Alf-well microplates by incubating the plates at 4uC overnight. Following blocking the wells with 2.5 skim milk in PBS (MPBS), 3-fold serially diluted whole cell lysate or pseudovirus-containing culture supernatant in lysis buffer (2 Triton X-100 from Sigma in PBS) were added to the ELISA plates. Captured JRFL gp160s were detected by using IgG1 2G12 as a primary antibody and HRP conjugated to anti-human F(ab’)2 (1: 2,500) as a secondary antibody, and ABTS (Roche) as substrate. The optical density at 405 nm (OD405nm) was determined after color development at RT for 20 min. 10457188 A similar Title Loaded From File capture ELISA set-up was used to determine structural integrity of loop deleted or replaced JRFL gp160 by measuring the binding of a panel of HIV-1-specific mAbs to each loop deleted or replaced Env mutant and WT captured on microplates. HIV-1-specific mAbs include CD4bs mAbs VRC01 and b12, CD4-induced (CD4i) mAbs X5 and 17b, and gp41-specific mAbs m47 (Ntrimer-specific, unpublished), 2F5 and 4E10. Glycan-specific mAb 2G12 was included in the assay to measure each loop deleted or replaced Env mutant expressed in 293 T cells. A standard curve used for data calibration was generated using recombinant JRFLgp120 as antigen and 2G12 as a primary antibody in the same capture ELISA.Transient Transfection of 293 T Cells and Flow CytometryThe day before transfection, 293 T cells were seeded in DMEM growth medium (DMEM containing 10 FBS and 1 pen-strep). 70?0 confluent 293 T cells were co-transfected with pSVIII expression plasmid containing JRFL wild type (WT) gene or its loop deletion or replacement mutants, and pcTAT using PEI as a transfection reagent in DMEM medium containing 10 FBS. 4?6 h post transfection, the medium was changed to DMEM growthImmunohistochemistry (IHC) Assay48 h post transfection, 293 T cells were fixed using 4 paraformaldehyde fixative solution by incubation at RT forImportance of HIV-1 Env Variable GHRH (1-29) chemical information LoopsTable 1. JRFL gp160 loop deletion or replacement mutants.JRFL Env loops V1V2 V2 V2 crown loop D V3 V3 crown CD4bl V4 VOriginal loop sequenceReplacing linker sequence and designated name of the constructVNATNTTNDSEGTMERGEIKNCSFNITTSIRDEVQKEYALFYKLDVVPIDNNNTSYRL GSGSG (DV1V2) NITTSIRDEVQKEYALFYKLDVVPIDNNNTSYRL FYKLD NFTNNAKT NTRKSIHIGPGRAFYTTGEIIG GPGR SSGGDPEIVMH NSTQLFNSTWNNNTEGSNNTEGNTITLP INENGTEIFR GSGSG (DV2) AAAAA (DV2C) AAAA (DlpD) GSGSG (DV3) AAAA (DV3C) GSGSG (DCD4bl) GSGSG (DV4); or GGGGSGGGGSGGGGSGSGSG (DV4fl) GSGSG (DV5); or GGGGSGSGSG (DV5fl)The amino acid (AA) sequences of the original loops and flexible linkers for replacement are shown. V2 and V3 crown sequences are underlined. CD4bl: CD4 binding loop. DV4fl and DV5fl: V4 and V5 loops replaced with flexible linkers of the same lengths. Designated names of resultant constructs are indicted in parentheses. doi:10.1371/journal.pone.0069789.tTable 2. Primers used to construct JRFL gp160 loop deletion mutants.Primer Name pSV3for pSV3rev delLpDF delLpDR Delv1v2F Delv1v2R V2crownF V2crownR delfullV2F delfullV2R V3crownF V3crownR Delfullv3F Delfullv3R V4delFnew V4delRnew V5delFnew V5delRnew DelCD4blF DelCD4blR V4FFL V4RFL V5FFL V5RFL JRFLCTfor JRFLCTrevPrimer Sequence (59 to 39) ACCATGCTCCTTGGGATGTTGATG TCTCAAGCGGTGGTAGCTGAAGAG GACGCTGCAGCAGCTATAATAGTACAGCTGAAAGAATC AGCTGCTGCAGCGTCAGATCTAATTACTACCTC GGTAGCGGATCAGGTATAAGTTGTGACACCTCAGTC ACCTGATCCGCTACCATCCTTGCAATTTAAAGTAAC GCTGCTGCAGCTGCTGTAGTACCAATAGATAATAATAATACC AGCAGCTGCAGCAGCAAGAGCATATTCTTTCTGCAC GGTTCAGGATCTGGCATAAGTTGTGACACC.Alf-well microplates by incubating the plates at 4uC overnight. Following blocking the wells with 2.5 skim milk in PBS (MPBS), 3-fold serially diluted whole cell lysate or pseudovirus-containing culture supernatant in lysis buffer (2 Triton X-100 from Sigma in PBS) were added to the ELISA plates. Captured JRFL gp160s were detected by using IgG1 2G12 as a primary antibody and HRP conjugated to anti-human F(ab’)2 (1: 2,500) as a secondary antibody, and ABTS (Roche) as substrate. The optical density at 405 nm (OD405nm) was determined after color development at RT for 20 min. 10457188 A similar capture ELISA set-up was used to determine structural integrity of loop deleted or replaced JRFL gp160 by measuring the binding of a panel of HIV-1-specific mAbs to each loop deleted or replaced Env mutant and WT captured on microplates. HIV-1-specific mAbs include CD4bs mAbs VRC01 and b12, CD4-induced (CD4i) mAbs X5 and 17b, and gp41-specific mAbs m47 (Ntrimer-specific, unpublished), 2F5 and 4E10. Glycan-specific mAb 2G12 was included in the assay to measure each loop deleted or replaced Env mutant expressed in 293 T cells. A standard curve used for data calibration was generated using recombinant JRFLgp120 as antigen and 2G12 as a primary antibody in the same capture ELISA.Transient Transfection of 293 T Cells and Flow CytometryThe day before transfection, 293 T cells were seeded in DMEM growth medium (DMEM containing 10 FBS and 1 pen-strep). 70?0 confluent 293 T cells were co-transfected with pSVIII expression plasmid containing JRFL wild type (WT) gene or its loop deletion or replacement mutants, and pcTAT using PEI as a transfection reagent in DMEM medium containing 10 FBS. 4?6 h post transfection, the medium was changed to DMEM growthImmunohistochemistry (IHC) Assay48 h post transfection, 293 T cells were fixed using 4 paraformaldehyde fixative solution by incubation at RT forImportance of HIV-1 Env Variable LoopsTable 1. JRFL gp160 loop deletion or replacement mutants.JRFL Env loops V1V2 V2 V2 crown loop D V3 V3 crown CD4bl V4 VOriginal loop sequenceReplacing linker sequence and designated name of the constructVNATNTTNDSEGTMERGEIKNCSFNITTSIRDEVQKEYALFYKLDVVPIDNNNTSYRL GSGSG (DV1V2) NITTSIRDEVQKEYALFYKLDVVPIDNNNTSYRL FYKLD NFTNNAKT NTRKSIHIGPGRAFYTTGEIIG GPGR SSGGDPEIVMH NSTQLFNSTWNNNTEGSNNTEGNTITLP INENGTEIFR GSGSG (DV2) AAAAA (DV2C) AAAA (DlpD) GSGSG (DV3) AAAA (DV3C) GSGSG (DCD4bl) GSGSG (DV4); or GGGGSGGGGSGGGGSGSGSG (DV4fl) GSGSG (DV5); or GGGGSGSGSG (DV5fl)The amino acid (AA) sequences of the original loops and flexible linkers for replacement are shown. V2 and V3 crown sequences are underlined. CD4bl: CD4 binding loop. DV4fl and DV5fl: V4 and V5 loops replaced with flexible linkers of the same lengths. Designated names of resultant constructs are indicted in parentheses. doi:10.1371/journal.pone.0069789.tTable 2. Primers used to construct JRFL gp160 loop deletion mutants.Primer Name pSV3for pSV3rev delLpDF delLpDR Delv1v2F Delv1v2R V2crownF V2crownR delfullV2F delfullV2R V3crownF V3crownR Delfullv3F Delfullv3R V4delFnew V4delRnew V5delFnew V5delRnew DelCD4blF DelCD4blR V4FFL V4RFL V5FFL V5RFL JRFLCTfor JRFLCTrevPrimer Sequence (59 to 39) ACCATGCTCCTTGGGATGTTGATG TCTCAAGCGGTGGTAGCTGAAGAG GACGCTGCAGCAGCTATAATAGTACAGCTGAAAGAATC AGCTGCTGCAGCGTCAGATCTAATTACTACCTC GGTAGCGGATCAGGTATAAGTTGTGACACCTCAGTC ACCTGATCCGCTACCATCCTTGCAATTTAAAGTAAC GCTGCTGCAGCTGCTGTAGTACCAATAGATAATAATAATACC AGCAGCTGCAGCAGCAAGAGCATATTCTTTCTGCAC GGTTCAGGATCTGGCATAAGTTGTGACACC.
