Mutations in the putative fusion peptide of Semliki Forest virus affect spike protein oligomerization and virus assembly.

The two transmembrane spike protein subunits of Semliki Forest virus (SFV) form a heterodimeric complex in the rough endoplasmic reticulum. This complex is then transported to the plasma membrane, where spike-nucleocapsid binding and virus budding take place. By using an infectious SFV clone, we have characterized the effects of mutations within the putative fusion peptide of the E1 spike subunit on spike protein dimerization and virus assembly. These mutations were previously demonstrated to block spike protein membrane fusion activity (G91D) or cause an acid shift in the pH threshold of fusion (G91A). During infection of BHK cells at 37 degrees C, virus spike proteins containing either mutation were efficiently produced and transported to the plasma membrane, where they associated with the nucleocapsid. However, the assembly of mutant spike proteins into mature virions was severely impaired and a cleaved soluble fragment of E1 was released into the medium. In contrast, incubation of mutant-infected cells at reduced temperature (28 degrees C) dramatically decreased E1 cleavage and permitted assembly of morphologically normal virus particles. Pulse-labeling studies showed that the critical period for 28 degrees C incubation was during virus assembly, not spike protein synthesis. Thus, mutations in the putative fusion peptide of SFV confer a strong and thermoreversible budding defect. The dimerization of the E1 spike protein subunit with E2 was analyzed by using either cells infected with virus mutants or mutant virus particles assembled at 28 degrees C. The altered-assembly phenotype of the G91D and G91A mutants correlated with decreased stability of the E1-E2 dimer.     

Role of spike protein conformational changes in fusion of Semliki Forest virus.

The alphavirus Semliki Forest virus (SFV) and a number of other enveloped animal viruses infect cells via a membrane fusion reaction triggered by the low pH within endocytic vesicles. In addition to having a low pH requirement, SFV fusion and infection are also strictly dependent on the presence of cholesterol in the host cell membrane. A number of conformational changes in the SFV spike protein occur following low-pH treatment, including dissociation of the E1-E2 dimer, conformational changes in the E1 and E2 subunits, and oligomerization of E1 to a homotrimer. To allow the ordering of these events, we have compared the kinetics of these conformational changes with those of fusion, using pH treatment near the fusion threshold and low-temperature incubation to slow the fusion reaction. Dimer dissociation, the E1 conformational change, and E1 trimerization all occur prior to the mixing of virus and cell membranes. Studies of cells incubated at 20 degrees C showed that as with virus fusion, E1 trimerization occurred in the endosome before transport to lysosomes. However, unlike the strictly cholesterol-dependent membrane fusion reaction, the E1 homotrimer was produced in vivo during virus uptake by cholesterol-depleted cells or in vitro by low-pH treatment of virus in the presence of artificial liposomes with or without cholesterol. Purified, lipid-free spike protein rosettes were assayed to determine the requirement for virus membrane cholesterol in E1 homotrimer formation. Spike protein rosettes were found to undergo E1 oligomerization upon exposure to low pH and target liposomes and showed an enhancement of oligomerization with cholesterol-containing membranes. The E1 homotrimer may represent a perfusion complex that requires cholesterol to carry out the final coalescence of the viral and target membranes.     

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.     

Spike protein oligomerization control of Semliki Forest virus fusion.

We have recently shown, using cleavage-deficient mutants of the p62-E1 membrane protein complex of Semliki Forest virus that p62 cleavage to E2 is necessary for the activation of the fusion function of the complex at pH 5.8 (a pH optimal for virus fusion) (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990). In this study, we show that the mutant precursor complexes can be induced to activate membrane fusion when treated with more acidic buffers (pH 5.0 and 4.5), which also appear to dissociate most of the p62-E1 complexes and change the conformation of the E1 subunit (the supposed fusion protein of Semliki Forest virus into a form which is resistant to trypsin digestion. These data suggest that p62 cleavage is not essential for membrane fusion per se but that the crucial event activating this process seems to be the apparent dissociation of the heterodimer, which in turn is facilitated by the spike precursor cleavage.     

Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry

The envelope of the Semliki Forest virus (SFV) contains two transmembrane proteins, E2 and E1, in a heterodimeric complex. The E2 subunit is initially synthesized as a precursor protein p62, which is proteolytically processed to the mature E2 form before virus budding at the plasma membrane. The p62 (E2) protein mediates binding of the heterodimer to the nucleocapsid during virus budding, whereas E1 carries the entry functions of the virus, that is, cell binding and low pH-mediated membrane fusion activity. We have investigated the significance of the cleavage event for the maturation and entry of the virus. To express SFV with an uncleaved p62 phenotype, BHK-21 cells were transfected by electroporation with infectious viral RNA transcribed from a full-length SFV cDNA clone in which the p62 cleavage site had been changed. The uncleaved p62E1 heterodimer was found to be used for the formation of virus particles with an efficiency comparable to the wild type E2E1 form. However, in contrast to the wild type virus, the mutant virus was virtually noninfectious. Noninfectivity resulted from impaired uptake into cells, as well as from the inability of the virus to promote membrane fusion in the mildly acidic conditions of the endosome. This inability could be reversed by mild trypsin treatment, which converted the viral p62E1 form into the mature E2E1 form, or by treating the virus with a pH 4.5 wash, which in contrast to the more mild pH conditions of endosomes, effectively disrupted the p62E1 subunit association. We conclude that the p62 cleavage is not needed for virus budding, but regulates entry functions of the E1 subunit by controlling the heterodimer stability in acidic conditions.     

pH-induced alterations in the fusogenic spike protein of Semliki Forest virus

The spike glycoproteins of Semliki Forest virus mediate membrane fusion between the viral envelope and cholesterol-containing target membranes under conditions of mildly acidic pH (pH less than 6.2). The fusion reaction is critical for the infectious cycle, catalyzing virus penetration from the acidic endosome compartment. To define the role of the viral spike glycoproteins in the fusion reaction, conformational changes in the spikes at acid pH were studied using protease digestion and binding assays to liposomes and nonionic detergent. A method was also developed to prepare fragments of both transmembrane subunit glycopolypeptides of the spike, E1 and E2, which lacked the hydrophobic anchor peptides. Unlike the intact spikes the fragments were monomeric and therefore useful for obtaining information on conformational changes in individual subunits. The results showed that both E1 and E2 undergo irreversible conformational changes at the pH of fusion, that the conformational change of E1 depends, in addition to acidic pH, on the presence of cholesterol, and that no major changes in the solubility properties of the spikes takes place. On the basis of these findings it was concluded that fusion involves both subunits of the spike and that E1 confers the stereo-specific sterol requirement. The results indicated, moreover, that acid-induced fusion of Semliki Forest virus differs in important respects from that of influenza virus, another well-defined model system for protein-mediated membrane fusion.     

Oligomerization-dependent folding of the membrane fusion protein of Semliki Forest virus.

The spikes of alphaviruses are composed of three copies of an E2-E1 heterodimer. The E1 protein possesses membrane fusion activity, and the E2 protein, or its precursor form, p62 (sometimes called PE2), controls this function. Both proteins are, together with the viral capsid protein, translated from a common C-p62-E1 coding unit. In an earlier study, we showed that the p62 protein of Semliki Forest virus (SFV) dimerizes rapidly and efficiently in the endoplasmic reticulum (ER) with the E1 protein originating from the same translation product (so-called heterodimerization in cis) (B.-U. Barth, J. M. Wahlberg, and H. Garoff, J. Cell Biol. 128:283-291, 1995). In the present work, we analyzed the ER translocation and folding efficiencies of the p62 and E1 proteins of SFV expressed from separate coding units versus a common one. We found that the separately expressed p62 protein translocated and folded almost as efficiently as when it was expressed from a common coding unit, whereas the independently expressed E1 protein was inefficient in both processes. In particular, we found that the majority of the translocated E1 chains were engaged in disulfide-linked aggregates. This result suggests that the E1 protein needs to form a complex with p62 to avoid aggregation. Further analyses of the E1 aggregation showed that it occurred very rapidly after E1 synthesis and could not be avoided significantly by the coexpression of an excess of p62 from a separate coding unit. These latter results suggest that the p62-E1 heterodimerization has to occur very soon after E1 synthesis and that this is possible only in a cis-directed reaction which follows the synthesis of p62 and E1 from a common coding unit. We propose that the p62 protein, whose synthesis precedes that of the E1 protein, remains in the translocon of the ER and awaits the completion of E1. This strategy enables the p62 protein to complex with the E1 protein immediately after the latter has been made and thereby to control (suppress) its fusion activity.     

An epitope of the Semliki Forest virus fusion protein exposed during virus-membrane fusion

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.     

Reversible acid-induced inactivation of the membrane fusion protein of Semliki Forest virus

Previously, it has been shown that the exposure of Semliki Forest virus (SFV) to a mildly acidic environment induces a rapid and complete loss of the ability of the virus to bind and fuse to target membranes added subsequently. In the present study, incubation of SFV at low pH followed by a specific reneutralization step resulted in a partial reversion of this loss of viral fusion capacity, as assessed in a liposomal model system. Also, the ability of the viral E1 fusion protein to undergo liposome-stimulated trimerization was restored. Furthermore, acid-treated and neutralized SFV largely retained infectivity. Exposure of SFV to low pH induced dissociation of the E1/E2 heterodimer, which was not reversed upon neutralization. It is concluded that the SFV E1 fusion protein, after acid-induced dissociation from E2, rapidly adopts an intermediate, nontrimeric conformation in which it is no longer able to interact with target membrane lipids. Neutralization restores the ability of E1 to interact with membranes. This interaction, however, remains strictly dependent on low pH.     

Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells

The Semliki Forest virus (SFV) directs the synthesis of a heterodimeric membrane protein complex which is used for virus membrane assembly during budding at the surface of the infected cell, as well as for low pH-induced membrane fusion in the endosomes when particles enter new host cells. Existing evidence suggests that the E1 protein subunit carries the fusion potential of the heterodimer, whereas the E2 subunit, or its intracellular precursor p62, is required for binding to the nucleocapsid. We show here that during virus uptake into acidic endosomes the original E2E1 heterodimer is destabilized and the E1 proteins form new oligomers, presumably homooligomers, with altered E1 structure. This altered structure of E1 is specifically recognized by a monoclonal antibody which can also inhibit penetration of SFV into host cells as well as SFV-mediated cell-cell fusion, thus suggesting that the altered E1 structure is important for the membrane fusion. These results give further support for a membrane protein oligomerization-mediated control mechanism for the membrane fusion potential in alphaviruses.     

Membrane and protein interactions of a soluble form of the Semliki Forest virus fusion protein.

Semliki Forest virus is an enveloped alphavirus that infects cells by a membrane fusion reaction triggered by the low pH present in endocytic vacuoles. Fusion is mediated by the E1 spike protein subunit. During fusion, several conformational changes occur in E1 and E2, the two transmembrane subunits of the spike protein. These changes include dissociation of the E1-E2 dimer, alteration of the trypsin sensitivity and monoclonal antibody binding patterns of E1, and formation of a sodium dodecyl sulfate (SDS)-resistant E1 homotrimer. A critical characteristic of Semliki Forest virus fusion is also its dependence on the presence of both cholesterol and sphingomyelin in the target membrane. We have here examined the conformational changes induced by low pH treatment of E1*, the water-soluble, proteolytically truncated ectodomain of the E1 subunit. Following low pH treatment, E1* was shown to bind efficiently to artificial liposomes. Similar to virus fusion, optimal E1*-liposome binding required low pH, cholesterol, and sphingomyelin. The E1 ectodomain, although monomeric in its neutral pH form, assembled into an SDS-resistant oligomer following treatment at low pH. This low pH-induced oligomerization required target membranes containing both cholesterol and sphingomyelin. Our results demonstrate that the E1 ectodomain responds to low pH similarly to the full-length E1 subunit. The ectodomain facilitates the characterization of conformational changes and membrane binding in the absence of virus fusion or other virus components.     

Membrane fusion mutants of Semliki Forest virus

Previous reports have indicated that the entry of Semliki Forest virus (SFV) into cells depends on a membrane fusion reaction catalyzed by the viral spike glycoproteins and triggered by the low pH prevailing in the endosomal compartment. In this study the in vitro pH-dependent fusion of SFV with nuclease-filled liposomes has been used to select for a new class of virus mutants that have a pH-conditional defect. The mutants obtained had a threshold for fusion of pH 5.5 as compared with the wild- type threshold of 6.2, when assayed by polykaryon formation, fusion with liposomes, or fusion at the plasma membrane. They were fully capable of infecting cells under standard infection conditions but were more sensitive to lysosomotropic agents that increase the pH in acidic vacuoles of the endocytic pathway. The mutants were, moreover, able to penetrate and infect baby hamster kidney-21 cells at 20 degrees C, indicating that the endosomes have a pH below 5.5. The results confirm the involvement of pH-triggered fusion in SFV entry, emphasize the central role played by acidic endosomal vacuoles in this reaction, shed further light on the mechanism of SFV inhibition by lysosomotropic weak bases, and demonstrate the usefulness of mutant viruses as biological pH probes of the endocytic pathway.     

Functions of the stem region of the Semliki Forest virus fusion protein during virus fusion and assembly

Membrane fusion of the alphaviruses is mediated by the E1 protein, a class II virus membrane fusion protein. During fusion, E1 dissociates from its heterodimer interaction with the E2 protein and forms a target membrane-inserted E1 homotrimer. The structure of the homotrimer is that of a trimeric hairpin in which E1 domain III and the stem region fold back toward the target membrane-inserted fusion peptide loop. The E1 stem region has a strictly conserved length and several highly conserved residues, suggesting the possibility of specific stem interactions along the trimer core and an important role in driving membrane fusion. Mutagenesis studies of the alphavirus Semliki Forest virus (SFV) here demonstrated that there was a strong requirement for the E1 stem in virus assembly and budding, probably reflecting its importance in lateral interactions of the envelope proteins. Surprisingly, however, neither the conserved length nor any specific residues of the stem were required for membrane fusion. Although the highest fusion activity was observed with wild-type E1, efficient fusion was mediated by stem mutants containing a variety of substitutions or deletions. A minimal stem length was required but could be conferred by a series of alanine residues. The lack of a specific stem sequence requirement during SFV fusion suggests that the interaction of domain III with the trimer core can provide sufficient driving force to mediate membrane merger.     

Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein

A complementary (cDNA) molecule encoding the structural proteins of Semliki Forest virus (SFV) has been inserted into a Simian virus 40- derived eucaryotic expression vector lacking introns. Introduction of the recombinant DNA into nuclei of baby hamster kidney cells results in the synthesis of authentic SFV membrane glycoproteins E1 and E2. The glycoproteins are both transported to the cell surface and induce cell- cell fusion after a brief treatment of the cells with low pH medium. The pH dependence of the fusion reaction was the same as that induced by virus particles (White, J., J. Kartenbeck, and A. Helenius, 1980, J. Cell Biol., 89:674-679). Transfection of cells with another recombinant DNA molecule in which the SFV cDNA is engineered into the same expression vector including an intron has been shown before to result in the expression of only the E2 protein on the cell surface, whereas the E1 protein is trapped in the rough endoplasmic reticulum (Kondor- Koch, C., H. Riedel, K. Soderberg, and H. Garoff, 1982, Proc. Natl. Acad. Sci. USA, 79:4525-4529). Such cells do not exhibit pH-dependent polykaryon formation, suggesting that the E1 protein is necessary for fusion activity. Immunoblotting experiments show that the RER-trapped E1 protein expressed from the DNA construction with an intron has a smaller apparent molecular weight than authentic E1, and that is has lost its amphipathic characteristics.     

Binding of Semliki Forest virus and its spike glycoproteins to cells.

We have studied the binding of the Semliki Forest virus and its isolated spike glycoproteins, in the form of water-soluble octameric complexes, to various cells at 5 degrees C. The number of viruses bound per cell increased strongly with increasing free concentrations of virus up to about 0.2 nM. At higher concentrations smaller increases in binding were observed but saturation was not achieved. The number of viruses bound at a given free concentration was widely different for different cells. For some cells the binding of the virus was maximal at pH 6.8 with little decrease at lower pH, for other cells it was maximal around pH 6.0. The spike protein complexes were used at 100 times higher molar concentrations than the virus. The binding increased strongly with increasing free concentrations up to about 50 nM and saturation was obtained at higher concentrations. Up to 1.3 X 10(6) spike protein complexes could be bound per cell but great variation could be seen between different cell types. For all cells maximal binding was found below pH 6.0. Together with earlier observations, our results suggest that the virus can bind to a cell by two different modes. Around neutral pH the virus binds to specific glycoproteins and at low pH unspecifically to the lipids of the plasma membrane. The possible physiological roles of these two types of binding are discussed.     

Biosynthesis, maturation, and acid activation of the Semliki Forest virus fusion protein.

The Semliki Forest virus spike protein has a potent membrane fusion activity which is activated in vivo by the low pH of endocytic vacuoles. The spike protein is composed of two transmembrane subunits, E1 and E2, plus E3, a peripheral polypeptide. Acid-induced conformational changes in the E1 or E2 subunits were analyzed by using monoclonal antibodies specific for the acid-treated spike protein. E1 and E2 reacted with the antibodies after treatment of wild-type or mutant virus at the pH of fusion. The E1 conformational change resembled fusion in its requirement for both low pH and cholesterol. Pulse-chase analysis and intracellular pH treatment were then used to determine the ability of the newly synthesized spike to undergo acid-induced conformational changes. p62, the precursor to E2 and E3, was shown to undergo a pH-dependent conformational change similar to that of E2 and was sensitive to acid very soon after biosynthesis. In contrast, a posttranslational maturation event was required for the conversion of E1 to the pH-sensitive form. E1 maturation occurred fairly late in the exocytic pathway, after the virus spike had passed the medial Golgi but before incorporation of the spike into a new virus particle.     

Structure-function relation of the NH2-terminal domain of the Semliki Forest virus capsid protein.

The capsid (C) protein of alphaviruses consists of two protein domains: a serine protease at the COOH terminus and an NH2-terminal domain which is thought to interact with RNA in the virus nucleocapsid (NC). The latter domain is very rich in positively charged amino acid residues. In this work, we have introduced large deletions into the corresponding region of a full-length cDNA clone of Semliki Forest virus, expressed the transcribed RNA in BHK-21 cells, and monitored the autoprotease activity of C, the formation of intracellular NCs, and the release of infectious virus. Our results show that if the gene region encoding the whole NH2-terminal domain is removed, the expressed C protein fragment cannot assemble into NCs and virus particles but it is still able to function as an autoprotease. Thus, these results underline the general importance of the NH2-terminal domain in the virus assembly process and furthermore show that the serine protease domain can function independently of the NH2 terminus. Surprisingly, analysis of additional C protein deletion variants showed that not all of the NH2-terminal domain is required for virus assembly, but large deletions involving up to one-third of its positively charged residues are still compatible with NC and virus formation. The fact that so much flexibility is allowed in the structure of the NH2-terminal domain of C suggests that most of this region is involved in nonspecific interactions with the encapsidated RNA, probably through its positively charged amino acid residues.     

Karyophilic properties of Semliki Forest virus nucleocapsid protein.

Semliki Forest virus capsid (C) protein molecules (Mr, 33,000) can be introduced efficiently into the cytoplasm of various target cells by electroporation, liposome, and erythrocyte ghost-mediated delivery (M. Elgizoli, Y. Dai, C. Kempf, H. Koblet, and M.R. Michel, J. Virol. 63:2921-2928, 1989). Here, we show that the transferred C protein molecules partition rapidly from the cytosolic compartment into the nucleus. Transport of the C protein molecules into the nucleus was reversibly arrested by metabolic inhibitors, indicating that the transfer process is energy dependent. Fractionation of isolated nuclei revealed that the delivered C protein preferentially associates with the nucleoli. This finding was confirmed by morphological studies, showing that in an in vitro system containing ATP isolated nuclei rapidly accumulated rhodamine-labeled C protein in their nucleoli. Furthermore, in this assay system, the lectin wheat germ agglutinin prevented transfer of C protein through nuclear pores. These results are in agreement with our observation that nucleoli contain measurable amounts of newly synthesized C protein as early as 5 h after infection of cells with SFV. Thereafter, nucleolar-associated C protein increased progressively during the course of infection.     

Structure of Semliki Forest virus core protein

Alphaviruses are enveloped, insect-borne viruses, which contain a positive-sense RNA genome. The protein capsid is surrounded by a lipid membrane, which is penetrated by glycoprotein spikes. The structure of the Sindbis virus (SINV) (the type virus) core protein (SCP) was previously determined and found to have a chymotrypsin-like structure. SCP is a serine proteinase which cleaves itself from a polyprotein. Semliki Forest virus (SFV) is among the most distantly related alphaviruses to SINV. Similar to SCP, autocatalysis is inhibited in SFCP after cleavage of the polyprotein by leaving the carboxy-terminal tryptophan in the specificity pocket. The structures of two different crystal forms (I and II) of SFV core protein (SFCP) have been determined to 3.0 Å and 3.3 Å resolution, respectively. The SFCP monomer backbone structure is very similar to that of SCP. The dimeric association between monomers, A and B, found in two different crystal forms of SCP is also present in both crystal forms of SFCP. However, a third monomer, C, occurs in SFCP crystal form I. While monomers A and B make a tail-to-tail dimer contact, monomers B and C make a head-to-head dimer contact. A hydrophobic pocket on the surface of the capsid protein, the proposed site of binding of the E2 glycoprotein, has large conformational differences with respect to SCP and, in contrast to SCP, is found devoid of bound peptide. In particular, Tyr184 is pointing out of the hydrophobic pocket in SFCP, whereas the equivalent tyrosine in SCP is pointing into the pocket. The conformation of Tyr184, found in SFCP, is consistent with its availability for iodination, as observed in the homologous SINV cores. This suggests, by comparison with SCP, that E2 binding to cores causes major conformational changes, including the burial of Tyr184, which would stabilize the intact virus on budding from an infected cell. The head-to-tail contacts found in the pentameric and hexameric associations within the virion utilize the same monomer surface regions as found in the crystalline dimer interfaces. Proteins 27:345–359, 1997. © 1997 Wiley-Liss, Inc.     

Role of cholesterol in fusion of Semliki Forest virus with membranes.

The low pH-triggered membrane fusion activity of Semliki Forest virus is dependent on the presence of cholesterol in the target membrane. When liposomes containing phospholipids and cholesterol analogs were used, fusion activity was observed with steroids which did not have a planar nucleus or an isooctyl side chain at C-17, but fusion activity was not observed when analogs which lacked the 3 beta-OH group were used. Binding of virus to liposomes at low pH was similarly, but not totally, dependent on the presence of a 3 beta-OH sterol.