result is consistent with the idea that binding of HR2 to the HR1 grooves is initiated near the hairpin turn of each monomer. Mutations at the distal portions also reduced fusion, albeit to a smaller extent. An intermediate of fusion (temperature-arrested state [TAS]) was formed, and the consequences of mutation were compared; a mutant that exhibited less fusion also showed slower kinetics from TAS. This suggests that formation of the bundle is a rate-limiting step downstream of the intermediate state. The rate of enlargement of a fusion pore also correlated with the extent and kinetics of fusion. The rate of pore enlargement was severely reduced by mutation. This supports our prior conclusion that formation of the 6HB occurs after pore creation and strongly suggests that the free energy released by bundle formation is directly used to promote pore growth.All class I viral fusion proteins are composed of three identical monomers that together fold into a six-helix bundle (6HB) in the protein''s final, postfusion state (33, 50). Each monomer of human immunodeficiency virus (HIV) Env (which is a class I fusion protein) consists of two subunits, gp120 and gp41. The three gp41 subunits of each Env trimer form the 6HB. The crystallographic structure of gp41 has been determined for its final state; its 6HB reveals that the three N-terminal segments of gp41—each segment referred to as heptad repeat 1 (HR1)—have folded into a central triple-stranded coiled coil of α-helices, and the three C-terminal segments (HR2) have packed, antiparallel, as α-helices into the three grooves of the coiled coil. The 6HB of HIV Env is thermally stable (43) and can confidently be considered a final structure.The crystallographic structures for both the initial and final states have been obtained for several other class I viral fusion proteins (2, 7, 22, 26, 43, 54). They reveal that the protein must undergo large-scale conformational changes in transiting from their initial state to final 6HB state, including changes in secondary structure. Although the initial structure of gp41 has not been determined, it is known that grooves are not exposed in its initial conformation; they become exposed as part of the gp41 conformational changes during fusion (13). The configurations of Env during which grooves are exposed are referred to as prebundles. It is assumed that gp41 undergoes large-scale reconfigurations during the fusion process.It is certain that formation of the 6HB is a central event in infection; 6HBs form in all class I proteins; peptides that prevent formation of the bundle block infection (6, 17, 20, 21, 36, 38, 51); and many residues of HIV''s 6HB are conserved, even though Env is a rapidly mutating protein. In fact, the residues that comprise the grooves are almost perfectly conserved, and the groove-packing residues of the C-terminal HR2 segments are highly conserved (8). The importance of the bundle is underscored by the finding that point mutations that weaken the associations between HR2 and their grooves are deleterious to fusion (23).Because each monomer of gp41 has a hairpin turn in the final structure, bundle formation requires that each monomer fold in the “middle,” like a jackknife. For HIV Env, HR1 is near the fusion peptide, and HR2 is close to the membrane-spanning domain (MSD); thus, bundle formation brings the fusion peptides and MSDs into proximity. At the time 6HBs were being identified as a common structure, it was assumed that their formation merely brought membranes into contact (8, 49), but it has since been shown that for HIV Env and for simian virus 5 not all of the fusion proteins that participate in fusion fold into 6HBs prior to membrane merger (35, 41) and, in fact, bundle formation is not completed until the viral envelope and cell membrane have become continuous upon the creation of a fusion pore (28).The amino acids at the “e” and “g” positions of HR1 comprise the grooves of the central triple-stranded coiled coil (Fig. ​(Fig.1).1). The “a” and “d” positions of the HR2 domains fit into these hydrophobic grooves to create the 6HB (Fig. ​(Fig.1).1). Alanine-scanning mutagenesis has been carried out for the “e” and “g” positions of HR1 to identify sites deleterious to fusion, and it has been shown that reductions or increases in bundle stability caused by mutation at these sites are not good predictors for reductions or increases in extents of fusion (23).Open in a separate windowFIG. 1.Depiction of a 6HB. Three HR1 segments, one from each gp41 monomer, combine to form a central triple-stranded coiled coil. Three HR2 segments of gp41 pack into the grooves to complete the bundle (six-circle figure). Residues at positions “a” and “d” of HR2 pack into the grooves. All were mutated, one at a time, to alanine. The “e” and “g” of HR1 create the groove. Residues at these positions were mutated to alanine, except for residue A558 (a “g” position) which is naturally alanine.In the present study, we mutated all “a” and “d” residues of HR2 and all “e” and “g” residues of HR1. We then measured the extent and rate of fusion, as well as the rate of pore growth, and compared results for the various mutants. From these comparisons, we were able to infer the order of interactions between residues of HR1 and HR2 in bundle formation.     

Transglycosylation of <Emphasis Type="Italic">L</Emphasis>-ascorbic acid

Cyclodextrin glucanotransferases (CGTs, EC 2.4.1.19) from mesophilic, thermophilic, and halophilic bacteria and maltase (EC 3.2.1.20) from the yeast Saccharomyces cerevisiae were used for transglycosylation of ascorbic acid with starch, maltodextrin, γ-cyclodextrin, and maltose. These compounds served as donors of glucosyl residues. CGT from thermophilic strains was shown to be the most potent in this respect (the degree of transglycosylation was as high as 60%).     

A study of low pH-induced refolding of Env of avian sarcoma and leukosis virus into a six-helix bundle

The fusion protein of avian sarcoma and leukosis virus is likely to fold into a six-helix bundle as part of its final configuration. A peptide, R99, inhibits fusion, probably by binding into the grooves of the triple-stranded coiled coil that becomes the central core of the six-helix bundle. The stages at which the envelope protein (Env) of avian sarcoma and leukosis virus subgroup A folds into a bundle during low pH-induced fusion were determined. Effector cells expressing Env were bound to target cells expressing the cognate receptor Tva, and intermediates of fusion were created. R99 was added and the extent of fusion inhibition was used to distinguish between a prebundle state with exposed grooves and a state in which the grooves were no longer exposed. The native conformation of Env was not sensitive to R99. But adding a soluble form of Tva to effector cells conferred sensitivity. Acidic pH applied at low temperature created an intermediate state of local hemifusion. Surprisingly, R99 caused these locally hemifused membranes to separate. This indicates that the grooves of Env were still exposed, that prebundle configurations of Env stabilized hemifused states, and that binding of R99 altered the conformation of Env. In the presence of an inhibitory lipid that blocks fusion before hemifusion, applying low pH at 37 degrees C created an intermediate in which R99 was without effect. This suggests that the six-helix bundle can form before hemifusion and that subsequent conformational changes, such as formation of the trimeric hairpin, are responsible for pore formation and/or growth.     

Tension of membranes expressing the hemagglutinin of influenza virus inhibits fusion.

The effects of membrane tension on fusion between cells expressing the hemagglutinin (HA) of influenza virus and red blood cells were studied by capacitance measurements. Inflation of an HA-expressing cell was achieved by applying a positive hydrostatic pressure to its interior through a patch-clamp pipette in the whole-cell configuration. Inflating cells to the maximum extent possible without lysis created a membrane tension and completely inhibited low-pH-induced fusion at room temperature. Fully inflated cells that were subsequently deflated to normal size resumed the ability to fuse in response to low pH. At the higher temperature of 32 degrees C, fusion conditions were sufficiently optimal that full inflation did not hinder fusion, and once formed, pores enlarged more rapidly than those of never inflated cells. It is suggested that under fusogenic conditions HA causes the formation of a dimple within the membrane in which it resides, and that membrane tension hinders fusion by preventing the formation of dimples. Because dimpling bends the bilayer portion of bound membranes so that they come into intimate contact, the damping of dimpling would suppress this initial step in the fusion process.     

Study of Colloidal Polysaccharides Produced by Iron Oxidizing Bacteria Leptospirillum ferriphilum CC

Iron and sulfur oxidizing bacteria produce capsular and colloidal extracellular polymeric substances. The properties and functional role of capsular polysaccharides are well studied. However, colloidal polysaccharides produced by iron oxidizing bacteria have not been sufficiently explored. In this paper, the physical and chemical properties of colloidal polysaccharides produced by the iron oxidizing bacterial isolate Leptospirillum ferriphilum CC have been studied. Colloidal polysaccharides were extracted and further investigated by optical polarized microscopy and analytical programs (LobVIEW15 and NOVA) that allowed determining the size, changes in shape, perimeter, degree of hydration, and crystallization of polysaccharides. Computer modeling of the experimental data has revealed that polysaccharides concentration does not contribute to the size of colloids but influence the number of particles.