Supplementary Materials and Methods




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Supplementary Materials and Methods


Retroviral vectors. All gammaretroviral vectors encoding secretable C peptides were derivatives of MP91 41. This vector contains LTRs from the myeloproliferative sarcoma virus (MPSV) and a modified untranslated leader derived from murine embryonal stem cell virus (MESV). The leader is devoid of gag-sequences and potential start codons (ATG) prior to the transgene, but contains an additional viral splice donor (SD) and a splice acceptor (SA). An elongated multiple cloning site (MCS) was introduced into the NotI site of MP91, and a woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) was inserted 3’ of the transgene, resulting in the vector M377. The transgenes encoding secretable HIV 1 fusion inhibitory C peptides were integrated into the multiple cloning site via NotI and SalI. The transgene cassettes were assembled in multi-step cloning procedures as described below. All site-directed mutagenesis reactions were performed according to the QuikChange Site-directed Mutagenesis Kit protocol from Stratagene (Heidelberg, Germany). Oligonucleotides were from Eurofins MWG Operon (Ebersberg, Germany). All oligonucleotide sequences are given in 5’ 3’ orientation. All final constructs were verified by DNA sequencing.

Modules of C46 transgene cassettes:



Name

Description

Amino acid sequence

SPtPA

Signal peptide from human tissue-type plasminogen activator (aa 1 23, accession no. P00750.1)

MDAMKRGLCCVLLLCGAVFVSPS

C46

HIV fusion inhibitory peptide derived from the HR2 of gp41 (aa 628 673 of HIV 1HxB2 env)

WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

L

Loop N terminally of the HR2 of gp41 (aa 605 627 of HIV 1HxB2 env)

TTAVPWNASWSNKSLEQIWNHTT

H

Hinge / membrane proximal region C terminally of the HR2 of gp41 (aa 674 685 of HIV 1HxB2 env)

NITNWLWYIKLF

M

c myc tag

EQKLISEEDL

GAr

Flexible glycine-alanine linker

GAGGAGGAGAGGAGAGAG

2xGAr

Flexible glycine-alanine linker

GAGGAGGAGAGGAGAGAGELGAGGAGGAGAGGAGAGAG

(G4S)4

Flexible glycine-serine linker

GGGGSGGGGSGGGGSGGGGS

IgC

Linker / hinge from human IgG2 (aa 99 117; accession no. P01859.2)

ERKCCVECPPCPAPPVAGP

IgS

Mutated IgG2 linker

ERKSSVESPPSPAPPVAGP

Fur

Minimal furin cleavage site

RAKRD

Furm

Mutated furin cleavage site

RAKRV

Furo

Optimized furin cleavage site

RSRAKRSV

GA

Flexible glycine-alanine linker

GGAGAGAG

GS

Flexible glycine-serine linker

GSGGGGSGGGGSG



C46 (internal number M851): The construct C46 was generated from C46-H-GAr described below by PCR. Using primers M699-out-for (GCC CCC GTC TGA ATT TTT GC) and C46-StopSalR (TCA TCA GTC GAC TCA TCA GAA CCA GTT CCA CAG GCT GG) a DNA fragment comprising just the signal peptide and the C46 peptide was amplified, and a stop codon was added at the 3’ end of the sequence. The PCR product was digested with NotI and SalI and ligated into the M377 vector backbone, resulting in construct C46-H-GAr.
C46-H-GAr (M788): The construct C46-H-GAr was generated from construct L-C46-H-GAr described below by removal of the loop (L) sequence using 3 step PCR. In a first PCR with primers M699-out-for (GCC CCC GTC TGA ATT TTT GC) and SPtPA-C46-rev (CGC GGT CCC ACT CCA TCC AGC TGG GCG AAA CGA AGA CTG C) a fragment of the vector backbone 5’ to the transgene as well as the SPtPA sequence was amplified. The primer SPtPA-C46-rev at the same time added several nucleotides overlapping with the C46 sequence to the 3’ end of the PCR product. In a second PCR reaction the a DNA fragment comprising the C46, H, GAr as well the c myc tag sequence was amplified from L-C46-H-GAr using oligonucleotides C46for (TGG ATG GAG TGG GAC CGC G) and M699-out-rev (CGA ATT CGA TCG ATG ACC GG) as primers. In a third PCR step, the two PCR products created in the two previous PCR reactions were combined and amplified using primers M699-out-for and M699-out-rev. This final PCR product was digested with NotI and SalI and ligated into the M377 vector backbone, resulting in construct C46-H-GAr.
L-C46-H-GAr (M699): For the construct L-C46-H-Gar, initially, the fragment L-C46-H was assembled by multi-step PCR and cloned into the pBluescript plasmid (Stratagene, Heidelberg, Germany) as follows: the loop sequence (L) was generated by annealing and subsequent single-cycle PCR using Pfx DNA polymerase (Stratagene, Heidelberg, Germany) with the overlapping oligonucleotides LoopFor (TAG GGA TCC GCG ATC GCA CCA CTG CCG TGC CCT GGA ACG CCA GCT GGA GCA ACA AGA) and LoopRev (GGT GGT GTG GTT CCA GAT CTG CTC CAG GCT CTT GTT GCT CCA GCT GGC G), thereby generating a BamHI and an AsiSI site at the 5’ end of the loop sequence. Secondly, the segment C46-H was amplified from the plasmid M87o-HIV-Ineo 44 by PCR using the oligonucleotides C46ForL (GCA GAT CTG GAA CCA CAC CAC CTG GAT GGA GTG GGA CCG CGA GAT C) and HIVlinkRev (CTA TTA CTG CAG GAA TTC GAA CAG CTT GAT GTA CCA CAG C). The C46ForL primer was designed in a way that its sequence overlapped with the Loop sequence, whereas the HIVlinkRev primer introduced an EcoRI and a PstI site, as well as two Stop codons at the 3’ end of the C46-H sequence. In a third step, the Loop fragment and the C46-H fragment were assembled by PCR using the LoopFor and HIVlinkRev oligonucleotides as primers. This PCR product (L-C46-H) was digested with BamHI, and ligated into the multiple cloning site of pBluescript via BamHI and EcoRV (blunt). The signal peptide from human tissue-type plasminogen activator was generated by annealing and subsequent Pfx DNA polymerase fill-up of the overlapping oligonucleotides SPtPAFor (TAG GGA TCC ATG GAT GCA ATG AAG AGA GGG CTC TGC TGT GTG CTG CTG CTG TGT GG) and SPtPARev (GTC TGC GCG ATC GCG GCT GGG CGA AAC GAA GAC TGC TCC ACA CAG CAG CAG CAC AC), introducing a 5’ BamHI and a 3’ AsiSI site into the signal peptide fragment. Using the BamHI and AsiSI sites the signal peptide was introduced in the 5’ region of the L-C46-H sequence in the pBluescript plasmid described above, resulting in construct pBlue-S-L-C46-H. A glycine-alanine repeat (GAr) was introduced into this plasmid using the annealing product of the oligonucleotides GAfor (AAT TAG GAG CCG GGG GTG CGG GTG GCG CGG GTG CGG GGG GTG CTG GGG CTG GGG CCG GAG) and GArev (AAT TCT CCG GCC CCA GCC CCA GCA CCC CCC GCA CCC GCG CCA CCC GCA CCC CCG GCT CCT). Annealing of these oligonucleotides created sticky ends corresponding to ends that result from EcoRI digestion. The oligonucleotides were designed in a way that after ligation of the annealing product into the EcoRI site of pBlue-S-L-C46-H only the 3’ EcoRI site was maintained, whereas the 5’ EcoRI site was disrupted (resulting in construct pBlue-S-L-C46-H-GAr). The remaining EcoRI site was used to incorporate a c myc tag (EQKLISEEDL), which was generated by annealing of oligonucleotides MycFor2 (AAT TAG AAC AGA AAT TAA TTT CAG AAG AGG ACT TAG) and MycRev2 (AAT TCT AAG TCC TCT TCT GAA ATT AAT TTC TGT TCT). The transgene cassette comprising the signal peptide, loop, C46, HIV-linker, GAr sequence and c myc tag was cloned into the M377 vector using NotI and SalI resulting in construct L-C46-H-GAr.
L-C46-H (M683): The construct L-C46-H-no linker is based on the plasmid pBlue-S-L-C46-H, described above. A c myc tag was introduced into the PstI site of M23-S-L-C46-H using the annealing product of oligonucleotides MycFor (AGA ACA GAA ATT AAT TTC AGA AGA GGA CTT GCT GCA) and MycRev (GCA AGT CCT CTT CTG AAA TTA ATT TCT GTT CTT GCA). The transgene cassette comprising the signal peptide, loop, C46, HIV-linker and c myc tag was cloned into the M377 vector using NotI and SalI, resulting in construct L-C46-H.
L-C46-H-2xGAr (M767): The construct L-C46-H-2xGAr was generated by introducing a second GAr into the construct pBlue-S-L-C46-H-GAr, described above. For this purpose, the annealing product of the oligonucleotides GAfor and GArev was ligated into the EcoRI site of pBlue-S-L-C46-H-Gar. Then the c myc tag generated from MycFor2 and MycRev2 oligonucleotides was incorporated into the EcoRI site as described above. The transgene cassette comprising the signal peptide, loop, C46, HIV-linker, 2xGAr and c myc tag was cloned into the M377 vector using NotI and SalI resulting in construct L-C46-H-2xGAr.
L-C46-H-(G4S)4 (M770): The construct L-C46-H-(G4S)4 was generated similarly to L-C46-H-GAr, but instead of the GAr a (G4S)4 linker was introduced into the intermediate construct pBlue-S-L-C46-H. For this, the annealing product of the oligonucleotides G4S4-Linker-for (AAT TAG GAG GAG GTG GGT CTG GAG GTG GAG GAT CTG GTG GAG GTG GGT CTG GAG GAG GTG GGT CTG) and G4S4-Linker-rev (AAT TCA GAC CCA CCT CCT CCA GAC CCA CCT CCA CCA GAT CCT CCA CCT CCA GAC CCA CCT CCT CCT) was ligated into the EcoRI site of pBlue-S-L-C46-H. The c myc tag generated from oligonucleotides MycFor2 and MycRev2 was then incorporated as described above, and the transgene cassette cloned into the M377 vector using NotI and SalI resulting in construct L-C46-H-(G4S)4.
L-C46-H-IgC (M769): The construct L-C46-H-IgC was generated similarly to L-C46-H-GAr, but instead of the GAr a linker from human IgG2 was introduced into the intermediate construct pBlue-S-L-C46-H. For this, the annealing product of the oligonucleotides IgG2-Linker-for (AAT TAG AGC GCA AGT GCT GCG TGG AGT GCC CTC CCT GCC CCG CCC CTC CCG TGG CCG GCC CCG) and IgG2-Linker-rev (AAT TCG GGG CCG GCC ACG GGA GGG GCG GGG CAG GGA GGG CAC TCC ACG CAG CAC TTG CGC TCT) was ligated into the EcoRI site of pBlue-S-L-C46-H. The c myc tag generated from oligonucleotides MycFor2 and MycRev2 was then incorporated as described above, and the transgene cassette cloned into the M377 vector using NotI and SalI resulting in construct L-C46-H-IgC.
L-C46-H-IgS (M784): The construct L-C46-H-IgS was generated similarly to L-C46-H-GAr, but instead of the GAr a linker from human IgG2 with all cysteine residues mutated to serines was introduced into the intermediate construct pBlue-S-L-C46-H. For this, the annealing product of the oligonucleotides IgG2-Linkermut-for (AAT TAG AGC GCA AGA GCA GCG TGG AGA GCC CTC CCA GCC CCG CCC CTC CCG TGG CCG GCC CCG) and IgG2-Linkermut-rev (AAT TCG GGG CCG GCC ACG GGA GGG GCG GGG CTG GGA GGG CTC TCC ACG CTG CTC TTG CGC TCT) was ligated into the EcoRI site of pBlue-S-L-C46-H. The c myc tag generated from oligonucleotides MycFor2 and MycRev2 was then incorporated as described above, and the transgene cassette cloned into the M377 vector using NotI and SalI resulting in construct L-C46-H-IgS.
L-C46-H-GAr glycosylation site mutants L1mut (M771), L2mut (M768), L3mut (M746), C46mut (M747), Hmut (M748), L1-2mut (M734), 5xmut (M766): N glycosylation sites in L-C46-H-GAr were mutated by site-directed mutagenesis using primer pairs of sense and antisense oligonucleotides and introducing alanine (A) residues instead of the serine (S) or threonine (T) residues of the respective N glycosylation site recognition sequences. The following oligonucleotides were used: QCGlycoForI-neu (sense; GCC CTG GAA CGC CGC CTG GAG CAA CAA GAG C) and QCGlycoRevI-neu (antisense; GCT CTT GTT GCT CCA GGC GGC GTT CCA GGG C) for L1mut (S35A); QCGlycoForII (sense; CAG CTG GAG CAA CAA GGC TCT GGA GCA GAT CTG GAA CC) and QCGlycoRevII (antisense; GGT TCC AGA TCT GCT CCA GAG CCT TGT TGC TCC AGC TG) for L2mut (S40A); QCGlycoForIII (sense; GCA GAT CTG GAA CCA CGC CAC CTG GAT GGA GT) and QCGlycoRevIII (antisense; ACT CCA TCC AGG TGG CGT GGT TCC AGA TCT GC) for L3mut (T48A); C46mutFor (sense; AGA TCA ACA ACT ACG CCA GCC TGA TCC ACA GCC TG) and C46mutRev (antisense; CAG GCT GTG GAT CAG GCT GGC GTA GTT GTT GAT CT) for C46mut (T61A); HingemutFor (sense; GGT TCA GAT CTA ACA TCG CCA ATT GGC TGT GGT AC) and HingemutRev (antisense; GTA CCA CAG CCA ATT GGC GAT GTT AGA TCT GAA CC) for Hmut (T100A); QCGlycoFor (sense; CGT GCC CTG GAA CGC CGC TTG GAG CAA CAA GGC TCT GGA GCA GAT CTG GAA CC) and QCGlycoRev (antisense; GGT TCC AGA TCT GCT CCA GAG CCT TGT TGC TCC AAG CGG CGT TCC AGG GCA CG) for L1-2mut (S35A/S40A). The construct 5xmut (S35A/S40A/T48A/T61A/T100A) was generated by multi-site-directed mutagenesis with oligonucleotides QCGlycoForIII, C46mutFor and HingemutFor, and using construct L1-2mut as a basis.
Fur (M818): The construct Fur was assembled by multiple PCR steps. First, a fragment comprising the signal peptide and C46 was amplified from construct C46-H-GAr using primers M699-out-for and C46-RRLL-rev (CTA CTA GAT ATC CAG CAG GCG GCG GAA CCA GTT CCA CAG GCT GG), thereby adding codons for the amino acids RRLL, an EcoRV restriction site, as well as two Stop codons to the 3’ end of the second C46 sequence. The resulting PCR product was digested with NotI and EcoRV and ligated into the pBluescript plasmid via NotI and EcoRV, generating plasmid pBlue-S-C46. In a second PCR step, the C46 peptide sequence was amplified from construct C46-H-GAr using primers C46for and C46-RRLL-rev (CTA CTA GAT ATC CAG CAG GCG GCG GAA CCA GTT CCA CAG GCT GG). The PCR product was ligated into the EcoRV site of pBlue-S-C46, and then the whole transgene cassette was cloned into the M377 vector via NotI and SalI, generating construct M377-S-C46-C46. The furin cleavage site Fur between the two C46 units was generated by site-directed mutagenesis using the primers C46-Furin-for (CCT GTG GAA CTG GTT CCG CGC CAA GAG GGA TTG GAT GGA GTG G) and C46-Furin-rev (CCA CTC CAT CCA ATC CCT CTT GGC GCG GAA CCA GTT CCA CAG G), resulting in the construct Fur.

Furo (M869): The construct Furo that comprising an optimized furin cleavage site (Furo) was generated from the construct Fur, containing only the basic cleavage site, by site-directed mutagenesis. First, the furin cleavage site Fur was C terminally elongated by amino acids SV, using the primers 818-RAKRSV-F (sense; CTG GTT CCG CGC CAA GAG GAG CGT GTG GAT GGA GTG GGA CCG CG) and 818-RAKRSV-R (antisense; CGC GGT CCC ACT CCA TCC ACA CGC TCC TCT TGG CGC GGA ACC AG). Then, the amino acids RS were added at the N terminus of the cleavage site using primers 818-RSRAKR-F (sense; CCA GCC TGT GGA ACT GGT TCA GGA GCC GCG CCA AGA GGA GCG TGT G) and 818-RSRAKR-R (antisense; CAC ACG CTC CTC TTG GCG CGG CTC CTG AAC CAG TTC CAC AGG CTG G). Furthermore, a new Stop-codon was introduced right 3’ to the C terminal C46 sequence by PCR using primers M699-out-for and C46-StopSalR. The PCR product was then ligated into the M377 vector via NotI and SalI, generating construct Furo.
FuroGA (M902): The construct FuroGA was cloned by 3 step PCR using Furo as template. In the first PCR a DNA fragment was amplified from Furo using primers M699-out-for and Furopt-GA8_R (TCC AGC GCC GGC ACC TGC ACC TCC CAC GCT CCT CTT GGC GCG GC). Thereby, the sequence for the eight amino acid GA repeat was added to the 3’ end of the Furo cleavage site. In a second PCR the with the Furo construct as template a DNA fragment was generated with the sequence of the GA repeat added to the 5’ end of the C terminal C46 unit using primers GA8-C46_F (GGA GGT GCA GGT GCC GGC GCT GGA TGG ATG GAG TGG GAC CGC G) and M699-out-rev. In a third PCR reaction the two DNA fragments were assembled taking advantage of the overlapping GA repeat sequences, and using primers M699-out-for and M699-out-rev. The final PCR product was ligated into the M377 vector using NotI and SalI, resulting in construct FuroGA.
GAFuro (M903): The construct GAFuro was cloned by 3 step PCR using Furo as a template. In the first PCR, a DNA fragment was amplified from Furo using primers M699-out-for and C46-GA8_R (TCC AGC GCC GGC ACC TGC ACC TCC GAA CCA GTT CCA CAG GCT GG). Thereby, the sequence encoding the eight amino acid GA repeat was added to the 3’ end of the N terminal C46 unit. In a second PCR a DNA fragment was generated with the sequence of the GA repeat added to the 5’ end of the Furo site using primers GA8-Furopt_F (GGA GGT GCA GGT GCC GGC GCT GGA AGG AGC CGC GCC AAG AGG AGC) and M699-out-rev with the Furo construct as template. In a third PCR reaction, the two DNA fragments were assembled taking advantage of the overlapping GA repeat sequences, and using primers M699-out-for and M699-out-rev. The final PCR product was ligated into the M377 vector using NotI and SalI, resulting in construct GAFuro.
GAFuroGA (M918): The construct GAFuroGA was cloned as described for construct FuroGA but using GAFuro as template for the PCR reactions.

Furm (M845): To generate Furm, the furin cleavage site RAKR  D in construct Fur was mutated to RAKV  D and thus destroyed. Site-directed mutagenesis was performed using the primers 818-FurinMutF (sense; GGT TCC GCG CCA AGG TGG ATT GGA TGG AGT GG) and 818-FurinMutR (antisense; CCA CTC CAT CCA ATC CAC CTT GGC GCG GAA CC).
GS (M863): The construct GS was cloned based on construct Furo. By site-directed mutagenesis using primers C46-BspEI-C46-F (sense; CCA GCC TGT GGA ACT GGT TCT CCG GAT GGA TGG AGT GGG ACC GCG) and C46-BspEI-C46-R (antisense; CGC GGT CCC ACT CCA TCC ATC CGG AGA ACC AGT TCC ACA GGC TGG) the Furo site was first replaced by a BspEI restriction site giving rise to construct M859. The GS linker was then incorporated into the newly established BspEI site using the annealing product of oligonucleotides G4S2-BspEI-F (sense; CCG GTG GAG GTG GAT CTG GAG GTG GAG GAT) and G4S2-BspEI-R (antisense; CCG GAT CCT CCA CCT CCA GAT CCA CCT CCA), which had sticky ends corresponding to ends that result from BspEI digestion, resulting in construct GS.
FuroGA N-glycosylation site mutants N33Q (M974), N95Q (M967) and N33Q/N95Q (M968): To generate the N glycosylation site mutants N33Q, N95Q, and N33Q/N95Q, site-directed mutagenesis was performed using plasmid FuroGA as a basis. The following oligonucleotides were used: OGSH11 (sense; ACC GCG AGA TCA ACC AGT ACA CCA GCC TGA T) and OGSH12 (antisense; ATC AGG CTG GTG TAC TGG TTG ATC TCG CGG T) for N33Q, OGSH7 (sense; ACC GCG AGA TTA ACC AGT ACA CCA GCC TGA T) and OGSH8 (antisense; ATC AGG CTG GTG TAC TGG TTA ATC TCG CGG T) for N95Q, and both oligonucleotide pairs for the double mutant N33Q/N95Q.
M945, M947, 987, M989, M991, M992, M993 and M994: To generate expression constructs with alternative linker sequences between the two C46 units (see also Supplemental Fig. S2), complementary oligonucleotides were annealed, phosphorylated, and cloned into the BspEI site of plasmid M859 (described above; precursor of construct “GS”). The following oligonucleotides were used: OGSH1 (sense; CCG GAA GGG GTG TGT TTC GTC GAG ATG CAT) and OGSH2 (antisense; CCG GAT GCA TCT CGA CGA AAC ACA CCC CTT) for M945, OGSH9 (sense; CCG GAG GTG CAG GTG CCG GCG CTG GAA GGG GTG TGT TTC GTC GAG ATG CAG GAG GTG CAG GTG CCG GCG) and OGSH10 (antisense; CCG GCG CCG GCA CCT GCA CCT CCT GCA TCT CGA CGA AAC ACA CCC CTT CCA GCG CCG GCA CCT GCA CCT) for M947, OGSH19 (sense; CCG GAG GTG CAG GTG CCG GCG CTG GAA GCC CTC TGG CCC AGG CAG TCA GAT CAT CTT CTC GAG GAG GTG CAG GTG CCG GCG) and OGSH20 (antisense; CCG GCG CCG GCA CCT GCA CCT CCT CGA GAA GAT GAT CTG ACT GCC TGG GCC AGA GGG CTT CCA GCG CCG GCA CCT GCA CCT) for M987, OGSH27 (sense; CCG GAA GCC CTC TGG CCC AGG CAG TCA GAT CAT CTT CTC GAG) and OGSH28 (antisense; CCG GCT CGA GAA GAT GAT CTG ACT GCC TGG GCC AGA GGG CTT) for M989, OGSH31 (sense; CCG GAG GTG CAG GTG CCG GCG CTG GAT CAA GAG GCT TGA CAT CAT TGG CTG ACA CTG GAG GTG CAG GTG CCG GCG) and OGSH32 (antisense; CCG GCG CCG GCA CCT GCA CCT CCA GTG TCA GCC AAT GAT GTC AAG CCT CTT GAT CCA GCG CCG GCA CCT GCA CCT) for M991, OGSH33 (sense; CCG GAT CAA GAG GCT TGA CAT CAT TGG CTG ACA CTG) and OGSH34 (antisense; CCG GCA GTG TCA GCC AAT GAT GTC AAG CCT CTT GAT) for M992, OGSH35 (sense; CCG GAG GTG CAG GTG CCG GCG CTG GAT CTG GCC GCA GTG TCC TGT CAT TCG AGT CAG GTG GAG GTG CAG GTG CCG GCG) and OGSH36 (antisense; CCG GCG CCG GCA CCT GCA CCT CCA CCT GAC TCG AAT GAC AGG ACA CTG CGG CCA GAT CCA GCG CCG GCA CCT GCA CCT) for M993, and OGSH37 (sense; CCG GAT CTG GCC GCA GTG TCC TGT CAT TCG AGT CAG GTT) and OGSH38 (antisense; CCG GAA CCT GAC TCG AAT GAC AGG ACA CTG CGG CCA GAT) for M994.
Furin (M901): Full-length cDNA for human furin was obtained from imaGenes GmbH (Berlin, Germany). The furin coding sequence was amplified by PCR using the primers NotI_Fur_F (5’-GCG GCC GCA TGG AGC TGA GGC CCT GGT TGC-3’) and FurHA_AgeI_R (5’-ACC GGT TCA TCA AGC GTA ATC TGG AAC ATC GTA TGG GTA GAG GGC GCT CTG GTC TTT GA-3’) whereby a C terminal HA tag was fused to the furin coding sequence. The PCR product was cloned into the retroviral vector M387 (a variant of M377 described above, but containing an additional IRES and puromycin resistance cassette) using NotI and AgeI.
Chemical Crosslinking. The crosslinking reactions of native or natively deglycosylated GAFuroGA, FuroGA, and its N glycosylation site mutants N33Q, N95Q, and N33Q/N95Q expressed in 293T cells were carried out with 1 mM of bis(sulfosuccinimidyl)suberate (BS3; Roche) for 30 minutes at 25°C. The reactions were quenched with 50 mM TRIS-HCl, pH 7.6 for 15 minutes at 25°C. After SDS-PAGE analysis and Western blotting using a human monoclonal antibody to gp41 (2F5; Polymun), the percentage of recovered protein after chemical crosslinking was determined using the program GelEval (Frogdance Software, University of Dundee). For that purpose, the band intensities of native and natively deglycosylated C46 peptides in the presence (+) and absence (-) of bis(sulfosuccinimidyl)suberate (BS3) were compared. The calculated ratios were normalized to the amount of actin in the corresponding samples.
Pulse-chase experiments. Pulse-chase experiments with membrane-anchored C46 were performed as described in Materials and Methods section. However, cell lysis was performed using RIPA buffer [150 mM NaCl, 1% NP40 (Igepal® CA-630; MP Biomedicals), 50 mM TRIS-HCl, pH 8.0, 0.5% Sodium Deoxycholate (DOC), 0.1% sodium dodecyl sulfate (SDS), 1 mM PMSF, protease inhibitor cocktail (Roche)]. Lysates were stored at  20°C.


Supplementary References


41. Schambach A, Wodrich H, Hildinger M, Bohne J, Krausslich HG, Baum C. Context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors. Mol Ther 2000; 2(5): 435-45.
44. Hermann FG, Martinius H, Egelhofer M, Giroglou T, Tonn T, Roth SD et al. Protein scaffold and expression level determine antiviral activity of membrane-anchored antiviral peptides. Hum Gene Ther 2009; 20(4): 325-36.


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