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A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B env Gene

A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B env Gene
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  A Native-Like SOSIP.664 Trimer Based on an HIV-1 Subtype B  env  Gene Pavel Pugach, a Gabriel Ozorowski, b Albert Cupo, a Rajesh Ringe, a Anila Yasmeen, a Natalia de Val, b Ronald Derking, c Helen J. Kim, b Jacob Korzun, a Michael Golabek, a Kevin de los Reyes, a Thomas J. Ketas, a Jean-Philippe Julien, b *  Dennis R. Burton, e,f  Ian A. Wilson, b,d Rogier W. Sanders, a,c P. J. Klasse, a Andrew B. Ward, b John P. Moore a Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA a ; Department of Integrative Structural andComputational Biology, IAVI Neutralizing Antibody Center and Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, LaJolla, California, USA b ; Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands c ; The Skaggs Institute forChemical Biology, The Scripps Research Institute, La Jolla, California, USA d ; Department of Immunology and Microbial Science, IAVI Neutralizing Antibody Center andCenter for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA e ; Ragon Institute of MGH, MIT and Harvard,Cambridge, Massachusetts, USA f  ABSTRACT Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike shouldexpose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies(non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are there-fore plausible vaccine candidates. Here, we report on the production and  in vitro  properties of a new SOSIP.664 trimer derivedfrom a subtype B  env   gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping)to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron mi-croscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP.664 trimers contain two subpopulations, which wepropose represent an equilibrium between the fully closed and a more open conformation. The latter is different from the fully open, CD4 receptor-bound conformation and may represent an intermediate state of the trimer. This new subtype B trimer addsto the repertoire of native-like Env proteins that are suitable for immunogenicity and structural studies. IMPORTANCE The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccinestrategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble proteinmimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via theintroduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known asSOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A  env   gene. Here, we describe the productionand characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method topurify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be usefulfor structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine. I mmunogens capable of inducing protective titers of broadly neutralizingantibodies(bNAbs)arebeingwidelysoughtforusein vaccine design strategies for human immunodeficiency virustype 1 (HIV-1) (1). The basis of this approach is that bNAbs can prevent globally diverse HIV-1 strains from infecting target cells.They do so via binding to the envelope glycoprotein (Env) com-plex on the virion surface, an event that is both necessary andsufficient to neutralize HIV-1 infectivity (2, 3). One of the more common strategies to induce bNAbs involves the design of solu-ble, recombinant protein mimics of the native Env complex, ameta-stablestructurecomprisingthreegp120andthreegp41sub-units.TheproductionofsolubleEnvtrimersinvolvesintroducingastopcodontotruncatethegp41ectodomain(gp41 ECTO )subunitpriortothetransmembraneregiontoyieldsolublegp140proteins(4–6). The fragility of the Env complex is, however, a substantialproblem from a protein engineering perspective, as the naturalnoncovalent interactions between the six subunits are not robustenoughtoallowsolubletrimerstobeexpressedandpurifiedwith-out the use of stabilizing changes.The most commonly used method to stabilize soluble gp140 Received  2 December 2014  Accepted  5 January 2015 Accepted manuscript posted online  14 January 2015 Citation  Pugach P, Ozorowski G, Cupo A, Ringe R, Yasmeen A, de Val N,Derking R, Kim HJ, Korzun J, Golabek M, de los Reyes K, Ketas TJ, Julien J-P,Burton DR, Wilson IA, Sanders RW, Klasse PJ, Ward AB, Moore JP. 2015. Anative-like SOSIP.664 trimer based on an HIV-1 subtype B  env   gene. J Virol89:3380–3395. doi:10.1128/JVI.03473-14. Editor:  R. W. DomsAddress correspondence to Andrew B. Ward,, orJohn P. Moore,*Present address: Jean-Philippe Julien, Program in Molecular Structure andFunction, The Hospital for Sick Children Research Institute and Departments of Biochemistry and Immunology, University of Toronto, Toronto, Ontario, Canada.P.P. and G.O. contributed equally to this article. This is paper number 29007 from The Scripps Research Institute.Copyright © 2015, American Society for Microbiology. All Rights Reserved.doi:10.1128/JVI.03473-14 3380 March 2015 Volume 89 Number 6Journal of Virology   onM ar  c h  3  0  ,2  0 1  5  b  y K r  e s  g eL i   b r  ar  y  ,T h  e S  c r i   p p s R  e s  e ar  c h I  n s  t  i   t   u t   eh  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   proteins has been to eliminate, by mutagenesis, the natural cleav-age site between gp120 and gp41 ECTO  (7–19). It is now clear that the resulting uncleaved gp140 (gp140 UNC ) proteins, when puri-fied by size exclusion chromatography (SEC), contain the rightnumber of gp120 and gp41 ECTO  subunits (i.e., 3 of each). How-ever, these various SEC-purified gp140 UNC  protein populationsmostly (90 to 100%) adopt nonnative configurations whether ornot they contain additional “trimerization motifs” at the C termi-nus of gp41 ECTO  (20–26; L. K. Pritchard, S. Vasiljevic, G. Ozo- rowski,G.E.Seabright,A.Cupo,R.W.Sanders,K.J.Doores,D.R.Burton, I. A. Wilson, A. B. Ward, J. P. Moore, and M. Crispin,submittedforpublication).Electronmicroscopy(EM),otherbio-physical measurements, and glycan profiling have shown or im-pliedthatthreesemidissociatedgp120moietiesremainlinkedtoapostfusionformoftrimericgp41 ECTO viatheuncleaved,intersub-unit peptide (20–22, 24, 25; Pritchard et al., submitted). Accord- ingly, this category of gp140 UNC  proteins does not form nativetrimers on a population basis and lacks the antigenic characteris-ticsofnativeEnvspikes;theyexpressmanynon-NAbepitopesbutnotthoseforvariouskeybNAbs,andtheirgp120subunitscontainaberrant inter- and intramolecular disulfide bonds (21, 24, 25, 27–32). Similar concerns apply to uncleaved, full-length gp140proteins expressed on the cell surface (27–30, 33). An alternative approach to soluble trimer design involves ac-tively promoting the natural gp120-gp41 ECTO  cleavage eventwhile also introducing specific stabilizing mutations, i.e., an engi-neered disulfide bond that covalently links the two subunits andan Ile-to-Pro change at residue 559 that helps maintain thegp41 ECTO  moieties in the prefusion form (4, 6). In addition, the truncation of gp41 ECTO  at residue 664 eliminates a hydrophobicregion that tends to cause trimer aggregation (34, 35). The result- ing trimers are designated SOSIP.664 gp140s. The paradigm of this particular trimer design is based on the BG505 subtype Apediatric founder virus (36–38). The BG505 SOSIP.664 trimershave antigenic properties and morphologies that mimic those of nativeEnvcomplexesandwerethetrimersusedfordeterminationof high-resolution X-ray crystallography and cryo-electron mi-croscopy(cryo-EM)Envstructures(23,38–41).Theirpotentialas immunogens is currently under evaluation.We now seek to increase the repertoire of native-like solubletrimersavailableforstructuralandimmunogenicitystudies.Here,we describe the B41 SOSIP.664 trimer based on a subtype B  env  gene. Such trimers appear fully native-like when viewed by nega-tive-stain electron microscopy (NS-EM) and have antigenicity properties comparable to those of their subtype A BG505 coun-terparts. We also report on how to overcome production issuesassociated with proteolytic clipping of the V3 region, which gen-erally arises with subtype B Env proteins (42–45), including viause of positive-selection affinity columns based on the trimer-specificbNAbPGT145.NS-EMimagesshowthatthepurifiedB41trimers contain two distinguishable subpopulations. We proposethat the subpopulations represent equilibrium between the fully closed (BG505-like) and a more open conformation that is anintermediate between the fully closed form and the fully open,CD4-bound configuration. MATERIALS AND METHODS Construct design.  The B41  env   gene was derived from a subtype Bfounder virus isolated from an HIV-1-infected serial plasma donor; itsmore formal designation is 9032-08.A1.4685, and its GenBank accessionnumber is EU576114 (46). To make the SOSIP.664 gp140 construct, we introduced the following sequence changes (HxB2 numbering system):A501C and T605C (gp120-gp41 ECTO  disulfide bond [4]), I559P in gp41 ECTO (trimerstabilization[6]),REKRtoRRRRRRingp120(cleavageenhancement[5]),tissueplasminogenactivator(TPA)leaderpeptide(in- crease of gene expression [38, 47]), and a stop codon to terminate the protein after gp41 ECTO  residue 664 (improvement of homogeneity andsolubility  [34, 35]). The resulting, codon-optimized B41 SOSIP.664  env  gene was obtained from GenScript (Piscataway, NJ) and cloned intopPPI4 using PstI and NotI (4). Trimer variants containing a D7324 epitope tag sequence at the C terminus of gp41 ECTO  were also made, aspreviously described, by adding the sequence GSAPTKAKRRVVQREKR after residue 664 in gp41 ECTO , followed by a stop codon (38). The result- ing trimers are designated B41 SOSIP.664-D7324.A monomeric B41 gp120 protein with the same sequence (other thanthe changes listed below) as that subunit of the trimer was designed by introducing a stop codon into the SOSIP.664 construct at residue 512,revertingtheoptimizedcleavagesitetothewildtype(RRRRRR  ¡  REKRatresidues 508 to 511), reverting the A501C change, and making an A500Ksubstitution to optimize the D7324 epitope that is present in the C5 do-main at the gp120 C terminus.TheB41gp160cloneforgeneratingEnv-pseudotypedvirusesforneu-tralization assays has been described elsewhere (46). Env protein expression by transient transfection.  Env proteins wereexpressed in wild-type, adherent HEK293T (referred to as 293T) cells by transient transfection of   env   genes using polyethyleneimine (PEI), essen-tially as described elsewhere (38, 48). The  furin  gene was cotransfectedwith all SOSIP.664 trimer-encoding  env   genes to maximize gp120-gp41 ECTO  cleavage (4, 5). This method involves culturing the transfected 293T cells in the presence of 10% fetal calf serum (FCS). Under theseconditions, we found that serum proteases clip the V3 region of gp120subunits of some of the B41 SOSIP.664 trimers, and of the B41 gp120monomers, damaging the proteins (see Results). To overcome this prob-lem,weusedthe293Fectinlipofectionsystemtotransientlytransfect293Fcells in suspension cultures with Env and Furin expression plasmids (4:1ratio) and cultured the transfected cells in serum-free medium, followingthe manufacturer’s (Invitrogen) recommendations (see Results). Env protein production from stable cell lines.  We have describedelsewhere how to make stable CHO and 293T cell lines that express fully cleaved BG505 SOSIP.664 trimers (49). We used the same method tomake lines that produce B41 SOSIP.664 trimers. To reduce the extent of V3 clipping, it was necessary to try to adapt the stable lines to growth atlower serum concentrations than the standard culture conditions (i.e.,with 10% FCS present). With the stable 293T line, we found that the FCSconcentrationcouldonlybereducedto5%withoutasubstantialdecreasein cell viability; at 5% FCS, some V3 clipping still occurred, so we discon-tinuedourworkwiththisline.However,thestableCHOcelllinecouldbesuccessfullyadaptedto1%serumandundertheseconditions,V3clippingwas negligible (see Results). The stable CHO cells were then propagatedunder hygromycin resistance in PRO-CHO-AT medium (Lonza) con-taining 1% FCS. B41 SOSIP.664 trimer and gp120 monomer purification.  We usedtwo methods to purify B41 SOSIP.664 trimers (and the D7324-taggedvariants) from the transfection supernatants. The first procedure is de-scribed in detail elsewhere (21, 38). Briefly, Env proteins were isolated via a bNAb 2G12 affinity column and MgCl 2  elution, and the trimer fractionwas purified by SEC on a Superdex 200 26/60 column (GE Healthcare)(21, 38); this method is referred to as 2G12/SEC. ThesecondmethodinvolvedanaffinitycolumnbasedonthePGT145bNAb that recognizes a trimer-specific epitope (25, 38, 50). PGT145 was coupled to CNBr-activated Sepharose 4B beads (GE Healthcare). Env-containing culture supernatants were flowed through the resulting col-umn, the beads were washed with 2 column volumes of buffer (0.5 MNaCl, 20 mM Tris [pH 8.0]), and the bound trimers were eluted using 1column volume of 3 M MgCl 2 . The eluted trimers were immediately buf- A Native-Like Subtype B SOSIP TrimerMarch 2015 Volume 89 Number 6  3381 Journal of Virology   onM ar  c h  3  0  ,2  0 1  5  b  y K r  e s  g eL i   b r  ar  y  ,T h  e S  c r i   p p s R  e s  e ar  c h I  n s  t  i   t   u t   eh  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   fer exchanged into 75 mM NaCl–10 mM Tris, pH 8.0, using SnakeSkindialysis tubing (molecular weight cutoff [MWCO]of 10,000 [10K])(Thermo Scientific). The purified trimers were then concentrated usingVivaspin columns with a 30-kDa cutoff (GE Healthcare). Unless other-wise indicated, the trimers eluted from the PGT145 column were thenfurther purified by SEC; this method is referred to as PGT145/SEC.WemadeB41gp120bytransfecting293Fcellsinserum-freemedium,followed by purification via the 2G12 affinity column and MgCl 2  elution.Further fractionation by SEC was not required. The resulting gp120 pro-teins were not V3 clipped (  5%).In all cases, the concentrations of purified proteins were determinedusing either a bicinchonic acid-based assay (BCA assay; Thermo Scien-tific, Rockford, IL) or UV 280  absorbance using theoretical extinction co-efficients (51). SDS-PAGE,BN-PAGE,andWesternblotting. Envproteinswerean-alyzedusingeitherthe10%Tris-glycine,4to12%bis-TrisNuPAGESDS-PAGE,orbluenativePAGE(BN-PAGE)systemfollowedbystainingwithCoomassiebluedye(38,52),accordingtothemanufacturer’srecommen- dations (Invitrogen). When reducing conditions were required, e.g., toassess gp120-gp41 ECTO  cleavage or V3 clipping, the Env proteins weremixed with 0.1 M dithiothreitol (DTT) prior to loading onto the SDS-PAGE gels. For assessments of V3 clipping, we tested more rigorous de-naturing and reducing conditions, specifically by boiling the samples in0.1MDTTandSDSfor10minpriortoanalysisonaTris-glycinegel.Thisprotocolyieldedanoutcomesimilartotheonedescribedabove,confirm-ingthatwewerenotunderestimatingtheextentofclippingduetoincom-plete gp120 denaturation (data not shown).Western blotting of SDS-PAGE gels was carried out as described pre-viously, using the anti-gp120 monoclonal antibody (MAb) ARP3119(NIBSCReagentRepository,UnitedKingdom)orapoolofserumderivedfrom individuals infected with subtype B HIV-1 strains (HIV-Ig) (49). ARP3119 recognizes a conserved, linear epitope (residues EDIISLW) inthe C1 region of gp120 and hence detects the 70-kDa fragment that isproduced when V3-clipped gp120 is fractionated on a reducing SDS-PAGE gel (the other, 50-kDa, fragment does not contain the ARP3119epitope). AntibodiesandFabs. Antibodyconcentrationsaregenerallyrecordedin micrograms per milliliter for neutralization assays and trimer bindingenzyme-linked immunosorbent assays (ELISAs) but as nanomolar con-centrations for isothermal titration calorimetry (ITC) experiments. Sincethe molecular mass of an average IgG molecule is approximately 150,000Da, the conversion factors for IgG are as follows: 1  g/ml  6.7 nM and1.0 nM  150 ng/ml.MAbs were obtained as gifts, or purchased, from the following sourc-es: John Mascola and Peter Kwong (VRC01 and F105), the InternationalAIDSVaccineInitiative(PGV04,PG9,PG16,PGT121,PGT126,PGT135,PGT145, b6, b12, and F240), Polymun Scientific (447-52D and 2G12),Michel Nussenzweig (8ANC195), James Robinson (39F, 17b, A32, 19b,14e, F91, and CO11), and Mark Connors (35O22). Neutralization assays.  To determine the extent of HIV-1 neutraliza-tion by MAbs, we used Env-pseudotyped viruses and a single-cycle infec-tion assay based on TZM-bl cells, as described previously  (38). All infec- tions were performed in duplicate. Uninfected cells were used to correctfor background luciferase activity. The infectivity of each mutant withoutinhibitor was set at 100%. Nonlinear regression curves were determinedand 50% inhibitory concentrations (IC 50 s) were calculated using a sig-moid function in Prism software version 5.0. Celia LaBranche and DavidMontefiori (Duke University Central Laboratory, Durham, NC) haveclassifiedtheB41(referredtoas9032-08.A1.4685)Env-pseudotypedvirusas having a tier 2 neutralization profile (46). D7324 capture ELISA.  ELISAs using 2G12/SEC-purified or PGT145/SEC-purified B41 SOSIP.664-D7324 trimers, or B41 gp120 monomers,were performed essentially as described previously (21, 38). The input trimer concentration was 300 ng/ml, and the corresponding gp120 con-centration (generally   900 ng/ml) was calibrated to yield similar bindingsignals for MAb 2G12. The sheep polyclonal Ab D7324 to a gp120 C5epitope used for coating the ELISA wells was obtained from Aalto Biore-agents, Dublin, Ireland.CorrelationsbetweentheneutralizationIC 50 andELISA50%effectiveconcentrations (EC 50 s) were investigated by nonparametric Spearman’srank correlation test with two-tailed  P   values for significance (Prism 6 forMac OSX; GraphPad). The  -level was set to 0.05. Differentialscanningcalorimetry(DSC). Thermaldenaturationwasprobed with a MicroCal VP-Capillary differential scanning calorimeter(Malvern Instruments) (38). Before the experiments were carried out, all samples were extensively dialyzed against phosphate-buffered saline(PBS). The protein concentration was subsequently adjusted to 0.1 to 0.3mg/ml, as described above. After loading of the protein sample into thecell, thermal denaturation was probed at a scan rate of 90°C/h. Buffercorrection, normalization, and baseline subtraction procedures were ap-plied before the data were analyzed using Origin 7.0 software. The datawere fitted using a non-two-state model, as the asymmetry of some of thepeaks suggested the presence of unfolding intermediates. ITC.  ITC binding experiments were carried out using a MicroCal Au-to-ITC200 instrument (GE Healthcare) and a protocol similar to onepreviously described elsewhere (50, 53). Briefly, Env proteins were dia- lyzed against Tris-saline buffer (150 mM NaCl, 20 mM Tris [pH 8.0])prior to conducting the titrations. The absorbance at 280 nm, derivedusing calculated extinction coefficients, served to determine and adjustthe input protein concentrations. The ligand present in the syringe wasPGT145 Fab, PGT151 Fab, or 19b IgG at a concentration between 10 and100   M, while the B41 SOSIP.664 trimer was present in the cell at aconcentration between 3 and 5   M. In each binding experiment, thereference power was 5  cal. For PGT151 and 19b binding experiments,the first injection of 0.5  l was followed by 15 injections of 2.5  l each, atintervals of 180 s. Because of the low binding enthalpy, PGT145 bindingexperimentswerecarriedoutwithafirstinjectionof0.5  l,followedby11injectionsof3.5  leach,atintervalsof180s.Origin7.0softwarewasusedto derive the stoichiometry of binding (  N  ), the affinity constant ( K  d  ) andthemolarreactionenthalpy(  H  ),byfittingtheintegratedtitrationpeaksvia a single-site binding model. (All measured and derived thermody-namic parameters of binding are reported in Table 2.) NS-EM.  B41 SOSIP.664 trimers were purified by either PGT145 or2G12 affinity chromatography, followed by SEC, and then prepared fornegative-stain electron microscopy (NS-EM) analysis as previously de-scribed for BG505 trimers (21, 38). Briefly, a 3-  l aliquot containing  0.01 mg/ml of trimer was applied for 5 s onto a carbon-coated 400 Cumesh grid that had been glow discharged at 20 mA for 30 s and then wasnegatively stained with 2% (wt/vol) uranyl formate for 60 s. Data werecollected using an FEI Tecnai T12 electron microscope operating at 120keV, with an electron dose of   25 electrons/Å 2 and a magnification of   52,000, which resulted in a pixel size of 2.05 Å at the specimen plane.Images were acquired with a Tietz TemCam-F416 CMOS camera using anominal defocus range of 900 to 1,300 nm. Image processing and 3D reconstruction.  Data processing methodswere adapted from those used previously  (38). Particles were picked au- tomatically using DoG Picker and put into a particle stack using the Ap-pion software package (54, 55). Initial, reference-free, two-dimensional (2D) class averages were calculated using particles binned by two via iter-ative multivariate statistical analysis (MSA)/multireference alignment(MRA) and sorted into classes (56). Particles corresponding to trimers were selected into a substack and binned by two before another round of reference-free alignment was carried out using the iterative MSA/MRAand Xmipp Clustering and 2D alignment software systems (57). The 2D class averages were visually inspected, and the classes weresegregated into one of three trimer structural groups designated “closed,”“open,” or “nonnative.” Trimers visually similar to those previously de-scribed for BG505 SOSIP.664 (38), namely, compact triangular propeller shapes with no additional density surrounding them, were classified as inthe closed conformation. Classes containing triangular propeller density  Pugach et al. 3382 March 2015 Volume 89 Number 6Journal of Virology   onM ar  c h  3  0  ,2  0 1  5  b  y K r  e s  g eL i   b r  ar  y  ,T h  e S  c r i   p p s R  e s  e ar  c h I  n s  t  i   t   u t   eh  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   of an intensity and size similar to the closed conformation group, butdisplayingone,two,orthreesmallerspheresofdensityatthedistalendsof the triangular density, were classified as in the open conformation (seeResults). We consider both these categories of trimer as having, overall, anative structure. In contrast, particles that did not clearly show a central,triangular mass, but instead resembled previously described images of uncleaved, non-SOSIP gp140 proteins (21), were classified as nonnativeforms. Any contaminating gp140 monomers, dimers, or noise particlesidentified were not included in further analysis. (Noise particles are in-consistencies in the carbon-coated nitrocelluose support and/or stainingpattern that are mistaken for protein by the automated particle pickingsoftware.) Such contaminants made up   1% of total particles, as SECpurification removed most gp140 monomers and dimers, and care wastakentoimageonlygridregionswithauniformstaindensity.Therelativeabundanceofeachsubpopulationwasthencalculatedasthepercentageof particlesbelongingtoaparticulargroupdividedbythetotalnumberofalltrimeric particles. To ensure that the presence or absence of additionaldensitywasnotstaindependent,threedatasetswerecollectedusingthreedistinct squares of variable stain thickness (thin, medium, and thick) onthe same grid. The calculated percentages for a specific subpopulationvaried by   5% between the three data sets (data not shown). In addition,a new substack was created from only the open and nonnative classes anda further round of reference-free alignment was carried out. The samequantities of open and nonnative particles were calculated from the re-sulting 2D class averages, confirming that the features are true and not aresult of misalignment. These quantities were consistent between the twoalignment software systems tested.To generate 3D NS-EM reconstructions, B41 SOSIP.664 trimers wereincubated with either VRC01 or b12 Fab fragments, in a 10-fold molarexcess compared to the trimer, at room temperature for 1 h. Grid prepa-rationanddatacollectionmethodsweresimilartothoseusedforunligan-ded trimers, with the addition of 10° tilt increments up to 50°. The tiltsprovided additional particle orientations to improve the image recon-structions.Fabswereclearlyvisualizedinthe2Dclassaveragesiftheywereboundto the trimer, allowing the percentage of Fab-bound versus unboundtrimers to be tabulated.  Ab initio  common lines models were calculatedfrom reference-free 2D class averages in EMAN2 (58) without imposing symmetry. One of those models was then refined against raw particles foran additional 30 cycles. EMAN (59) was used for all 3D reconstructions. The resolutions of the final models were determined using a Fourier shellcorrelation (FSC) cutoff of 0.5.The cryo-EM model of the BG505 SOSIP.664 trimer complex withPGV04 (PDB code: 3J5M) was fit manually into the EM densities andrefined by using the UCSF Chimera “Fit in map” function (60). Reconstruction data codes.  The reconstruction data reported in thispaper have been deposited in the Electron Microscopy Data Bank ( (EMDB ID code EMDB-6256). RESULTS BiochemicalandbiophysicalcharacterizationofB41SOSIP.664trimers.  The B41  env   gene, previously described as 9032-08.A1.4685, is derived from a subtype B founder virus (FiebigstageIII)isolatedfromaserialplasmadonor(46).Weintroduced the sequence changes necessary to create SOSIP.664 trimers (seeMaterials and Methods) and transiently transfected the modified env  geneinto293Tcells,cotransfectingthe  furin genetomaximizecleavageofgp120fromgp41 ECTO (4,5).Inpilotexperiments(data notshown),weusedacombinationofendpointstojudgethattheB41 SOSIP.664 construct had suitable properties for additionalstudies, specifically, high-level Env expression (SDS-PAGE gels),efficienttrimerformation(BN-PAGEgels),andahighpercentageof native-like trimers (NS-EM).For more detailed studies, the B41 SOSIP.664 construct wasexpressedtransientlyin293Tcellsunderstandardconditionsthatinvolved the use of culture media containing 10% FCS. The se-creted Env proteins were first affinity purified using the 2G12bNAb, followed by SEC via a Superdex 200 26/60 column to iso-late trimers (Fig. 1). A BN-PAGE analysis of the B41 SOSIP.664Env proteins eluted from the 2G12 column showed that  40%were trimers, while dimers, monomers, and aggregates were eachpresent at relative abundances of   20% (Fig. 1A). The SEC col-umn removed the unwanted Env forms, yielding pure (  95%)trimers(Fig.1BandC).Acomparativereducingversusnonreduc- ing SDS-PAGE gel analysis showed that the purified trimers werefully (  95%) cleaved into their gp120 and gp41 ECTO  subunits(data not shown). These results broadly mirror our experienceswith BG505 SOSIP.664 trimers (38). However, in contrast to our experiences with BG505, we observed some degradation productswhen the B41 SOSIP.664 trimers were analyzed by reducing SDS-PAGE followed by Coomassie blue staining or Western blotting(Fig. 1D). The anti-gp120 MAb ARP3119 detected a band of   70kDa that is characteristic of a degradation event arising when theV3 region of gp120 is proteolytically clipped at a scissile site nearthe tip of its  -hairpin loop (42–45). The other gp120 fragment generatedbyV3clipping,abandof   50kDa,isnotrecognizedby MAb ARP3119. This fragment and other less prominent bands(i.e., other degradation products) were detected when an HIV-Ig(subtype B) preparation was used to probe the blots (data notshown). Of note is that the extents of V3 clipping varied betweenB41SOSIP.664trimerpreparationsbutneverexceededmorethan  25%, as judged by the relative intensities of the 70-kDa and FIG 1  Biochemical characterization of B41 SOSIP.664 trimers. The  env   and  furin  genes were expressed in 293T cells in the presence of 10% FCS. (A) Env proteins purified on a 2G12 affinity column were analyzed on a Coomassieblue-stained BN-PAGE gel. The bands corresponding to aggregates, trimers,dimers, and monomers are indicated. The molecular mass marker proteinswere thyroglobulin (669 kDa) and ferritin (440 kDa). (B) The same Env pro-teins were analyzed by SEC on a Superdex 200 26/60 column. (C) Coomassieblue-stained BN-PAGE analysis of B41 SOSIP.664 trimers purified via 2G12/SEC columns. (D) BG505 and B41 SOSIP.664 trimers were produced in 293Tcells in the presence of 10% FCS, purified via 2G12/SEC columns, and thenanalyzed by reduced SDS-PAGE followed by Western blotting with MAbARP3119.The70-kDafragmentofgp120thatischaracteristicofV3clippingisindicated with an arrow. A Native-Like Subtype B SOSIP TrimerMarch 2015 Volume 89 Number 6  3383 Journal of Virology   onM ar  c h  3  0  ,2  0 1  5  b  y K r  e s  g eL i   b r  ar  y  ,T h  e S  c r i   p p s R  e s  e ar  c h I  n s  t  i   t   u t   eh  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   120-kDa bands in semiquantitative assessments. In contrast, mo-nomeric B41 gp120 proteins were much more extensively V3clipped (usually   50%) when produced and analyzed under thesameconditions(Fig.2C).Comparedtoothersubtypes,Envpro- teinsfromsubtypeBareknowntobegenerallyquitevulnerabletoV3 clipping, because a scissile site for thrombin family proteases( 2 ) is present in their consensus V3 sequence (GPGR  2 AF) thatis absent from the corresponding non-B consensus sequence (GPGQAF) (42–45). Hence, the subtype A BG505 SOSIP.664 trimersare not vulnerable to V3 clipping (Fig. 1D) (49). Production of non-V3-clipped B41 SOSIP.664 trimers inlow-serum medium.  To eliminate, or at least minimize, V3 clip-ping,wefirstexploreddifferentcultureconditionsforB41SOSIP.664 trimer production. An alternative approach, based on the useof a PGT145 affinity column to positively select nonclipped trim-ers, is described below. As noted above, V3 clipping is commonly mediated by tryptic proteases of the thrombin family. Prior stud-ieshaveshownthatthemostcommonsourceoftheseproteasesisthe FCS component of culture media (44, 45). While an alterna- tive source may be proteases released from cells that are damagedor killed during transient transfection, we noted that our initialclones of stably transfected CHO and 293T cells, cultured in 10%FCS, also yielded trimers that were  20% V3 clipped in the ab-sence of significant cell damage or death (data not shown). WethereforeproducedB41SOSIP.664proteinsbytransienttransfec-tion of 293F cells in serum-free medium. The resulting trimers,purified by 2G12/SEC, were fully intact with no evidence for V3clipping in a Western blot analysis, implying that FCS was indeedthe source of the proteolytic activity (Fig. 2A). When the non-clipped, 293F cell-derived trimers were treated with thrombin, asubpopulation (  20%) became clipped (Fig. 2A). Hence, 293F cells do not produce Env variants that are intrinsically resistant toV3 clipping, but rather it is their ability to be cultured in serum-free medium that yields nonclipped B41 gp120 monomers andSOSIP.664 trimers.To facilitate the production of B41 SOSIP.664 trimers on alargerscale,wemadestable293TandCHOlinesusingprocedurespreviously described for the corresponding BG505 trimers (49).We then attempted to adapt these lines to growth in media con-taining lower serum concentrations, and we succeeded with theCHO line (see Materials and Methods). The B41 SOSIP.664 trim-ers produced when this line was cultured in 1% serum were only minimally (  5%) V3 clipped (Fig. 2B). We expect that we willeventually be able to adapt the CHO line to serum-free cultureconditions, which should completely eliminate all V3 clipping. Neutralization of the parental B41 virus.  To gain an under-standingoftheantigenicityofB41Env,wefirstassessedtheability ofMAbsagainstmultipleepitopestoneutralizetheB41Env-pseu-dotyped virus in a TZM-bl cell-based assay. The Duke University Central Laboratory has independently classified this virus as hav-ing a tier 2 neutralization profile (46). The bNAbs tested targeted CD4bs (b12, VRC01, and PGV04) as well as the CD4-IgG2 pro-tein, the N332 glycan-dependent V3 cluster (PGT121 andPGT126), the N332 glycan-dependent outer domain cluster(PGT135 and 2G12), the quaternary structure-dependent V1V2epitopes(PG9,PG16,andPGT145),andthegp120-gp41interface(PGT151,35O22,and8ANC195).bNAbstothemembrane-prox-imal external region (MPER) were not included in the test panel,as this region is not present in the B41 SOSIP.664 construct. Withthe exception of 35O22, all tested bNAbs neutralized the B41 vi-rus, although with a wide range of potencies (Table 1).Several MAbs that are generally found to be nonneutralizingfortier2virusesdidnotneutralizeB41(IC 50  50  g/ml),includ-ing b6, F91, and F105 to CD4bs; 17b to a CD4-induced epitope;447-52D, 39F, CO11, 19b, and 14e to V3; and F240 to gp41 ECTO (Table 1). We confirmed that their epitopes were present on atleast one form of B41 Env protein (e.g., gp120 monomers orgp41 ECTO ), showing that their inability to neutralize the virus wasnot due to a sequence-dependent lack of the epitope (data notshown, but see below). AntigenicityofB41SOSIP.664trimersbyELISA. For antige-nicity studies by ELISA, we used D7324-tagged trimers and, forcomparison, gp120 monomers. Both proteins were produced in293F cells in the absence of serum and purified by 2G12 affinity chromatography and SEC. Thus, they were not significantly V3clipped. The MAbs tested are listed in Table 1. The amounts of  gp120 monomers and SOSIP.664-D7324 trimers captured ontotheELISAwellswerecalibratedtoyieldcomparable2G12bindingcurvesthatservetonormalizethedataderivedusingtheothertestMAbs (Fig. 3A).bNAbs such as PG9, PG16, and PGT145 against quaternary structure-dependentorinfluencedepitopesarevaluableforgaug-ing whether soluble trimers adopt an appropriate conformation.The relevant epitopes span the V1V2 domains of two gp120 FIG2  PreventingproteolyticclippingofB41SOSIP.664trimers.(A)Thetrimerswereproducedin293Fcellsinserum-freemediumandtreatedwiththrombin(100  g/ml)forthespecifiedtimesbeforeanalysisbyreducingSDS-PAGEandWesternblottingwithMAbARP3119.(B)B41SOSIP.664trimerswereproducedeither transiently in 293T cells in the presence of 10% FCS, in stable CHO cell lines in the presence of 1% FCS, or transiently in 293F cells under serum-freeconditions. Trimers were purified via the 2G12 affinity column and SEC and then analyzed by SDS-PAGE and Western blotting with MAb ARP3119. (C) B41gp120 monomers or SOSIP.664 trimers were produced in 293T cells in the presence of 10% serum, purified via either a 2G12 or a PGT145 affinity column, andanalyzed by reducing SDS-PAGE and Western blotting with MAb ARP3119. The leftmost lane shows molecular mass markers. In all three panels, the 70-kDafragment of gp120 that is characteristic of V3 clipping is indicated with an arrow. Pugach et al. 3384 March 2015 Volume 89 Number 6Journal of Virology   onM ar  c h  3  0  ,2  0 1  5  b  y K r  e s  g eL i   b r  ar  y  ,T h  e S  c r i   p p s R  e s  e ar  c h I  n s  t  i   t   u t   eh  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om 
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