subtype b patient isolates were obtained through the nih aids reagent program, division of aids, niaid, nih from the panel of 60 international hiv - 1 isolates ( catalog # 11412 ), contributor dr. robert gallo for hiv - 1 mn virus ( catalog # 317 ), and contributor dr. nelson michael for hiv - 1 bk132 ( gs 009 ) virus ( catalog # 7691 ) and hiv - 1 bz167 / gs 010 ( 89bz _ 167 ) virus ( catalog # 7692 ). for clarity, hiv - 1 mn virus was labeled as ptb - 3, hiv - 1 bk132 ( gs 009 ) virus was labeled as ptb - 1, and hiv - 1 bz167 / gs 010 ( 89bz _ 167 ) virus was labeled as ptb - 2. pnl4 - 3 [ semq ] was constructed by replacing the vif drmr with semq in nl4 - 3. since the first 19 amino acids of vif overlap with the c - terminal region of in, substitution of drmr with semq changed the 5 terminal amino acids of in from rqdad to krdad. these substitutions were previously shown to have a minimal effect on virus replication. plasmid plv - gfp is a ubiquitin - c - driven egfp - expressing vector ( catalog # 14884, addgene, watertown, ma, usa ). the multiple cloning site 5 ′ of the ires - puromycin cassette in the lentiviral vector plvx - ires - puro ( catalog # 632183; clontech, mountain view, ca, usa ) was utilized to construct the following vectors. vector peyfpip expresses eyfp and puro. vector pa3g ( dk ) was created by insertion of a3g - d128k lacking a stop codon followed by an in - frame insertion of p2a fused to eyfp lacking a start codon. insertion of the 5 ′ direct repeat fragment of a3g - d128k ( nt 1 to 1, 083 ) followed by an in - frame insertion of p2a fused to eyfp lacking a start codon created vector pa3dr ( dk ). insertion of the 3 ′ direct repeat fragment of a3g - d128k ( nt 183 to 1, 152 ) followed by an in - frame insertion of p2a fused to eyfp lacking a start codon created vector p3gdr ( dk ). to create vectors pa33g ( dk ) and pa3x3g ( dk ), the following fragments were joined by ligation in the same reading frame: the 5 ′ direct repeat of a3g - d128k ( 1 to 1, 083 nt of a3g for the a33g ( dk ) vector and the same 1 to 1, 083 nt plus three in - frame stop codons, tga - taa - tga for the a3x3g ( dk ) vector ); 3 ′ direct repeat of a3g - d128k ( nt 184 to 1, 152 ); and p2a fused to eyfp ( lacking a start codon ). this vector has an ∼900 - bp direct repeat of a3g - d128k ( nt 184 to 1, 083 ). to create pcmv - a3g ( dk ), pcr3. 1 ( + ) ( catalog # v79020; invitrogen, carlsbad, ca, usa ) was digested with nhe i and xba i, and a3g - d128k was amplified from pa3g ( dk ) with nhe i - containing forward primer and xba i - containing reverse primers for complementary sticky - end ligation. to create pcmv - a3g ( dk ) - p2a - eyfp, the nhe i and xba i cloning sites were used to insert a3g - d128k lacking a stop codon, and the xba i and apa i sites were used to ligate an in - frame fragment of p2a fused to eyfp ( lacking a start codon ). the structures of all final plasmids were confirmed by sequencing ( macrogen, rockville, md, usa ). cell lines 174xcem ( nih aids reagent program, division of aids, niaid: 174xcem cells from dr. peter cresswell; cd4 / cxcr4 / ccr5 / gpr15 [ catalog # 272 ] ), h9 ( t - lymphoid cell line, american type culture collection, catalog # htb - 176 ), pm1 ( nih aids reagent program, division of aids, niaid: pm1 from dr. marvin reitz; derived from hut 78 cells; catalog # 3038 ), and cemss ( nih aids reagent program, division of aids, niaid: cem - ss from dr. peter l. nara; catalog # 776 ) were grown in rpmi 1640 medium ( cellgro, manassas, va, usa ) supplemented with 10 % fetal calf serum ( hyclone, logan, ut, usa ) and 1 % penicillin - streptomycin ( final concentration: penicillin, 50 u / ml; and streptomycin, 50 μg / ml; lonza, walkersville, md, usa ). hek293t cells ( american type culture collection; catalog # crl - 3216 ) and tzm - bl cells ( nih aids reagent program, division of aids, niaid, nih: tzm - bl from dr. john c. kappes, dr. xiaoyun wu, and tranzyme [ catalog # 8129 ] ) were grown in dmem ( cellgro ) supplemented with 10 % fetal calf serum ( hyclone ) and 1 % penicillin - streptomycin stock ( final concentration: penicillin, 50 u / ml; and streptomycin, 50 μg / ml; lonza ). all cells were maintained in humidified 37°c incubators with 5 % co2. umbilical cord blood ( ucb ) cd34 cells were isolated and verified as described previously from cord blood generously donated from the cleveland cord blood center ( cleveland, oh, usa ). all approved human protocols are available upon request at the scripps research institute. all transfections were performed using transit - lt1 transfection reagent ( mirus bio, madison, wi, usa ) according to the manufacturer ’ s instructions. to generate virus, 293t cells were seeded at 3 × 10 cells per 100 - mm dish and transfected with pc - help ( 5 μg ), phcmv - g ( 4 μg ), and vectors of interest ( 10 μg ). twenty - four to 30 h post - transfection, virus was harvested, filtered with 0. 45 - μm filters, and stored at −80°c. for generating a33g ( dk ) or a3x3g ( dk ) virus, pvif2 - ha ( 2 – 4 μg ) was also included in the transfection to degrade any expressed a3g - d128k in the producer cells as a result of direct repeat deletion during transfection. direct repeat deletion during transfection has been previously measured to be ∼5 %. additionally, reverse transcription inhibitor 3 ′ - azido - 3 ′ - deoxythymidine ( azt; 4 μm, final concentration, sigma, st. louis, mo, usa ) was added to culture media to block potential reinfection of the producer cells. p24 ca amounts were determined by using the hiv - 1 p24 elisa kit ( xpressbio, frederick, md, usa ) according to the manufacturer ’ s instructions. the sensitivity of p24 detection was > 1. 7 pg / ml. to test whether the p2a tag on a3g - d128k would affect a3g antiviral activity, phl [ wt ] ( 3. 3 μg ) was co - transfected with pcmv - a3g ( dk ) or pcmv - a3g ( dk ) - p2a - eyfp ( 0. 67 μg ), and virus was harvested 48 h post - transfection. normalized p24 ca amounts were used to transduce 4 × 10 293t cells in a 96 - well plate, and luciferase activity was measured 48 h post - transduction using a 96 - well luminometer ( lumistar galaxy, bmg labtech, cary, nc, usa ). data were plotted as the percent inhibition of luciferase activity normalized to the hl [ wt ] control. to determine the infectivity of a3g ( dk ), a3dr ( dk ), 3gdr ( dk ), and a3x3g ( dk ) viruses, normalized p24 ca amounts were used to transduce target 293t cells ( 4 × 10 cells per 6 - well plate ). forty - eight hours post - transduction, the proportion of eyfp cells was analyzed by flow cytometry using the facscalibur system ( bd biosciences, san jose, ca, usa ) and flowjo software ( ashland, or, usa ). to determine the frequency of a3g ∼900 - bp direct repeat deletion and a3g - d128k gene reconstitution, virus produced from vector a3x3g ( dk ) was used to transduce 293t target cells and placed on puromycin selection. genomic dnas from independent cell pools were isolated ( ∼2 × 10 puromycin - resistant cells per pool ); and, using primers flanking a3g ( dk ), pcr amplification was performed to detect the proportion of undeleted a3x3g ( dk ) ( 2, 190 bp ) or deleted a3g ( dk ) ( 1, 290 bp ) proviruses. primers cmvfor ( 5 ′ - cagagctcgtttagtgaacc - 3 ′ ) and a3gendrev ( 5 ′ - gattctggagaatggcccgc - 3 ′ ) flank a3g ( dk ). in addition, individual puromycin - resistant 293t cell clones were isolated from cells transduced with virus from vector pa33g ( dk ) and analyzed to determine direct repeat deletion frequency by pcr amplification using primers cmvfor and gendrev4431 ( 5 ′ - ggctgtgctcatctagtccatc - 3 ′ ) ( undeleted, 1, 864 bp; deleted, 964 bp ). cem, pm1, or cemss cells ( 2 × 10 cells per well in 96 - well plate ) were spin - infected with a3x3g ( dk ) virus ( 40 ng p24 ca ) for 2 h, 1, 200 × g, at room temperature in a beckman allegra 21r centrifuge ( rotor s2096 ).