Antagonism of the proinflammatory and pronociceptive actions of canonical and biased agonists of protease-activated receptor-2.

BACKGROUND AND PURPOSE: Diverse proteases cleave protease-activated receptor-2 (PAR2) on primary sensory neurons and epithelial cells to evoke pain and inflammation. Trypsin and tryptase activate PAR2 by a canonical mechanism that entails cleavage within the extracellular N-terminus revealing a tethered ligand that activates the cleaved receptor. Cathepsin-S and elastase are biased agonists that cleave PAR2 at different sites to activate distinct signalling pathways. Although PAR2 is a therapeutic target for inflammatory and painful diseases, the divergent mechanisms of proteolytic activation complicate the development of therapeutically useful antagonists. EXPERIMENTAL APPROACH: We investigated whether the PAR2 antagonist GB88 inhibits protease-evoked activation of nociceptors and protease-stimulated oedema and hyperalgesia in rodents. KEY RESULTS: Intraplantar injection of trypsin, cathespsin-S or elastase stimulated mechanical and thermal hyperalgesia and oedema in mice. Oral GB88 or par2 deletion inhibited the algesic and proinflammatory actions of all three proteases, but did not affect basal responses. GB88 also prevented pronociceptive and proinflammatory effects of the PAR2-selective agonists 2-furoyl-LIGRLO-NH2 and AC264613. GB88 did not affect capsaicin-evoked hyperalgesia or inflammation. Trypsin, cathepsin-S and elastase increased [Ca(2+) ]i in rat nociceptors, which expressed PAR2. GB88 inhibited this activation of nociceptors by all three proteases, but did not affect capsaicin-evoked activation of nociceptors or inhibit the catalytic activity of the three proteases. CONCLUSIONS AND IMPLICATIONS: GB88 inhibits the capacity of canonical and biased protease agonists of PAR2 to cause nociception and inflammation.

Investigation of the presence of ghrelin in the central nervous system of the rat and mouse.

Ghrelin and ghrelin receptor agonist have effects on central neurons in many locations, including the hypothalamus, caudal brain stem, and spinal cord. However, descriptions of the distributions of ghrelin-like immunoreactivity in the CNS in published work are inconsistent. We have used three well-characterized anti-ghrelin antibodies, an antibody to the unacylated form of ghrelin, and a ghrelin peptide assay in rats, mice, ghrelin knockout mice, and ghrelin receptor reporter mice to re-evaluate ghrelin presence in the rodent CNS. The stomach served as a positive control. All antibodies were effective in revealing gastric endocrine cells. However, no specific staining could be found in the brain or spinal cord. Concentrations of antibody 10 to 30 times those effective in the stomach bound to nerve cells in rat and mouse brain, but this binding was not reduced by absorbing concentrations of ghrelin peptide, or by use of ghrelin gene knockout mice. Concentrations of ghrelin-like peptide, detected by enzyme-linked immunosorbent assay in extracts of hypothalamus, were 1% of gastric concentrations. Ghrelin receptor-expressing neurons had no adjacent ghrelin immunoreactive terminals. It is concluded that there are insignificant amounts of authentic ghrelin in neurons in the mouse or rat CNS and that ghrelin receptor-expressing neurons do not receive synaptic inputs from ghrelin-immunoreactive nerve terminals in these species.

Identification of subunits of voltage-gated calcium channels and actions of pregabalin on intrinsic primary afferent neurons in the guinea-pig ileum.

BACKGROUND: The intrinsic primary afferent neurons (IPANs) in the intestine are the first neurons of intrinsic reflexes. Action potential currents of IPANs flow partly through calcium channels, which could feasibly be targeted by pregabalin. The aim was to determine whether pregabalin-sensitive α2δ1 subunits associate with calcium channels of IPANs and whether α2δ1 subunit ligands influence IPAN neuronal properties. METHODS: We used intracellular electrophysiological recording and in situ hybridisation to investigate calcium channel subunit expression in guinea-pig enteric neurons. KEY RESULTS: The α subunits of N (α1B) and R (α1E) type calcium channels, and the auxiliary α2δ1 subunit, were expressed by IPANs. This is the first discovery of the α2δ1 subunit in enteric neurons; we therefore investigated its functional role, by determining effects of the α2δ1 subunit ligand, pregabalin, that inhibits currents carried by channels incorporating this subunit. Pregabalin (10 µmol L(-1)) reduced the action potential duration. The effect was not increased with increase in concentration to 100 µmol L(-1). If N channels were first blocked by ω-conotoxin GVIA (0.5 µmol L(-1)), pregabalin had no effect on the residual inward calcium current. Reduction of the calcium current by pregabalin substantially inhibited the after-hyperpolarising potential (AHP) and increased neuron excitability. CONCLUSION & INFERENCES: Intrinsic primary afferent neurons express functional N (α1B) channel-forming subunits that are associated with α2δ1 modulatory subunits and are inhibited by pregabalin, plus functional R (α1E) channels that are not sensitive to binding of pregabalin to α2δ subunits. The positive effects of pregabalin in irritable bowel syndrome (IBS) patients might be partly mediated by its effect on enteric neurons.

Functional and in situ hybridization evidence that preganglionic sympathetic vasoconstrictor neurons express ghrelin receptors.

Agonists of ghrelin receptors can lower or elevate blood pressure, and it has been suggested that the increases in blood pressure are caused by actions at receptors in the spinal cord. However, this has not been adequately investigated, and the locations of neurons in the spinal cord that express ghrelin receptors, through which blood pressure increases may be exerted, are not known. We investigated the effects within the spinal cord of two non-peptide ghrelin receptor agonists, GSK894490 and CP464709, and two peptide receptor agonists, ghrelin and des-acyl ghrelin, and we used polymerase chain reaction (PCR) and in situ hybridization to examine ghrelin receptor expression. I.v. application of the non-peptide ghrelin receptor agonists caused biphasic changes in blood pressure, a brief drop followed by a blood pressure increase that lasted several minutes. The blood pressure rise, but not the fall, was antagonized by i.v. hexamethonium. Application of these agonists or ghrelin peptide directly to the spinal cord caused only a blood pressure increase. Des-acyl ghrelin had no significant action. The maximum pressor effects of agonists occurred with application at spinal cord levels T9 to T12. Neither i.v. nor spinal cord application of the agonists had significant effect on heart rate or the electrocardiogram. Ghrelin receptor gene expression was detected by PCR and in situ hybridization. In situ hybridization localized expression to neurons, including autonomic preganglionic neurons of the intermediolateral cell columns at all levels from T3 to S2. The numbers of ghrelin receptor expressing neurons in the intermediolateral cell columns were similar to the numbers of nitric oxide synthase positive neurons, but there was little overlap between these two populations. We conclude that activation of excitatory ghrelin receptors on sympathetic preganglionic neurons increases blood pressure, and that decreases in blood pressure caused by ghrelin agonists are mediated through receptors on blood vessels.

Adenovirus-mediated G(alpha)(q)-protein antisense transfer in neurons replicates G(alpha)(q) gene knockout strategies.

Antisense approaches are increasingly used to dissect signaling pathways linking cell surface receptors to intracellular effectors. Here we used a recombinant adenovirus to deliver G-protein alpha(q) antisense into rat superior cervical ganglion (SCG) neurons and neuronal cell lines to dissect G(alpha)(q)-mediated signaling pathways in these cells. This approach was compared with other G(alpha)(q) gene knockdown strategies, namely, antisense plasmid and knockout mice. Infection with adenovirus expressing G(alpha)(q) antisense (G(alpha)(q)AS AdV) selectively decreased immunoreactivity for the G(alpha)(q) protein. Expression of other G(alpha) protein subunits, such as G(alpha)(oA/B,) was unaltered. Consistent with this, modulation of Ca(2+) currents by the G(alpha)(q)-coupled M(1) muscarinic receptor was severely impaired in neurons infected with G(alpha)(q)AS AdV whereas modulation via the G(alpha)(oA)-coupled M(4) muscarinic receptor was unchanged. In agreement, activation of phospholipase C and consequent mobilization of intracellular Ca(2+) by UTP receptors was lost in NG108-15 cells infected with G(alpha)(q)AS AdV but not in cells infected with the control GFP-expressing adenovirus. Results obtained with this recombinant AdV strategy qualitatively and quantitatively replicated results obtained using SCG neurons microinjected with G(alpha)(q) antisense plasmids or SCG neurons from G(alpha)(q) knockout mice. This combined antisense/recombinant adenoviral approach can therefore be useful for dissecting signal transduction mechanisms in SCG and other neurons.

Mechanisms of enveloped virus entry into animal cells.

The ability of viruses to transfer macromolecules between cells makes them attractive starting points for the design of biological delivery vehicles. Virus-based vectors and sub-viral systems are already finding biotechnological and medical applications for gene, peptide, vaccine and drug delivery. Progress has been made in understanding the cellular and molecular mechanisms underlying virus entry, particularly in identifying virus receptors. However, receptor binding is only a first step and we now have to understand how these molecules facilitate entry, how enveloped viruses fuse with cells or non-enveloped viruses penetrate the cell membrane, and what happens following penetration. Only through these detailed analyses will the full potential of viruses as vectors and delivery vehicles be realised. Here we discuss aspects of the entry mechanisms for several well-characterised viral systems. We do not attempt to provide a fully comprehensive review of virus entry but focus primarily on enveloped viruses.

Membrane fusion activity of Semliki Forest virus in a liposomal model system: specific inhibition by Zn2+ ions.

Semliki Forest virus (SFV) has been shown previously to fuse efficiently with cholesterol- and sphingolipid-containing liposomal model membranes in a low-pH-dependent manner. Several steps can be distinguished in this process, including low-pH-induced irreversible binding of the virus to the liposomes, facilitated by target membrane cholesterol, and subsequent fusion of the viral membrane with the liposomal bilayer, specifically catalyzed by target membrane sphingolipid. Binding and fusion are mediated by the heterodimeric viral envelope glycoprotein E2/E1. At low pH the heterodimer dissociates, and the E1 monomers convert to a homotrimeric structure, the presumed fusion-active conformation of the viral spike. In this paper, we demonstrate that SFV-liposome fusion is specifically inhibited by Zn2+ ions. The inhibition is at the level of the fusion reaction itself, since virus-liposome binding was found to be unaffected. Zn2+ did not inhibit E2/E1 dissociation, but severely inhibited exposure of an acid-specific epitope on E1, E1 homotrimer formation, and acquisition of trypsin-resistance. It is concluded that virus--liposome binding solely requires low-pH-induced E2/E1 heterodimer dissociation, while fusion depends on further rearrangements in the E1 spike protein. As these rearrangements occur subsequent to the binding step, their precise course, including the formation of a fusion complex, may be influenced by interaction of E1 with target membrane lipids.

Promiscuous use of CC and CXC chemokine receptors in cell-to-cell fusion mediated by a human immunodeficiency virus type 2 envelope protein.

The CC chemokine receptors CCR5, CCR2, and CCR3 and the CXC chemokine receptor CXCR4 have been implicated as CD4-associated cofactors in the entry of primary and cell line-adapted human immunodeficiency virus type 1 (HIV-1) strains. CXCR4 is also a receptor for T-cell-line-adapted, CD4-independent strains of HIV-2. With the exception of this latter example, little has been reported on the entry cofactors used by HIV-2 strains. Here we show that a CD4-dependent, T-cell-line-adapted HIV-2 strain uses CXCR4 and, to a lesser extent, CCR3 for fusion with and infectious entry into cells. In a cell-to-cell fusion assay, the envelope protein of this virus can utilize a wider repertoire of chemokine receptors to induce fusion. These include CCR1, CCR2, CCR3, CCR4, CCR5, CXCR2, and CXCR4. Kinetic analysis indicated that cell lines expressing the receptors that support infection, CXCR4 and CCR3, form syncytia more rapidly than do cell lines expressing the other receptors. Nevertheless, although less efficient, fusion with CXCR2 expressing cells was specific, since it was inhibited by antibodies against CXCR2. The extensive use of chemokine receptors in cell-to-cell fusion has implications for understanding the molecular basis of CD4-chemokine receptor-induced lentivirus fusion and may have relevance for syncytium formation and the direct cell-to-cell transfer of virus in vivo.

SFV infection in CHO cells: cell-type specific restrictions to productive virus entry at the cell surface.

Alphaviruses, such as Semliki Forest virus, normally enter cells by penetration from acidic organelles of the endocytic pathway. The virions are internalised intact from the cell surface before undergoing acid-induced fusion in endosomes. To investigate the possibility that endocytosis might play a role in delivering virions to specific sites for replication, we compared SFV infection of baby hamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells following either normal virus fusion in endosomes or experimentally-induced fusion at the cell surface. Whereas baby hamster kidney cells were infected efficiently following fusion in endosomes or at the plasma membrane, Chinese hamster ovary cells were only infected following fusion from endocytic organelles. Virions fused at the plasma membrane of CHO cells failed to initiate viral RNA and protein synthesis. Similar results were observed when CHO cells were challenged with a rhabdovirus, vesicular stomatitis virus. These data suggest that in certain cell types a barrier, other than the plasma membrane, can prevent infection by alpha- and rhabdoviruses fused at the cell surface. Moreover, they suggest the endocytic pathway provides a mechanism for bringing viral particles to a site, or sites, in the cell where replication can proceed.

Activation of murine lymphocytes by lipopolysaccharide incorporated in fusogenic, reconstituted influenza virus envelopes (virosomes).

We have studied the in vitro activation of murine lymphocytes with LPS incorporated in the membranes of both phospholipid vesicles (liposomes) and vesicles composed of fusogenic, reconstituted influenza virus envelopes (virosomes). The incorporation of Salmonella minnesota rough-LPS in liposomes reduced the potency of LPS to stimulate splenocyte proliferation and cell surface kappa-light chain expression on 70 Z/3 pre-B cells by over 100-fold. Salmonella minnesota rough-LPS inserted into virosomes was at least 10-fold more potent than free LPS, both when prebound virosomes were allowed to be taken up by the cells at neutral pH and when the virosomes were fused into the plasma membrane by low pH treatment. Inactivation of the virosomes by low pH pretreatment reduced the potency of the virosomal LPS to the level of liposome-incorporated LPS. The association of the various LPS forms with the cells was quantitated using radio-iodinated LPS. Correcting for uptake, virosomal LPS remained 2- to 10-fold more potent than free LPS in stimulating B lymphocytes and at least 100-fold more active than liposomal LPS or fusion-inactivated virosomes. After low pH-induced fusion with the plasma membrane, the majority (80%) of the prebound virosomes had fused with the cells, compared with about 8% after neutral uptake. From these results we conclude that LPS inserted into the plasma or endosomal membranes efficiently activates murine lymphocytes. The fusion data suggest that the incorporation into endosomal membranes might be a more effective stimulus.

An internalization signal in the simian immunodeficiency virus transmembrane protein cytoplasmic domain modulates expression of envelope glycoproteins on the cell surface.

A Tyr to Cys mutation at amino acid position 723 in the cytoplasmic domain of the simian immunodeficiency virus (SIV) transmembrane (TM) molecule has been shown to increase expression of envelope glycoproteins on the surface of infected cells. Here we show that Tyr-723 contributes to a sorting signal that directs the rapid endocytosis of viral glycoproteins from the plasma membrane via coated pits. On cells infected by SIVs with a Tyr at position 723, envelope glycoproteins were transiently expressed on the cell surface and then rapidly endocytosed. Similar findings were noted for envelope molecules expressed in the absence of other viral proteins. Immunoelectron microscopy demonstrated that these molecules were localized in patches on the cell surface and were frequently associated with coated pits. In contrast, envelope glycoproteins containing a Y723C mutation were diffusely distributed over the entire plasma membrane. To determine if an internalization signal was present in the SIV TM, chimeric molecules were constructed that contained the CD4 external and membrane spanning domains and a SIV TM cytoplasmic tail with a Tyr or other amino acids at SIV position 723. In Hela cells stably expressing these molecules, chimeras with a Tyr-723 were rapidly endocytosed, while chimeras containing other amino acids at position 723, including a Phe, were internalized at rates only slightly faster than a CD4 molecule that lacked a cytoplasmic domain. In addition, the biological effects of the internalization signal were evaluated in infectious viruses. A mutation that disrupted the signal and as a result, increased the level of viral envelope glycoprotein on infected cells, was associated with accelerated infection kinetics and increased cell fusion during viral replication. These results demonstrate that a Tyr-dependent motif in the SIV TM cytoplasmic domain can function as an internalization signal that can modulate expression of the viral envelope molecules on the cell surface and affect the biological properties of infectious viruses. The conservation of an analogous Tyr in all human and simian immunodeficiency viruses suggests that this signal may be present in other primate lentiviruses and could be important in the pathogenesis of these viruses in vivo.

Fusion of Semliki Forest virus with cholesterol-containing liposomes at low pH: a specific requirement for sphingolipids.

Semliki Forest virus (SFV) utilizes a membrane fusion strategy to introduce its genome into the host cell. After binding to cell-surface receptors, virus particles are internalized through receptor-mediated endocytosis and directed to the endosomal cell compartment. Subsequently, triggered by the acid pH in the lumen of the endosomes, the viral envelope fuses with the endosomal membrane. As a result of this fusion reaction the viral RNA gains access to the cell cytosol. Low-pH-induced fusion of SFV, in model systems as well as in cells, has been demonstrated previously to be strictly dependent on the presence of cholesterol in the target membrane. In this paper, we show that fusion of SFV with cholesterol-containing liposomes depends on sphingomyelin (SM) or other sphingolipids in the target membrane, ceramide representing the sphingolipid minimally required for mediating the process. The action of the sphingolipid is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. The 3-hydroxyl group on the sphingosine backbone plays a key role in the SFV fusion reaction, since 3-deoxy-sphingomyelin does not support the process. This, and the remarkably low levels of sphingolipid required for half-maximal fusion (1-2 mol%), suggest that the sphingolipid does not play a structural role in SFV fusion, but rather acts as a cofactor, possibly through activation of the viral fusion protein. Domain formation between cholesterol and sphingolipid, although it may facilitate SFV fusion, is unlikely to play a crucial role in the process.

Cellular cytoplasmic delivery of a polypeptide toxin by reconstituted influenza virus envelopes (virosomes).

In this paper it is demonstrated that reconstituted influenza virus envelopes (virosomes) fuse efficiently with membranes of the endosomal cell compartment of cultured cells, after internalization through receptor-mediated endocytosis. As a consequence, molecules, encapsulated in the virosomal interior, are transferred to the cell cytoplasm. This process was monitored on the basis of delivery of subunit A of diphtheria toxin (DTA), initially encapsulated in the virosomal lumen. Virosome-mediated cytoplasmic delivery of DTA resulted in a virtually complete inhibition of cellular protein synthesis. DTA delivery was blocked by factors inhibiting the pH-dependent fusion activity of viral hemagglutinin, i.e., 20 mM NH4Cl, preincubation of the virosomes at low pH, and anti-hemagglutinin antibodies. Quantitation of the extent of virosome-mediated delivery of biologically active DTA demonstrated that a lower bound of approximately 10% of the virosomes entering the cells deposited their aqueous contents into the cytosol. This is in good agreement with the final extent of virosome fusion of 40%, as determined by a fluorescence lipid mixing assay based on the dilution of pyrene-labeled phosphatidylcholine from the virosomal into the endosomal membrane. At an added concentration of 1 microM virosomal lipid per 50,000 cells, depending on the condition of the experiment, a minimum of 4000 virosomes per cell were found to be internalized. Virosomes could also be induced to fuse with the cell plasma membrane by a transient lowering of the extracellular pH, as detected by appearance of virosome-encapsulated DTA in the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane.

Enveloped animal viruses, such as Semliki Forest virus (SFV), utilize a membrane fusion strategy to deposit their genome into the cytosol of the host cell. SFV enters cells through receptor-mediated endocytosis, fusion of the viral envelope occurring subsequently from within acidic endosomes. Fusion of SFV has been demonstrated before to be strictly dependent on the presence of cholesterol in the target membrane. Here, utilizing a variety of membrane fusion assays, including an on-line fluorescence assay involving pyrene-labeled virus, we demonstrate that low-pH-induced fusion of SFV with cholesterol-containing liposomal model membranes requires the presence of sphingomyelin or other sphingolipids in the target membrane. The minimal molecular characteristics essential for supporting SFV fusion are encompassed by a ceramide. The action of the sphingolipids is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. Complex formation of the sphingolipids with cholesterol is unlikely to be important for the induction of SFV--liposome fusion, as sphingolipids that do not interact appreciably with cholesterol, such as galactosylceramide, effectively support the process. The remarkably low levels of sphingomyelin required for half-maximal fusion (1-2 mole%) suggest that sphingolipids do not play a structural role in the SFV fusion process, but rather act as a cofactor, possibly activating the viral fusion protein in a specific manner.

Assembly and entry mechanisms of Semliki Forest virus.

The alphavirus Semliki Forest (SFV) is an enveloped virus with a positive single-stranded RNA genome. The genome is complexed with 240 copies of a capsid protein into a nucleocapsid structure. In the membrane the virus carries an equal number of copies of a membrane protein heterodimer. The latter oligomers are grouped into clusters of three. These structures form the spikes of the virus and carry its entry functions, that is receptor binding and membrane fusion activity. The membrane protein heterodimer is synthesized as a p62E1 precursor protein which upon transport to the cell surface is cleaved into the mature E2E1 form. Recent studies have given much new information on the assembly and entry mechanism of this simple RNA virus. Much of this work has been possible through the construction of a complete cDNA clone of the SFV genome which can be used for in vitro transcription of infectious RNA. One important finding has been to show that a spike deletion variant and a capsid protein deletion variant are budding-negative when expressed separately but can easily complement each other when transfected into the same cell. This shows clearly that enveloped viruses use different budding strategies: one which depends on a nucleocapsid-spike interaction as exemplified by SFV and another one which is based on a direct core-lipid bilayer interaction as shown before to be the case with retroviruses. Another important finding concerns the activation process of the presumed fusion protein of SFV, the E1 subunit. In the original p62E1 heterodimer E1 is completely inactive. Activation proceeds in several steps. First p62 cleavage activates the potential for low pH inducible fusion. Next the low pH which surrounds incoming virus in endosomes induces dissociation of the heterodimeric structure. This is followed by a rearrangement of E1 subunits into homotrimers which are fusion active.

Evaluation of viral membrane fusion assays. Comparison of the octadecylrhodamine dequenching assay with the pyrene excimer assay.

Membrane fusion, in particular the fusion of enveloped viruses, is often measured with an assay based on octadecylrhodamine (R18) fluorescence dequenching. We have studied the association of R18 with membranes and used the R18 assay to measure virus fusion in model systems and in cultured cells. The results were compared with those of an assay based on the decrease in excimer fluorescence of pyrene-labeled phospholipids. For liposomes made from premixed R18 and phosphatidylcholine (PC), R18 fluorescence quenching was proportional to the concentration of the probe up to about 4 mol %. No quenching was found at very low concentrations of R18. However, various artificial and biological membranes labeled by the addition of R18 from an ethanolic solution showed significant quenching at such low R18 concentrations. Thus, some of the R18 was not randomly distributed but likely was associated with the surface of the membranes in the form of highly quenched clusters or micelles. Moreover, in influenza virus membranes, R18 appeared highly quenched at very low concentrations, indicative of the probe interacting with viral proteins. In contrast, pyrene-labeled PC incorporated in either liposomes or reconstituted viral membranes (virosomes) showed an excimer/monomer fluorescence ratio proportional to the concentration of probe. When intracellular membrane fusion was investigated with R18-labeled influenza virus or Semliki Forest virus (SFV), fluorescence dequenching was observed in the absence of fusion, most likely due to spontaneous probe exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the M2 protein in influenza virus membrane fusion: effects of amantadine and monensin on fusion kinetics.

We have investigated the effects of the anti-influenza drug amantadine (AMT) and the proton-ionophore monensin on the membrane fusion activity of influenza virus in a liposomal model system, using a kinetic fluorescence lipid mixing assay. Fusion of influenza virus A/turkey/Oregon/71 (H7N3) with liposomes was slowed down in the presence of 2 microM AMT. The effect of AMT was not observed with an AMT-resistant mutant virus. Fusion inhibition by AMT was reversed by the proton-ionophore monensin. In fact, 1 microM monensin stimulated fusion of AMT-sensitive or -resistant virus, irrespective of the presence of AMT. The effects of AMT and monensin increased with increasing temperature. They were not observed at 25 degrees, but were very prominent at 45 degrees. Monensin did not influence the fusion rates of reconstituted viral envelopes (virosomes), which lack the nucleocapsid and the M1 protein. These results suggest that intraviral low pH facilitates influenza virus fusion, possibly by weakening interactions of the C-terminus of the viral hemagglutinin with the M1 protein and/or the viral nucleocapsid. The effect of AMT on the fusion capacity of influenza virus may contribute to the anti-influenza action of the drug in the early stages of cellular infection. However, the limited extent of the fusion inhibition suggests that the fusion step is unlikely to be the primary target of AMT.

Membrane fusion of Semliki Forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein.

This paper presents a kinetic analysis of low-pH-induced fusion of Semliki Forest virus (SFV) with cholesterol-containing unilamellar lipid vesicles (liposomes), consisting otherwise of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. Fusion is monitored continuously with a lipid mixing assay, involving virus bio-synthetically labeled with the fluorophore pyrene. At pH 5.55, 37 degrees C, SFV-liposome fusion occurs on the time scale of seconds. Extensive fusion (up to 60% of the virus) requires an excess of liposomes, while a low-pH preincubation of the virus alone results in inactivation of its fusion capacity. The onset of fusion after acidification of virus-liposome mixtures is preceded by a pH- and temperature-dependent lag phase. Early in this lag phase, a conformational change in the E2E1 spike glycoprotein occurs, involving formation of a trypsin-resistant E1 homotrimer, exposing a conformation-specific epitope (E1"). These changes are followed by a rapid, cholesterol-dependent binding of the virus to the liposomes (as assessed by sucrose density gradient analysis), subsequent fusion starting only after an additional delay. This sequence of events strongly suggests that the E1 homotrimeric structure represents the fusion-active conformation of the SFV spike, the actual fusion complex possibly involving a higher order oligomer of E1 trimers.

Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein.

Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated fusion process of the alphavirus Semliki Forest virus (SFV). The spike protein of SFV is composed of three copies of the protein heterodimer E2E1. This structure is resistant to solubilization in mild detergents such as Nonidet P-40 (NP40). We have recently shown that the spike structure is reorganized during virus entry into acidic endosomes (J. M. Wahlberg and H. Garoff, J. Cell Biol. 116:339-348, 1992). The original NP40-resistant heterodimer is dissociated, and the E1 subunits form new NP40-resistant protein oligomers. Here, we show that the new oligomer is represented by an E1 trimer. From studies that use an in vitro assay for fusion of SFV with liposomes, we show that the E1 trimer is efficiently expressed during virus-mediated membrane fusion. Time course studies show that both E1 trimer formation and fusion are fast processes, occurring in seconds. It was also possible to inhibit virus binding and fusion with a monoclonal antibody directed toward the trimeric E1. These results give support for a model in which the E1 trimeric structure is involved in the SFV-mediated fusion reaction.