The Dynamic Envelope of a Fusion Class II Virus
2008; Elsevier BV; Volume: 283; Issue: 39 Linguagem: Inglês
10.1074/jbc.m801470200
ISSN1083-351X
AutoresShang-Rung Wu, Lars Haag, Mathilda Sjöberg, Henrik Garoff, Lena Marmstål Hammar,
Tópico(s)HIV Research and Treatment
ResumoIn alphaviruses, here represented by Semliki Forest virus, infection requires an acid-responsive spike configuration to facilitate membrane fusion. The creation of this relies on the chaperon function of glycoprotein E2 precursor (p62) and its maturation cleavage into the small external E3 and the membrane-anchored E2 glycoproteins. To reveal how the E3 domain of p62 exerts its control of spike functions, we determine the structure of a p62 cleavage-impaired mutant virus particle (SQL) by electron cryomicroscopy. A comparison with the earlier solved wild type virus structure reveals that the E3 domain of p62SQL forms a bulky side protrusion in the spike head region. This establishes a gripper over part of domain II of the fusion protein, with a cotter-like connection downward to a hydrophobic cluster in its central β-sheet. This finding reevaluates the role of the precursor from being only a provider of a shield over the fusion loop to a structural playmate in formation of the fusogenic architecture. In alphaviruses, here represented by Semliki Forest virus, infection requires an acid-responsive spike configuration to facilitate membrane fusion. The creation of this relies on the chaperon function of glycoprotein E2 precursor (p62) and its maturation cleavage into the small external E3 and the membrane-anchored E2 glycoproteins. To reveal how the E3 domain of p62 exerts its control of spike functions, we determine the structure of a p62 cleavage-impaired mutant virus particle (SQL) by electron cryomicroscopy. A comparison with the earlier solved wild type virus structure reveals that the E3 domain of p62SQL forms a bulky side protrusion in the spike head region. This establishes a gripper over part of domain II of the fusion protein, with a cotter-like connection downward to a hydrophobic cluster in its central β-sheet. This finding reevaluates the role of the precursor from being only a provider of a shield over the fusion loop to a structural playmate in formation of the fusogenic architecture. When an enveloped virus infects its target cell, the mechanism usually involves a step with hairpin refolding of the viral fusion protein to promote merging of virus and target membranes. Opposite to the class I fusion proteins, which are trimers from the start and in which the fusion-related refolding involves formation and backfolding of α-helical bundles, the class II fusion proteins elaborate on homotrimer formation to mediate membrane fusion with target membrane, as discussed by Kielian (1Kielian M. Virology. 2006; 344: 38-47Crossref PubMed Scopus (156) Google Scholar, 2Kielian M. Rey F.A. Nat. Rev. Microbiol. 2006; 4: 67-76Crossref PubMed Scopus (442) Google Scholar) and others (3White J.M. Hoffman L.R. Arevalo J.H. Wilson I.A. Chiu W. Burnett R.M. Garcea R.L. Structural Biology of Viruses. Oxford University Press, Oxford1997: 80-104Google Scholar, 4Malashkevich V.N. Singh M. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8502-8506Crossref PubMed Scopus (51) Google Scholar, 5Lau W.L. Ege D.S. Lear J.D. Hammer D.A. DeGrado W.F. Biophys. J. 2004; 86: 272-284Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In the alphaviruses, here represented by Semliki Forest virus (SFV), 3The abbreviations used are: SFVSemliki Forest virusEMelectron microscopycryo-EMelectron cryomicroscopyMES2-(N-morpholino)ethanesulfonic acidWTwild typeELISAenzyme-linked immunosorbent assayMOPS3-(N-morpholino)propanesulfonic acidmAbmonoclonal antibodyERendoplasmic reticulumC, E1, E2, and E3structural proteins of Semliki Forest virus. the fusion proteins are of class II and essentially lack helical motifs. Homotrimers of the fusion protein E1, formed in relation to the membrane fusion process, are stable associations (6Wahlberg J.M. Bron R. Wilschut J. Garoff H. J. Virol. 1992; 66: 7309-7318Crossref PubMed Google Scholar, 7Bron R. Wahlberg J.M. Garoff H. Wilschut J. EMBO J. 1993; 12: 693-701Crossref PubMed Scopus (161) Google Scholar, 8Klimjack M.R. Jeffrey S. Kielian M. J. Virol. 1994; 68: 6940-6946Crossref PubMed Google Scholar, 9Garoff H. Wilschut J. Liljestrom P. Wahlberg J.M. Bron R. Suomalainen M. Smyth J. Salminen A. Barth B.U. Zhao H. Arch. Virol. Suppl. 1994; 9: 329-338PubMed Google Scholar, 10Andersson H. Barth B.U. Ekstrom M. Garoff H. J. Virol. 1997; 71: 9654-9663Crossref PubMed Google Scholar, 11Gibbons D. Ahn A. Chatterjee P. Kielian M. J. Virol. 2000; 74: 7772-7780Crossref PubMed Scopus (57) Google Scholar, 12Gibbons D. Erk I. Reilly B. Navaza J. Kielian M. Rey F. Lepault J. Cell. 2003; 114: 573-584Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Gibbons D. Reilly B. Ahn A. Vaney M. Vigouroux A. Rey F. Kielian M. J. Virol. 2004; 78: 3514-3523Crossref PubMed Scopus (22) Google Scholar). The strong interaction would be the driving force for completion of fusion, after low pH and membrane contact trigger. However, it would be suicidal for virus transmission if the fusion protein were allowed to create such a configuration prematurely. The SFV assembly pathway handles this problem by providing a chaperon protein, the precursor of glycoprotein E2, in SFV named p62. The p62 precedes the E1 in the proprecursor sequence (p62–6K-E1; see Scheme 1) and waits in the ER to form dimers with the nascent E1, thereby allowing transport to the Golgi compartment and forestalling E1 self-aggregation (10Andersson H. Barth B.U. Ekstrom M. Garoff H. J. Virol. 1997; 71: 9654-9663Crossref PubMed Google Scholar, 15Barth B.U. Wahlberg J.M. Garoff H. J. Cell Biol. 1995; 128: 283-291Crossref PubMed Scopus (31) Google Scholar). The E2 itself, if translocated into ER by a cleavable signal sequence, is not sufficient for the purpose. This was shown with an E3 deletion mutant, where the N-terminal E3 domain of the precursor was exchanged for a cleavable artificial signal sequence to preserve the membrane topology of authentic E2. In this E3 deletion mutant, expressed via a recombinant vaccinia virus, the heterodimerization of the spike proteins was abolished, and the E1 was completely retained in the ER (14Lobigs M. Zhao H.X. Garoff H. J. Virol. 1990; 6443: 46-55Google Scholar). In the early Golgi compartment, the p62-E1 heterodimers may form trimers of dimers (16Mulvey M. Brown D.T. Virology. 1996; 219: 125-132Crossref PubMed Scopus (44) Google Scholar) that in the trans-Golgi undergo furin-dependent maturation cleavage (9Garoff H. Wilschut J. Liljestrom P. Wahlberg J.M. Bron R. Suomalainen M. Smyth J. Salminen A. Barth B.U. Zhao H. Arch. Virol. Suppl. 1994; 9: 329-338PubMed Google Scholar, 17Jain S.K. DeCandido S. Kielian M. J. Biol. Chem. 1991; 266: 5756-5761Abstract Full Text PDF PubMed Google Scholar, 18Salminen A. Wahlberg J.M. Lobigs M. Liljestrom P. Garoff H. J. Cell Biol. 1992; 116: 349-357Crossref PubMed Scopus (124) Google Scholar), transport to the plasma membrane, and assembly with nucleocapsid components into infectious virus particles. The furin cleavage of p62 into the small external glycoprotein E3 and the membrane-anchored spike glycoprotein E2 is not a prerequisite for transport to the plasma membrane and virus assembly, since virions are formed in furin-deficient cells (19Zhang X. Fugere M. Day R. Kielian M. J. Virol. 2003; 77: 2981-2989Crossref PubMed Scopus (75) Google Scholar), as well as with cleavage-impaired p62 mutants (18Salminen A. Wahlberg J.M. Lobigs M. Liljestrom P. Garoff H. J. Cell Biol. 1992; 116: 349-357Crossref PubMed Scopus (124) Google Scholar, 19Zhang X. Fugere M. Day R. Kielian M. J. Virol. 2003; 77: 2981-2989Crossref PubMed Scopus (75) Google Scholar, 20Glasgow G.M. Sheahan B.J. Atkins G.J. Wahlberg J.M. Salminen A. Liljestrom P. Virology. 1991; 185: 741-748Crossref PubMed Scopus (50) Google Scholar, 21Berglund P. Sjoberg M. Garoff H. Atkins G.J. Sheahan B.J. Liljestrom P. Nat. Biotechnol. 1993; 11: 916-920Crossref Scopus (257) Google Scholar, 22Tubulekas I. Liljestrom P. J. Virol. 1998; 72: 2825-2831Crossref PubMed Google Scholar). Such "nonmature" particles, or virion equivalents, are less sensitive to pH trigger for fusion than the maturation-cleaved wild type (WT) particles with E2 and are noninfectious under normal cell conditions. It is therefore assumed that the p62 protects the prespike structure against acid-induced reorganization with fusion loop exposure during the low pH passage through Golgi compartments. By the maturation cleavage in late trans-Golgi, the pH-sensitive virus shell architecture, prompted for merging interaction with a target membrane, is established. Semliki Forest virus electron microscopy electron cryomicroscopy 2-(N-morpholino)ethanesulfonic acid wild type enzyme-linked immunosorbent assay 3-(N-morpholino)propanesulfonic acid monoclonal antibody endoplasmic reticulum structural proteins of Semliki Forest virus. By what means does the E3 domain of the precursor constitute such major control? A first essential feature of the p62 would be to establish control over the nascent E1 to prevent E1 homotrimer formation during spike assembly. Although it would be expected that it exerts this function by a tighter E1 binding, or even an extra contact, compared with the E2 itself, it has never been shown that such a contact exists. The sequence of the about 66-residue-long E3 domain is relatively well conserved within the alphaviruses and implies conserved structural elements (Fig. 1A); The N-terminal domain is the signal sequence that was directly glycosylated after translocation into ER and retained as the N terminus of the precursor (23Garoff H. Huylebroeck D. Robinson A. Tillman U. Liljestrom P. J. Cell Biol. 1990; 111: 867-876Crossref PubMed Scopus (44) Google Scholar). This sequence is followed by a region dominated by a highly conserved pattern of Pro and Cys. In the C-terminal half of the molecule, the sequence indicates two α-helices with amphipathic patterns that would allow hairpin back-folding along each other's. After the helical domain follows a conserved Cys, making an even total number of cysteines, and at the C terminus a cluster of basic residues form the furin cleavage site, of which the outermost two may be removed after cleavage (24Mayne J.T. Rice C.M. Strauss E.G. Hunkapiller M.W. Strauss J.H. Virology. 1984; 134: 338-357Crossref PubMed Scopus (32) Google Scholar). In summary, the E3 domain that is the first part of the p62 entering into ER may comprise the retained signal sequence with its carbohydrate moiety, a cross-linked domain with a protruding hairpin pair of α-helices, and the furin cleavage region linking to the E2 sequence. In the whole virus structure, revealed by electron cryomicroscopy (cryo-EM), the trimeric spikes protrude above a protein shell layer that surrounds the virus at some distance above its membrane (25Smith T.J. Cheng R.H. Olson N.H. Peterson P. Chase E. Kuhn R.J. Baker T.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10648-10652Crossref PubMed Scopus (113) Google Scholar, 26Fuller S.D. Berriman J.A. Butcher S.J. Gowen B.E. Cell. 1995; 81: 715-725Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 27Ferlenghi I. Gowen B. de Haas F. Mancini E.J. Garoff H. Sjoberg M. Fuller S.D. J. Mol. Biol. 1998; 283: 71-81Crossref PubMed Scopus (37) Google Scholar, 28Mancini E.J. Clarke M. Gowen B.E. Rutten T. Fuller S.D. Mol. Cell. 2000; 5: 255-266Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 29Haag L. Garoff H. Xing L. Hammar L. Kan S.T. Cheng R.H. EMBO J. 2002; 21: 4402-4410Crossref PubMed Scopus (33) Google Scholar, 30Mukhopadhyay S. Zhang W. Gabler S. Chipman P.R. Strauss E.G. Strauss J.H. Baker T.S. Kuhn R.J. Rossmann M.G. Structure. 2006; 14: 63-73Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 31Wu S.R. Haag L. Hammar L. Wu B. Garoff H. Xing L. Murata K. Cheng R.H. J. Biol. Chem. 2007; 282: 6752-6762Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Modeling of the solved crystal structure of the ectodomain of fusion protein E1 (32Lescar J. Roussel A. Wien M.W. Navaza J. Fuller S.D. Wengler G. Rey F.A. Cell. 2001; 105: 137-148Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 33Roussel A. Lescar J. Vaney M.C. Wengler G. Wengler G. Rey F.A. Structure. 2006; 14: 75-86Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) into cryo-EM-derived whole virion structures implies that E1 constitutes the protein shell by intermolecular E1-E1 interactions and internal interactions between its domain III and I (31Wu S.R. Haag L. Hammar L. Wu B. Garoff H. Xing L. Murata K. Cheng R.H. J. Biol. Chem. 2007; 282: 6752-6762Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 32Lescar J. Roussel A. Wien M.W. Navaza J. Fuller S.D. Wengler G. Rey F.A. Cell. 2001; 105: 137-148Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 33Roussel A. Lescar J. Vaney M.C. Wengler G. Wengler G. Rey F.A. Structure. 2006; 14: 75-86Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 34Pletnev S.V. Zhang W. Mukhopadhyay S. Fisher B.R. Hernandez R. Brown D.T. Baker T.S. Rossmann M.G. Kuhn R.J. Cell. 2001; 105: 127-136Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). E1 is part of the spikes by heterodimeric interactions with E2. The fusion loop is thereby hidden by E2 under the rim of the spike wing (31Wu S.R. Haag L. Hammar L. Wu B. Garoff H. Xing L. Murata K. Cheng R.H. J. Biol. Chem. 2007; 282: 6752-6762Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 32Lescar J. Roussel A. Wien M.W. Navaza J. Fuller S.D. Wengler G. Rey F.A. Cell. 2001; 105: 137-148Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 33Roussel A. Lescar J. Vaney M.C. Wengler G. Wengler G. Rey F.A. Structure. 2006; 14: 75-86Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). A similar organization is deduced from studies on sugar deletion mutants of Sindbis virus (34Pletnev S.V. Zhang W. Mukhopadhyay S. Fisher B.R. Hernandez R. Brown D.T. Baker T.S. Rossmann M.G. Kuhn R.J. Cell. 2001; 105: 127-136Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). There seems to be no direct contact between the E1 molecules within the spike, which is held together only by pairwise E2-E2 contacts (31Wu S.R. Haag L. Hammar L. Wu B. Garoff H. Xing L. Murata K. Cheng R.H. J. Biol. Chem. 2007; 282: 6752-6762Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Thus, the E1 protein in the virion is inter- and intramolecularly self-associated but in an alternative fashion to the fusion-related configuration of stable trimers for which the atomic structure is determined (12Gibbons D. Erk I. Reilly B. Navaza J. Kielian M. Rey F. Lepault J. Cell. 2003; 114: 573-584Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 13Gibbons D. Reilly B. Ahn A. Vaney M. Vigouroux A. Rey F. Kielian M. J. Virol. 2004; 78: 3514-3523Crossref PubMed Scopus (22) Google Scholar). The further domains of E1 that are required for homotrimerization are controlled by E2 (31Wu S.R. Haag L. Hammar L. Wu B. Garoff H. Xing L. Murata K. Cheng R.H. J. Biol. Chem. 2007; 282: 6752-6762Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 33Roussel A. Lescar J. Vaney M.C. Wengler G. Wengler G. Rey F.A. Structure. 2006; 14: 75-86Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Then, while the rearrangement of the virus envelope to form fusion-related E1 homotrimers seems complex enough as it is, what are the location and crucial interactions of the E3 domain that hold back the fusion-related refolding? Venien-Bryan and Fuller (35Venien-Bryan C. Fuller S.D. J. Mol. Biol. 1994; 236: 572-583Crossref PubMed Scopus (45) Google Scholar) early found that the E3 was primarily interacting with the E2 in the spike structure, and Paredes et al. (36Paredes A.M. Heidner H. Thuman-Commike P. Prasad B.V. Johnston R.E. Chiu W. J. Virol. 1998; 72: 1534-1541Crossref PubMed Google Scholar), who studied a cleavage-impaired Sindbis virus mutant, proposed that the E3 domain in the nonmature virion would "protrude midway between the center of the spike complex and the tips." In that constellation, the E3 domain would not be able to shield the fusion loop, at least not in the same spike. We approach the question of how the p62 exerts its action by searching the location of the E3 domain in the whole particle structure of an SFV mutant, here referred to as SQL. In SQL, the furin cleavage site of p62 is impaired by amino acid substitutions (21Berglund P. Sjoberg M. Garoff H. Atkins G.J. Sheahan B.J. Liljestrom P. Nat. Biotechnol. 1993; 11: 916-920Crossref Scopus (257) Google Scholar, 22Tubulekas I. Liljestrom P. J. Virol. 1998; 72: 2825-2831Crossref PubMed Google Scholar) and thereby gives rise to an acid-resistant virion. This is noninfectious at around pH 6, where the WT virus is prone to fuse. However, it may turn fusion-competent at a lower pH, indicating that the structure of the functional machinery remains in working order (37Lobigs M. Wahlberg J.M. Garoff H. J. Virol. 1990; 64: 5214-5218Crossref PubMed Google Scholar). The location of the E3 domain is then deduced from comparison of the SQL structure with our recently reported SFV WT structure (31Wu S.R. Haag L. Hammar L. Wu B. Garoff H. Xing L. Murata K. Cheng R.H. J. Biol. Chem. 2007; 282: 6752-6762Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Our analyses give details on the mutant virion structure and essentially confirm the location of the E3 domain suggested by the Paredes studies on Sindbis virus (36Paredes A.M. Heidner H. Thuman-Commike P. Prasad B.V. Johnston R.E. Chiu W. J. Virol. 1998; 72: 1534-1541Crossref PubMed Google Scholar). A similar location would also be deduced from the structure of the same SFV mutant studied by Ferlenghi et al. (27Ferlenghi I. Gowen B. de Haas F. Mancini E.J. Garoff H. Sjoberg M. Fuller S.D. J. Mol. Biol. 1998; 283: 71-81Crossref PubMed Scopus (37) Google Scholar). Furthermore, a direct contact of the p62 with a hydrophobic cluster in the E1 molecule is demonstrated. The properties of this contact and implications for how the functional spike architecture is created are discussed. Wild-type and Mutant Virus—The SFV WT used in this study was generated from the plasmid pSFV4 (38Liljestrom P. Lusa S. Huylebroeck D. Garoff H. J. Virol. 1991; 65: 4107-4113Crossref PubMed Google Scholar), and the mutant SQL was generated from a corresponding plasmid where the sequence RHRR in the p62 cleavage site is replaced with SHQL (21Berglund P. Sjoberg M. Garoff H. Atkins G.J. Sheahan B.J. Liljestrom P. Nat. Biotechnol. 1993; 11: 916-920Crossref Scopus (257) Google Scholar). The mutant virus is noninfectious, but cleavage with chymotrypsin removes the block and results in wild type-like infectivity (21Berglund P. Sjoberg M. Garoff H. Atkins G.J. Sheahan B.J. Liljestrom P. Nat. Biotechnol. 1993; 11: 916-920Crossref Scopus (257) Google Scholar). Virus Purification by Tartrate Gradient Centrifugation and Quality Control—The SFV4 was propagated and purified, essentially as described previously (29Haag L. Garoff H. Xing L. Hammar L. Kan S.T. Cheng R.H. EMBO J. 2002; 21: 4402-4410Crossref PubMed Scopus (33) Google Scholar, 39Hammar L. Markarian S. Haag L. Lankinen H. Salmi A. Cheng R.H. J. Biol. Chem. 2003; 278: 7189-7198Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The SFV SQL was activated for infection by chymotrypsin treatment but otherwise handled in the same way as the wild type virus. Briefly, monolayers of BHK-21 cells were grown in 225-cm2 T-flasks using Glasgow's modified Eagle's minimal essential medium (Invitrogen), supplemented with 5% fetal calf serum, 10% tryptose phosphate broth, 2 mm glutamine, 20 mm HEPES (Sigma), and 20 μg/ml cholesterol. The SQL stock was activated on ice by α-chymotrypsin for 2 h, when the reaction was quenched with aprotinin (21Berglund P. Sjoberg M. Garoff H. Atkins G.J. Sheahan B.J. Liljestrom P. Nat. Biotechnol. 1993; 11: 916-920Crossref Scopus (257) Google Scholar). At 90% confluence, the cells were infected with SFV WT or the chymotrypsin-activated SQL and further incubated at 37 °C. Virus was harvested from the cell supernatant at 18 h postinfection. The virus-containing supernatant was cleared from cell debris, and the virus was pelleted at 17,000 × g at 4 °C for 18 h. The virus pellet was soaked in TNM buffer (50 mm Tris-HCl, 50 mm NaCl, 10 mm MgCl2, pH 7.4) and applied to isopycnic centrifugation using a 10–30% (w/w) potassium tartrate density gradient in 0.1 m MOPS, pH 7.4, run at 100,000 × g for 5 h at 4 °C. The virus was eluted from the gradient, diluted with TNM buffer, and pelleted by centrifugation at 100,000 × g for 5 h at 4 °C. The final virus pellet was soaked in TNM buffer and kept at 4 °C to avoid structural damage. For comparison of E3 content, a parallel experiment was done by the traditional scheme in the literature (i.e. using sucrose to create the density gradient). The quality of the virus was controlled by SDS-PAGE, ELISA, and negative stain EM. For structural analyses, the samples were applied on cryogrids, plunge-frozen into liquid ethane, and transferred to liquid nitrogen and subsequently to liquid helium before microscopy imaging. ELISA for pH-dependent Epitope Exposure—ELISA experiments were done using Protein A (GE Healthcare) affinity-purified mAb E1f against the E1 glycoprotein (39Hammar L. Markarian S. Haag L. Lankinen H. Salmi A. Cheng R.H. J. Biol. Chem. 2003; 278: 7189-7198Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The SFV WT and mutant SQL were purified using tartrate gradient ultracentrifugation, as described above (29Haag L. Garoff H. Xing L. Hammar L. Kan S.T. Cheng R.H. EMBO J. 2002; 21: 4402-4410Crossref PubMed Scopus (33) Google Scholar). Equal amounts (∼85 ng/well) of purified SFV WT and SQL were coated overnight at 4 °C onto 96-well ELISA plates (high binding enzyme immunoassay/radioimmunoassay plate; Corning Glass). After blocking with TBS-B (10 mm Tris-HCl, 150 mm NaCl, 5% bovine serum albumin, pH 7.4) for 1 h, the mAb E1f, suspended in a series of buffers of different pH, was added (∼50 ng/well). The pH of these buffers was obtained by mixing appropriate volumes of 50 mm Tris and 50 mm MES, both containing 50 mm NaCl. After a 1-h incubation, the excess of antibody was removed by washes with TBS-b (10 mm Tris-HCl, 150 mm NaCl supplemented with 0.5% bovine serum albumin, pH 7.4) before a 1-h incubation with an Fc-specific, peroxidase conjugated, anti-mouse reporter antibody (A-2554; Sigma). Finally, the wells were washed with TBS-b, and bound reporter antibody was developed using ortho-phenylenediamine dihydrochloride (Dako A/S) according to the manufacturers' recommendations. The reaction was quenched when adequate color intensity was reached or after ∼15 min, and the light absorbance at 490 nm was measured in a PowerwaveX spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT). Cryo-EM and Image Processing—The SFV WT and SFV SQL specimens were prepared for electron cryomicroscopy and recorded under low dose conditions in a JEM3200F field emission gun transmission electron microscope equipped with the top entry type liquid helium cold stage at a magnification of 40,000 and an accelerating voltage of 300 kV. Micrographs were digitized and further bin-averaged to give a step size of 14 μm, corresponding to 3.5 Å/pixel at the specimen. Particle images were manually boxed out using the RobEM package. The parameters of orientation and origin were determined by model-based polar Fourier transform routines (40Baker T.S. Cheng R.H. J. Struct. Biol. 1996; 116: 120-130Crossref PubMed Scopus (324) Google Scholar, 41Cheng R.H. Kuhn R.J. Olson N.H. Rossmann M.G. Choi H.K. Smith T.J. Baker T.S. Cell. 1995; 80: 621-630Abstract Full Text PDF PubMed Scopus (284) Google Scholar). The resolution of the final reconstruction was determined using a 0.5 cutoff of the Fourier shell correlation coefficient between the "odd" and "even" image reconstructions. The numbers of particles included in the reconstructions are 834 (SFV WT) and 1,580 (SQL). Image reconstruction procedures were carried out essentially as described previously (31Wu S.R. Haag L. Hammar L. Wu B. Garoff H. Xing L. Murata K. Cheng R.H. J. Biol. Chem. 2007; 282: 6752-6762Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), using the initial SFV model provided by Dr. Bomu Wu. We used the Iris explorer software (NAG, Inc., Downers Grove, IL), supplemented with custom-made modules 4L. Bergman unpublished data. for the three-dimensional visualization, along with Pymol™ (DeLano Scientific LLC; available on the World Wide Web). The pH Profile of Fusion Loop Exposure—A functional difference of the SFV WT virus and the SQL is the threshold for fusion activation. Here, we tested the exposure of the fusion loop relative pH as the capacity to bind mAb E1f, an antibody that recognizes the fusion loop sequence (39Hammar L. Markarian S. Haag L. Lankinen H. Salmi A. Cheng R.H. J. Biol. Chem. 2003; 278: 7189-7198Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Thus, coated in an ELISA plate at the same protein load, the particles were exposed to mAb E1f at different pH, and the bound antibody, reflecting the accessible sequence, was measured. The pH profiles obtained showed an optimum for the WT at pH 6.2, similar to a previously reported value (39Hammar L. Markarian S. Haag L. Lankinen H. Salmi A. Cheng R.H. J. Biol. Chem. 2003; 278: 7189-7198Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), whereas the peak for the mutant appeared first at below pH 5 (Fig. 1B). The maximal response was lower with the mutant than with the WT virus, reaching its optimum at about 40% of the WT value. This may partly reflect that the antibody is less stable at the lower pH and/or that the mutant particle is too rigid to allow access to the same number of sites as the WT particle. Therefore, we present the result normalized to optimal mAb E1f binding value, simply to demonstrate the difference in the pH response profile. Spike Homogeneity—A problem with structure determination by cryo-EM is the purity and homogeneity of the material studied. Therefore, the virus material was extensively purified, and loosely attached components were removed. In SFV, the E3 may partly stay associated to the WT virions after maturation cleavage. This varied with the purification efficiency (Fig. 1C). At the final stages of the extensive purification of SFV WT particles for cryo-EM, the glycoprotein E3 was essentially removed. Such E3-depleted particles have been the source for our recently presented structure of SFV (29Haag L. Garoff H. Xing L. Hammar L. Kan S.T. Cheng R.H. EMBO J. 2002; 21: 4402-4410Crossref PubMed Scopus (33) Google Scholar, 31Wu S.R. Haag L. Hammar L. Wu B. Garoff H. Xing L. Murata K. Cheng R.H. J. Biol. Chem. 2007; 282: 6752-6762Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 42Haag, L. (2006) The Dynamic Envelope of Semliki Forest Virus: Molecular Reorganizations during Pre-fusion Stages, Ph.D. thesis, Karolinska Institute, Huddinge, SwedenGoogle Scholar). As medium for the density gradient centrifugation, tartrate has been preferred to sucrose, since it is easier to remove and therefore allows a better situation for the imaging. A similar homogeneity problem is imposed by the incomplete maturation cleavage of the glycoprotein E2 precursor, p62, in the WT virus. This cleavage is not essential for the transport of the envelope proteins to the plasma membrane and assembly of virions (18Salminen A. Wahlberg J.M. Lobigs M. Liljestrom P. Garoff H. J. Cell Biol. 1992; 116: 349-357Crossref PubMed Scopus (124) Google Scholar, 21Berglund P. Sjoberg M. Garoff H. Atkins G.J. Sheahan B.J. Liljestrom P. Nat. Biotechnol. 1993; 11: 916-920Crossref Scopus (257) Google Scholar), which is why traces usually remain in the WT virus preparations. This may also vary with the condition of the producer cells. Under the conditions used in this study, we found that 5 ± 3%, n = 12 (mean ± S.D.), of the heterodimers contained p62. It is uncertain whether this inhomogeneity in spike composition was arbitrary distributed. However, its impact on the final reconstruction of the WT structure, after averaging and refinement, would be minor. In the mutant SQL, the p62 equivalent, p62SQL, remained uncleaved (no E2 was present), and the composition of the spikes would be homogeneous. Three-dimensional Reconstruction of WT and Mutant of Semliki Forest Virus—The structure of the cleavage-impaired SFV mutant SQL determined in this work was achieved at a resolution computed to 17 Å. The WT particle structure at neutral pH was earlier obtained at a resolution computed to 11 Å (31Wu S.R. Haag L. Hammar L. Wu B. Garoff H. Xing L. Murata K. Cheng R.H. J. Biol. Chem. 2007; 282: 6752-6762Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). For an accurate comparison of the two particles, the reconstruction of SFV WT was here band pass-filtered to 17 Å resolution. With a diameter of 703 Å, the SQL particles are only slightly larger than the WT ones, reaching a diameter of 700 Å. As judged from the one-dimensional radial density distribution plot (Fig. 1D) and whole particle renderings (not shown), the general organization of the two icosahedral particles is essentially similar, except for the details in the spike morphology described below. Revealing the Location of the E3 Domain—Common and discriminating details between the SFV WT and SQL particles are revealed by superimposing three-dimensional reconstructions of the two structures. They overlap well in the limb and shell domains of the envelope above the membrane and in the center of the spikes. The part of the head do
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