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© The Rockefeller University Press,
0021-9525/1998//91 $5.00
The Journal of Cell Biology, Volume 140, Number 1,
, 1998 91-99
Article |
A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit
Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.
Abbreviations used in this paper: RT, reverse-transcriptase; SFV, Semliki Forest virus; wt, wild-type.
MEMBRANE fusion is a critical and ubiquitous cellular process involved in the formation and trafficking of endocytic and exocytic vesicles, and in fusions between cells. It is becoming apparent that membrane lipid composition is strategically involved in the control of membrane budding, protein transport, and membrane fusion (Pfanner et al., 1990; De Camilli et al., 1996; Kearns et al., 1997), but the role of specific lipids and their interactions with cellular proteins are not yet clear. A striking example of a defined lipid requirement in membrane fusion is the Semliki Forest virus (SFV)1 fusion protein, which mediates the cholesterol and sphingolipid- dependent fusion of the virus membrane with the cellular membrane during virus infection.
SFV is a member of the alphaviruses, enveloped positive– stranded RNA viruses with highly ordered icosahedral structures in which the spike and capsid proteins interact in a one-to-one association (reviewed in Strauss and Strauss, 1994; Kielian, 1995). The spike proteins also form extensive lateral interactions, and constitute a protein layer that almost completely covers the virus lipid bilayer (Cheng et al., 1995; Fuller et al., 1995). This simple virus structure protects the virus RNA genome, mediates virus fusion with the cell membrane to release the nucleocapsid and initiate infection, and is efficiently assembled during the budding of progeny virions from the host cell plasma membrane. SFV is a highly developed system to study virus membrane fusion and budding, two key steps in infection by all enveloped viruses.
SFV infects cells by cell surface receptor binding, uptake via receptor-mediated endocytosis, and low pH-triggered fusion of the virus membrane with that of the endosome. Virus membrane fusion is carried out by the 80 spike proteins on the surface of the virus. The spike proteins are trimers containing a complex of the E1, E2, and E3 glycopolypeptides, (E1/E2/E3)3. E1 and E2 are type 1 transmembrane proteins of 
50 kD, and E3 is a peripheral polypeptide of 10 kD. E1 is the fusion-active spike protein subunit, and a soluble ectodomain form of E1 has been shown to bind membranes in a low pH-dependent reaction (Klimjack et al., 1994). Upon exposure to low pH, the SFV spike protein undergoes a defined series of conformational changes (Garoff et al., 1994; Kielian, 1995). The normally tight dimeric interaction between the E1 and E2 transmembrane subunits is weakened, E1 exposes new antigenic epitopes, forms a trypsin-resistant homotrimer, and associates with the target membrane. E1 contains the putative virus fusion peptide, a highly conserved hydrophobic domain between amino acids 79–97 (Garoff et al., 1980). The importance of these conformational changes and the E1 fusion peptide is supported by the fact that a mutation within the fusion peptide, glycine 91 to aspartate, blocks both the formation of the E1 homotrimer and membrane fusion (Levy-Mintz and Kielian, 1991; Kielian et al., 1996).
In addition to the requirement for low pH, SFV fusion has a striking requirement for specific lipids in the target membrane. Fusion requires cholesterol, with optimal concentrations of one cholesterol molecule per two phospholipids (White and Helenius, 1980). Fusion is also dependent on the presence of small amounts (2 mol%) of sphingolipid in the target membrane (Nieva et al., 1994; Moesby et al., 1995). The SFV cholesterol requirement does not involve the general membrane fluidizing properties of cholesterol, but seems to be specific for the sterol 3β-hydroxyl group (Kielian and Helenius, 1984). The molecular mechanism by which cholesterol affects fusion is unclear at present, but cholesterol is required for the hydrophobic interaction of the virus with the target membrane before fusion (Kielian and Helenius, 1984; Bron et al., 1993; Nieva et al., 1994). In addition, cholesterol is specifically required for both the membrane binding of the E1 ectodomain, and its acid-dependent conformational changes such as epitope exposure, homotrimerization, and acquisition of trypsin resistance (Kielian and Helenius, 1985; Kielian et al., 1990; Klimjack et al., 1994).
To evaluate the role of cholesterol in vivo, we used the observation that insects are cholesterol auxotrophs (Nes and McKean, 1977), and that insect cells, unlike mammalian or avian cells, can be cholesterol-depleted without deleterious effects (Silberkang et al., 1983). Previously, we depleted the C6/36 mosquito cell line to <2% of the control level of cholesterol, and showed that the depleted cells are unaltered in their endocytic uptake and acidification properties, and are permissive for infection by vesicular stomatitis virus, an unrelated, cholesterol-independent virus (Phalen and Kielian, 1991). However, as predicted from the in vitro studies, depleted cells are blocked in SFV fusion and infection, and the block is specifically reversed by cholesterol addition. The depleted C6/36 cells are reduced about 5,000-fold in SFV infection efficiency compared to controls (Marquardt and Kielian, 1996). Using transfection of viral RNA or very high multiplicity infection, we then evaluated the involvement of cholesterol in the SFV exit pathway (Marquardt et al., 1993). Unexpectedly, our studies demonstrated that cholesterol is also involved in the efficient production of progeny virus. Thus, cholesterol seems to play two critical roles in the SFV life cycle, one involving virus fusion and one involving a late step in the formation or release of virus particles.
Given that SFV growth is strongly inhibited in cholesterol-depleted cells, we have now exploited this cell culture system to select for virus mutations that would permit growth in the absence of cholesterol. Such mutants were termed srf mutants, for sterol requirement in function. We previously showed that the srf phenotype allows more efficient virus exit from sterol-depleted cells (Marquardt et al., 1993). Characterization and molecular analysis of a srf mutant here reveals that the mutant's infection efficiency, membrane fusion, growth rate, and exit were all markedly less cholesterol-dependent than those of the parental virus. We demonstrate that the mutant has a single amino acid substitution on the E1 spike protein subunit, proline 226 to serine, which conferred these cholesterol-independent properties. This mutant thus identifies a novel region of E1, separate from the fusion peptide, that is involved in the cholesterol requirement for SFV fusion and exit.
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Isolation of SFV Mutants with Altered Sterol Requirements
A plaque-purified wild-type (wt) stock of SFV (Kielian et al., 1984) was mutagenized to 20% viability by treatment with 1-methyl-3-nitro-1-nitrosoguanidine (Kielian et al., 1984). The mutagenized stock was grown for 6 h at 37°C on BHK cells at a multiplicity of 0.5 pfu/cell to allow expression of mutant phenotypes. A starting titer of 107 pfu of this virus stock was then selected by growth on one plate of chlorocholestene-repleted cells at 0.5 pfu/cell for 48 h in MEM containing 0.2% BSA and 10 mM Hepes, pH 7.0. The selection was repeated by two more passages on chlorocholestene- repleted cells at a starting multiplicity of 
0.1 pfu/cell. At this point, the selected virus stock was found to have 100-fold greater infectivity on chlorocholestene-repleted cells than the parental wt virus. Potential mutants were then isolated by limiting dilution on chlorocholestene-repleted cells grown in 96-well tissue culture trays. Cells were infected with 10-fold serial dilutions of virus, and cultured for 2 d at 28°C in serum-free medium (OptiMEM; GIBCO BRL, Gaithersburg, MD) supplemented with 0.2% BSA and 100 U/ml penicillin and 100 µg/ml streptomycin. Medium from the wells was harvested and stored on ice. Wells containing virus able to replicate efficiently in the absence of cholesterol were identified by staining cells with a polyclonal antibody to the SFV spike protein (Kielian et al., 1990), peroxidase-conjugated second antibody, and 3-amino-9-ethylcarbozole substrate, and visualizing by light microscopy. Wells with strongly positive-infected cells were identified as containing potential virus mutants, and the medium from such positive wells at viral dilutions yielding 60% positive wells was then used for two additional rounds of limiting dilution. Five potential mutants were isolated, three of which grew to sufficient titers to permit further expansion by three to four successive low multiplicity passages on chlorocholestene-repleted cells. These isolates were termed srf-1, -2 and -3, for sterol requirement in function. Other selections based on nonmutagenized virus or low pH-induced virus–plasma membrane fusion did not result in isolation of mutants.
Preparation of Radiolabeled Virus
Wt SFV and srf mutants were radiolabeled and purified by infecting control, depleted, or chlorocholestene-repleted C6/36 cells for 6 h at multiplicities of 150 pfu/cell, labeling for 18 h in methionine/cysteine free DME containing 150 mCi/ml [35S]methionine/cysteine, followed by pelleting and purification on discontinuous 10–20%/25–50% sucrose gradients containing a 600 µl 50% sucrose cushion (Kielian et al., 1984).
Assays of Virus–Cell Interactions
Binding of radiolabeled wt SFV or srf mutants to C6/36 or BHK cells was measured as previously described (Phalen and Kielian, 1991). Endocytosis of radiolabeled virus was followed by measuring lysosomal degradation and the release of acid-soluble radioactivity (Phalen and Kielian, 1991). Growth curves of wt and mutants on control or depleted C6/36 cells were performed at multiplicities of 1 pfu/cell, and titered on BHK cells (Marquardt and Kielian, 1996). Primary infection of C6/36 cells was quantitated by an infectious center assay using immunofluorescence with a polyclonal antibody to the SFV spike protein to detect infected cells (Marquardt and Kielian, 1996). Fusion of prebound virus with control or sterol-modified cells was induced by treatment for 1 min, pH 5.5, at 28°C. Cells were then incubated overnight in medium containing 20 mM NH4Cl to prevent secondary infection, and infected cells quantitated by immunofluorescence (Marquardt and Kielian, 1996). Exit of newly synthesized virus from wt and mutant-infected C6/36 cells was followed by pulse–chase analysis as previously described, using increased multiplicity and radiolabel to assay wt virus in cholesterol-depleted cells (Marquardt et al., 1993; Marquardt and Kielian, 1996). Fluorograms were quantitated using a PhosphorImager and ImageQuant software from Molecular Dynamics, Inc. (Sunnyvale, CA).
Virus Sequence Analysis
Viral RNA was prepared by the method of Ou et al. (1981), using purified wild-type virus propagated on BHK cells, and pelleted srf-3 virus propagated on chlorocholestene-repleted C6/36 cells. 1 µg of viral RNA was reverse transcribed in a 20-µl reaction using oligo dT as a primer and 40 U of AMV reverse transcriptase, as previously described (Kielian et al., 1996). 2–4 µl of the resultant cDNA were then amplified using 500 ng each of primers within E1 and E2 in a 50-µl reaction containing Vent polymerase (New England Biolabs, Inc., Beverly, MA) (Kielian et al., 1996). The amplified DNA was purified using a QIAquick kit (Qiagen Inc., Chatsworth, CA), and both strands of the DNA encoding E1 and 6K were sequenced by the automated sequencing facility at the Albert Einstein College of Medicine (Kielian et al., 1996). Several independent RT-PCR products were sequenced to control for possible errors during transcription and amplification. The E1 sequence from srf-1 was obtained using the virus resulting from infection of one 75-cm2 flask of cholesterol-depleted C6/36 cells. All of the viral RNA was used for reverse transcription, and 2–4 µl of resulting cDNA were used for amplification and sequencing as above. The E1 sequence from srf-2 was obtained using total cellular RNA from a 100-mm plate of cholesterol-depleted C6/36 cells infected at 1 pfu/ cell at 28°C for 24 h. Total cellular RNA was extracted using the RNAzol method and 8 µg RNA was reverse transcribed, as described previously (Kielian et al., 1996), and used for amplification and sequencing as above.
Infectious SFV Clones
The mutation in srf-3 was mapped by transferring a unique 2.1-kb NdeI fragment containing srf-3 nucleotides 8929–11033 into the wt infectious SFV clone. cDNA from srf-3 was prepared as described above, and PCR-amplified using oligos 3' to the E1 sequence and within E2 to generate an 2.5-kb product that was then digested with NdeI. The wild-type infectious clone pSP6-SFV4 was digested to completion with NdeI, the large fragment self-ligated and propagated, treated with NdeI and phosphatase, and ligated with the srf-3 NdeI fragment to regenerate the complete infectious clone. Individual clones were transcribed using SP6 polymerase (Duffus et al., 1995), and the RNAs tested for primary and secondary infection on control and cholesterol-depleted C6/36 cells. 50–100 ng of RNA was mixed with 40 µg lipofectin in 250 µl of OptiMEM (GIBCO BRL). Depleted or control C6/36 cells grown on 12 mm coverslips in 24-well trays were incubated with this mixture for 1 h, and then cultured for 18 h in OptiMEM/0.2%BSA with or without 20 mM NH4Cl to prevent secondary infection (Marquardt et al., 1993). The cells were then fixed and virus infection assayed by indirect immunofluorescence using an antibody to the SFV spike protein (Marquardt and Kielian, 1996). A clone that produced efficient secondary infection of both control and depleted C6/36 cells was chosen, and used to make a virus stock by lipofection of the transcribed RNA into depleted C6/36 cells. The clone and the recombinant virus stock were referred to as srf-3/ic. The entire NdeI fragment of the srf-3/ic was sequenced on both strands by automated sequencing. Sequences were compared to the original published sequence of SFV spike proteins (Garoff et al., 1980), the sequence of pSP6-SFV-4 in the database, and the sequence of our plasmid isolate of pSP6-SFV-4, termed wt/ic. In addition, we performed RT-PCR and sequence analysis of RNA from the parent virus to srf-3, which is our plaque-purified strain of SFV (Kielian et al., 1984). srf-3 and srf-3/ic contained a unique but silent change at valine 3 of 6K (GUG-GUU), and a change from the published sequence of E1 asparagine 323 to aspartic acid (AAC-GAC). Sequence analysis of both wt/ic and the srf-3 parent strain showed that they also have Asp 323, however, and thus the Asn 323 sequence in the database is probably due to an early sequencing error. The only amino acid change in the NdeI fragment between srf-3/srf-3/ic and the parent virus/published sequences was due to a single base change, from a CCC to UCC in the srf-3 RNA, producing the E1 proline 226 to serine substitution. Mutation of C to U has been previously observed in other alphavirus mutants isolated following nitrosoguanidine treatment (Lindqvist et al., 1986).
The E1 P226S mutation was introduced de novo into the wild-type infectious clone by PCR mutagenesis using the overlap extension method (Levy-Mintz and Kielian, 1991). The sequence of the 5'-3' mutagenic oligo was GCCCTTCAtCaGGCATGG, and the mutagenized fragment was subcloned into pSP6-SFV4 using the NdeI sites as described above. The P226S mutagenesis also introduced an EcoNI site which was used initially to screen the clones. A clone, termed SFV4-P226S, was selected, and a virus stock was generated by RNA transcription and lipofection as above. The entire NdeI fragment from SFV4 P226S was sequenced and confirmed that no additional changes were introduced during mutagenesis.
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100-fold increase in infectivity on chlorocholestene-enriched cells compared to that of wt virus. Because SFV does not produce plaques on C6/36 cells (Brown and Condreay, 1986), potential mutants in this stock were isolated by limiting dilution on C6/ 36 cells enriched with chlorocholestene, resulting in three putative mutant isolates. These were termed srf-1, srf-2 and srf-3 for the purposes of phenotypic characterization, although the procedure used to isolate them did not guarantee that they were independent mutants.
Cholesterol Independence of srf Mutants
To determine the cholesterol requirements of the srf mutants, we first compared their growth kinetics in control, chlorocholestene-enriched, and sterol-depleted C6/36 cells, measuring the production of progeny virus by plaque assays on BHK cells. Growth of all four viruses on cholesterol-containing cells was rapid and efficient, resulting in titers of 109 pfu/ml by 12 h after infection (Fig. 1 A). The srf mutants did not exhibit a significant growth advantage, arguing that they were simply not better able to replicate in mosquito cells. As expected, the wt virus grew much less efficiently on chlorocholestene-enriched cells compared to control cells, with titers about four logs lower at 12 h and three logs lower at 48 h (Fig. 1 B). In contrast, all three mutants grew more efficiently than wt on chlorocholestene-enriched cells, showing titers four to five logs higher than wt by 24 h. Although the mutants grew more slowly on chlorocholestene-enriched cells than control cells, the final yield of mutant virus was comparable in the two cell types. In cholesterol-depleted, nonenriched cells, growth of wt virus was severely impaired, producing titers of only 105 pfu/ml even after a 48-h infection of depleted cells (Fig. 1 C). Surprisingly, the mutants were able to replicate on cholesterol-depleted cells that were not enriched with chlorocholestene. Although the growth kinetics were slower than on control cells, final titers of 109 pfu/ml were obtained. Thus, the three srf mutants were dramatically altered in their cholesterol requirements for growth. Although the mutants were originally selected for growth on chlorocholestene-enriched cells, in fact they appeared to be relatively sterol independent. This may reflect a lack of chlorocholestene incorporation in the enriched cell membrane, as discussed below. The srf-3 mutant was selected for further study.
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We compared the cholesterol dependence of wt SFV and srf exit, using pulse–chase analysis in control and cholesterol-depleted cells (Marquardt et al., 1993; Marquardt and Kielian, 1996). wt SFV exit was inhibited in cholesterol-depleted cells compared to control cells, with total radiolabeled spike proteins released in virus particles by 90 min chase averaging 24% in control cells and 2% in depleted cells (n = 7). In contrast, srf-3 exit averaged 32% from control cells and 16% from depleted cells (n = 6). srf-1, -2, and -3 showed similar increases in exit from cholesterol-depleted cells. Electron microscopy showed that srf virions budding from sterol-depleted cells appeared morphologically similar to srf or wt virions released from control cells (data not shown; Marquardt et al., 1993). Thus, the srf mutants are increased in both their ability to fuse with and exit from cholesterol-depleted cells. These data also suggest that the cholesterol requirement for wt virus fusion may be more stringent than that for virus exit (see also Marquardt and Kielian, 1996). To test the genetic stability of the srf mutants, srf-3 was passaged on either depleted C6/36 cells, chlorocholestene-enriched C6/36 cells, control C6/36 cells, or BHK cells. The srf phenotype was then assayed by comparing its infectivity on C6/36 cells with and without cholesterol (Table II). srf-3 showed dramatically increased infectivity on depleted cells irrespective of its passage history, and thus the srf-3 mutation is stable for at least one passage in the absence of selective pressure. In parallel, we assayed wt virus that had been passaged on control C6/36 cells, and found that its infectivity was strongly cholesterol-dependent (Table II), in agreement with the results using BHK-grown virus (Fig. 2 A). Thus, the srf-3 phenotype appears to be due to a virus mutation, rather than to differences in lipid and carbohydrate composition between mammalian and mosquito grown virus (Kielian, 1995).
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In general, it is not known if members of other virus families have specific lipid requirements for membrane fusion or virus egress. In vitro and/or in vivo studies of influenza virus, a myxovirus, and vesicular stomatitis virus, a rhabdovirus, suggest that both viruses have fusion mechanisms independent of either cholesterol or sphingolipid (White et al., 1982; Eidelman et al., 1984; Phalen and Kielian, 1991; Cleverley et al., 1997). Cholesterol does seem to enhance fusion of Sendai virus, a paramyxovirus whose fusion protein bears structural and sequence similarities to that of influenza (Hsu et al., 1983; Kundrot et al., 1983). Many medically important viruses such as HIV-1 have not yet been tested for lipid requirements in fusion or infection. It is interesting that during budding, HIV-1 appears to select for significantly increased concentrations of both cholesterol and sphingomyelin in the virus membrane compared to the cell plasma membrane (Aloia et al., 1993). It is not known if this selectivity represents an actual lipid requirement for HIV-1 budding.
There are several precedents for protein–cholesterol interactions important in membrane biology. Caveolin, an integral component of specialized membrane domains known as caveolae (Rothberg et al., 1992), binds cholesterol (Murata et al., 1995) and may be involved in transport of sterol from the endoplasmic reticulum to the plasma membrane (Smart et al., 1996). The caveolin residues involved in sterol binding are not yet identified. Several proteins involved in cellular cholesterol metabolism have membrane-spanning domains that may act as cholesterol sensors, although the specific cholesterol–interaction site has yet to be identified (Brown and Goldstein, 1997). The thiol-activated cytolysins are bacterial toxins that act by binding cholesterol in the target cell membrane, inserting into the membrane, and oligomerizing to form a pore (Alouf and Geoffroy, 1991). Similar to the SFV E1 protein, these toxins appear to require the sterol 3β-hydroxyl group for their activity, and at least one, lysteriolysin O, has an acid pH optimum for cholesterol binding and pore formation (Portnoy et al., 1992). The crystal structure of a member of this family, perfringolysin O, was recently determined (Rossjohn et al., 1997). The putative cholesterol-binding site is composed of noncontiguous amino acid residues, and involves interactions with aliphatic side chains and hydrogen bonding between the sterol 3β-hydroxyl and glutamate and arginine residues. Cholesterol oxidase is an enzyme that specifically oxidizes the sterol 3β-hydroxyl group. The crystal structure of the enzyme with a bound sterol substrate shows that the binding site is a solvent-sealed internal cavity in which the sterol rings have extensive contacts and the sterol hydroxyl is hydrogen-bonded to a flovin adenine dinucleotide cofactor and a bound water molecule (Li et al., 1993). Thus, work from other systems suggests that sterol binding is likely to involve noncontiguous residues that interact with the sterol ring structure and hydrogen bond with the sterol hydroxyl. The SFV E1 protein does not show significant regions of sequence identity with these other cholesterol-binding proteins. It remains to be seen if similar binding sites or motifs for cholesterol interaction exist among these molecules.
What might be the mechanism by which the gain of serine 226 confers srf-3 cholesterol independence? One intriguing possibility is that the serine hydroxyl group may act as a substitute for the critical 3β-hydroxyl group of cholesterol. The mutation could act by "filling" a cholesterol-binding site either within the P226 region or in an interacting domain of E1. If this is the case, the mutation could cause a normally cholesterol-requiring step in fusion to be less cholesterol dependent. During low pH-triggered fusion, E1 undergoes at least three separate conformational changes: exposure of new epitopes, formation of a homotrimer, and hydrophobic association with the target membrane bilayer (Kielian, 1995; Kielian et al., 1996). Preliminary data indicate that srf-3 E1 is less cholesterol dependent than wt for both epitope exposure and homotrimer formation (Chatterjee, P., M. Vashishtha, and M. Kielian, unpublished data). Further studies will focus on the role of the serine hydroxyl and on the potential interactions of this region with other spike protein domains.
The most surprising finding of this study is that a single point mutation in E1 substitutes for the specific cholesterol requirement in both SFV fusion and exit. The exact step in the SFV exit pathway that requires cholesterol has not been determined, but our data suggest that the most likely site is at a point after arrival of the spike protein at the cell surface (Marquardt et al., 1993). It is unlikely that E1's fusion activity is directly required for virus exit. During virus budding, the forming virus particle must pinch off in a membrane fission reaction, but unlike virus membrane fusion, this occurs at neutral pH. In addition, virus mutants that are completely blocked in membrane fusion (Duffus et al., 1995) or have a dramatically acid-shifted pH threshold for fusion (Salminen et al., 1992; Duffus et al., 1995) can still assemble into virus particles. Computer reconstructions of alphaviruses indicate that almost all of the surface of the particle is covered by a spike protein shell, with very little of the lipid bilayer exposed (Cheng et al., 1995; Fuller et al., 1995). However, in spite of the fact that the majority of the cholesterol in the virus membrane bilayer appears inaccessible, cholesterol-depleted virus is highly destabilized. The requirement for cholesterol in both efficient wt virus exit and virus particle stability suggests that cholesterol is important in the normal spike protein interactions involved in formation of the alphavirus particle. The phenotype of the srf-3 mutant suggests that these interactions are mediated by spike protein domains that are also required for membrane fusion.
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This work was supported by grants to M. Kielian from the American Cancer Society (RPG-93-013-05VM), the Hirschl Charitable Trust, and the Pew Biomedical Scholars Program, by the Jack K. and Helen B. Lazar fellowship in Cell Biology, and by Cancer Center Core Support Grant NIH/NCI P30-CA13330. M. Vashishtha was supported by a fellowship from the Martin Foundation, T. Phalen was supported by National Institutes of Health (NIH) training grant 15T32 HL-07675, and M.T. Marquardt was supported by NIH training grant 2T32 CA09173-15 and by funds from the Albert Einstein College of Medicine.
Submitted: 12 August 1997
Revised: 8 October 1997
Address all correspondence to Margaret Kielian, Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. Tel.: (718) 430-3638. Fax: (718) 430-8574. E-mail: kielian{at}aecom.yu.edu
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Aloia RC, Tian H & Jensen FC. Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes, Proc Natl Acad Sci USA, 1993, 90, 5181–5185.
Alouf, J.E., and C. Geoffroy. 1991. The family of the antigenically-related cholesterol-binding ("sulfhydryl-activated") cytolytic toxins. In Sourcebook of Bacterial Toxins. J.E. Alouf and J.H. Freer, editors. Academic Press, London. 147–186.
Bron R, Wahlberg JM, Garoff H & Wilschut J. Membrane fusion of Semliki Forest virus in a model system: correlation between fusion kinetics and structural changes in the envelope glycoprotein, EMBO (Eur Mol Biol Organ) J, 1993, 12, 693–701.[Medline]
Brown, D.T., and L.D. Condreay. 1986. Replication of alphaviruses in mosquito cells. In The Togaviridae and Flaviviridae. S. Schlesinger and M.J. Schlesinger, editors. Plenum Press, New York. 171–207.
Brown MS & Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, Cell, 1997, 89, 331–340.[Medline]
Cheng RH, Kuhn RJ, Olson NH, Rossman MG, Choi H-K, Smith TJ & Baker TS. Nucleocapsid and glycoprotein organization in an enveloped virus, Cell, 1995, 80, 621–630.[Medline]
Cleverley DZ, Geller HM & Lenard J. Characterization of cholesterol-free insect cells infectible by baculoviruses: effects of cholesterol on VSV fusion and infectivity and on cytotoxicity induced by influenza M2 protein, Exp Cell Res, 1997, 233, 288–296.[Medline]
De Camilli P, Emr SD, McPherson PS & Novick P. Phosphoinositides as regulators in membrane traffic, Science, 1996, 271, 1533–1539.[Abstract]
Duffus WA, Levy-Mintz P, Klimjack MR & Kielian M. Mutations in the putative fusion peptide of Semliki Forest virus affect spike protein oligomerization and virus assembly, J Virol, 1995, 69, 2471–2479.[Abstract]
Eidelman O, Schlegel R, Tralka TS & Blumenthal R. pH-dependent fusion induced by Vesicular Stomatitis virus glycoprotein reconstituted into phospholipid vesicles, J Biol Chem, 1984, 259, 4622–4628.
Fuller SD, Berriman JA, Butcher SJ & Gowen BE. Low pH induces swiveling of the glycoprotein heterodimers in the Semliki Forest virus spike complex, Cell, 1995, 81, 715–725.[Medline]
Garoff H, Frischauf A-M, Simons K, Lehrach H & Delius H. Nucleotide sequence of cDNA coding for Semliki Forest virus membrane glycoproteins, Nature, 1980, 288, 236–241.[Medline]
Garoff H, Wilschut J, Liljestrom P, Wahlberg JM, Bron R, Suomalainen M, Smyth J, Salminen A, Barth BU & Zhao H. Assembly and entry mechanisms of Semliki Forest virus, Arch Virol, 1994, 9, 329–338.
Helenius A, Marsh M & White J. Inhibition of Semliki Forest virus penetration by lysosomotropic weak bases, J Gen Virol, 1982, 58, 47–61.
Hsu MC, Scheid A & Choppin PW. Fusion of Sendai virus with liposomes: Dependence on the viral fusion protein (F) and the lipid composition of liposomes, Virology, 1983, 126, 361–369.[Medline]
Kearns BG, McGee TP, Mayinger P, Gedvilaite A, Phillips SE, Kagiwada S & Bankaitis VA. Essential role for diacylglycerol in protein transport from the yeast Golgi complex, Nature, 1997, 387, 101–105.[Medline]
Kielian M. Membrane fusion and the alphavirus life cycle, Adv Virus Res, 1995, 45, 113–151.[Medline]
Kielian M & Helenius A. pH-induced alterations in the fusogenic spike protein of Semliki Forest virus, J Cell Biol, 1985, 101, 2284–2291.
Kielian M, Jungerwirth S, Sayad KU & DeCandido S. Biosynthesis, maturation, and acid-activation of the Semliki Forest virus fusion protein, J Virol, 1990, 64, 4614–4624.
Kielian M, Klimjack MR, Ghosh S & Duffus WA. Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus, J Cell Biol, 1996, 134, 863–872.
Kielian MC & Helenius A. The role of cholesterol in the fusion of Semliki Forest virus with membranes, J Virol, 1984, 52, 281–283.
Kielian MC, Keranen S, Kaariainen L & Helenius A. Membrane fusion mutants of Semliki Forest virus, J Cell Biol, 1984, 98, 139–145.
Klimjack MR, Jeffrey S & Kielian M. Membrane and protein interactions of a soluble form of the Semliki Forest virus fusion protein, J Virol, 1994, 68, 6940–6946.
Kundrot CE, Spangler EA, Kendall DA, MacDonald RC & MacDonald RI. Sendai virus-mediated lysis of liposomes requires cholesterol, Proc Natl Acad Sci USA, 1983, 80, 1608–1612.
Levy-Mintz P & Kielian M. Mutagenesis of the putative fusion domain of the Semliki Forest virus spike protein, J Virol, 1991, 65, 4292–4300.
Li J, Vrielink A, Brick P & Blow DM. Crystal structure of cholesterol oxidase complexed with a steroid substrate: Implications for flavin adenine dinucleotide dependent alcohol oxidases, Biochemistry, 1993, 32, 11507–11515.[Medline]
Liljeström P, Lusa S, Huylebroeck D & Garoff H. In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000- molecular-weight membrane protein modulates virus release, J Virol, 1991, 65, 4107–4113.
Lindqvist BH, DiSalvo J, Rice CM, Strauss JH & Strauss EG. Sindbis virus mutant ts20 of complementation group E contains a lesion in glycoprotein E2, Virology, 1986, 151, 10–20.[Medline]
Marquardt MT & Kielian M. Cholesterol-depleted cells that are relatively permissive for Semliki Forest virus infection, Virol, 1996, 224, 198–205.[Medline]
Marquardt MT, Phalen T & Kielian M. Cholesterol is required in the exit pathway of Semliki Forest virus, J Cell Biol, 1993, 123, 57–65.
Moesby L, Corver J, Erukulla RK, Bittman R & Wilschut J. Sphingolipids activate membrane fusion of Semliki Forest virus in a stereospecific manner, Biochemistry, 1995, 34, 10319–10324.[Medline]
Murata M, Peränen J, Schreiner R, Wieland F, Kurzchalia TV & Simons K. VIP21/caveolin is a cholesterol-binding protein, Proc Natl Acad Sci USA, 1995, 92, 10339–10343.
Nes, W.R. and M.L. McKean. 1977. Occurence, physiology, and ecology of sterols. In Biochemistry of Steroids and Other Isopentenoids. W.R. Nes and M.L. McKean. University Park Press, Baltimore, MD. 411–533.
Nieva JL, Bron R, Corver J & Wilschut J. Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane, EMBO (Eur Mol Biol Organ) J, 1994, 13, 2797–2804.[Medline]
Ou J-H, Strauss EG & Strauss JH. Comparative studies of the 3'-terminal sequences of several alphavirus RNAs, Virology, 1981, 109, 281–289.[Medline]
Pfanner N, Glick BS, Arden SR & Rothman JE. Fatty acylation promotes fusion of transport vesicles with Golgi cisternae, J Cell Biol, 1990, 110, 955–961.
Phalen T & Kielian M. Cholesterol is required for infection by Semliki Forest virus, J Cell Biol, 1991, 112, 615–623.
Portnoy DA, Chakraborty T, Goebel W & Cossart P. Molecular determinants of Listeria monocytogenes pathogenesis, Infect Immun, 1992, 60, 1263–1267.
Rossjohn J, Feil SC, McKinstry WJ, Tweten RK & Parker MW. Structure of a cholesterol-binding, thiol-activate cytolysin and a model of its membrane form, Cell, 1997, 89, 685–692.[Medline]
Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR & Anderson RGW. Caveolin, a protein component of caveolae membrane coats, Cell, 1992, 68, 673–682.[Medline]
Salminen A, Wahlberg JM, Lobigs M, Liljeström P & Garoff H. Membrane fusion process of Semliki Forest virus II: cleavage-dependent reorganization of the spike protein complex controls virus entry, J Cell Biol, 1992, 116, 349–357.
Silberkang M, Havel CM, Friend DS, McCarthy BJ & Watson JA. Isoprene synthesis in isolated embryonic Drosophilacells. I. Sterol- deficient eukaryotic cells, J Biol Chem, 1983, 258, 8303–8311.
Smart EJ, Ying YS, Donzell WC & Anderson RGW. A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane, J Biol Chem, 1996, 271, 29427–29435.
Strauss JH & Strauss EG. The alphaviruses: gene expression, replication, and evolution, Microbiol Rev, 1994, 58, 491–562.
White J & Helenius A. pH-dependent fusion between the Semliki Forest virus membrane and liposomes, Proc Natl Acad Sci USA, 1980, 77, 3273–3277.
White J, Kartenbeck J & Helenius A. Membrane fusion activity of influenza virus, EMBO (Eur Mol Biol Organ) J, 1982, 1, 217–222.[Medline]
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