four days later, the cells were placed on puromycin selection ( 0. 2 μg / ml; gibco ) and allowed to expand with gradual increases in puromycin to a final concentration of 0. 5 μg / ml. flow cytometry was used to determine when > 95 % of the cells expressed eyfp. we sorted some cell pools first for eyfp cells using the bd facsaria ii system ( bd biosciences ) before placing them on puromycin selection. cem / d128k cell pool a consisted of ∼1. 6 × 10 independent transduced cells, whereas cell pools b, c, and d contained ∼1. 4 × 10 independent transduced cells. pm1 / d128k cell pool a consisted of ∼800 independent transduced cells, whereas pools b and c ranged from 3, 200 to 5, 600 independent transduced cells. for isolation of cemss / d128k cell clones, puromycin - resistant cell pools were further diluted to single - cell clones and analyzed by western blot for expression of a3g - d128k. cem / d128k, pm1 / d128k, and cemss / d128k cell lines were plated at 2. 5 × 10 cells per well in 6 - well plates in triplic ate. the densities of viable cells were determined on days 2, 3, 4, and 5 by using a trypan blue exclusion assay ( gibco ) on a cellometer auto t4 cell counter ( nexelcom biosciences, lawrence, ma, usa ). cell lystates were prepared using cellytic m ( sigma ) solution containing protease inhibitor cocktail ( roche, mannheim, germany ), followed by centrifugation at 10, 000 × g for 10 min in a heraeus biofuge fresco centrifuge to remove cellular debris. the cell lysates were mixed with nupage lds sample buffer ( invitrogen ) containing β - mercaptoethanol and heated for 5 min at 95°c, and the samples were analyzed on 4 % – 20 % tris - glycine gels ( invitrogen ) using standard western blotting techniques. for detection of a3g, the rabbit anti - a3g antiserum apoc17 was used at a dilution of 1: 5, 000, obtained through the nih aids reagent program, division of aids, niaid ( from dr. klaus strebel; catalog # 10082 ). for detection of a3f, the rabbit polyclonal anti-human apobec3f ( a3f ) antibod y ( c18 ) was used at a dilution of 1: 500, obtained through the nih aids reagent program, division of aids, niaid ( from dr. michael h. malim; catalog # 11474 ). for detection of α - tubulin, mouse anti - α - tubulin antibod y ( catalog # t9026, sigma ) was used at a 1: 10, 000 dilution. for detection of p2a, the rabbit anti - p2a antibod y ( catalog # abs31, sigma ) was used at a 1: 10, 000 dilution. for detection of p24 ca, antibod y against hiv - 1 p24 ( monoclonal, 1: 10, 000 dilution ) was obtained through the nih aids reagent program, division of aids, niaid: anti-hiv - 1 p24 gag monoclonal ( # 24 - 3 ) from dr. michael malim ( catalog # 6458 ). an irdye 800cw - labeled goat anti-rabbit secondary antibod y ( catalog # 926 - 32211, li - cor, lincoln, ne, usa ) was used at a 1: 10, 000 dilution to detect rabbit primary antibod ies, and an irdye 680 - labeled goat anti-mouse secondary antibod y ( catalog # 926 - 68070, li - cor ) was used at a 1: 10, 000 dilution to detect mouse primary antibod ies. protein bands were visualized and quantified using an odyssey infrared imaging system ( li - cor ). parental cem, cemss, and pm1 cells, as well as cem / d128k, cemss / d128k, and pm1 / d128k cells, were seeded at 1 × 10 cells in 100 μl media and infected with nl4 - 3 virus ( 70 pg p24 ca ) or equivalent virus from subtype ae, subtype c, hiv - 2, siv, or supernatants from multi - round infection experiments. for hiv - 2 and sivmac239, virus produced from transfected 293t cells that produced the same amount of luciferase activity upon infection of tzm - bl cells with 70 pg p24 ca - containing nl4 - 3 virus were used to infect the cem / d128k and pm1 / d128k cell lines. note that sivmac239 can use the alternate coreceptor bob / gpr15 to infect the cem cell line, which is cd4 / cxcr4 / ccr5 / bob. the virus - cell mixtures were incubated at 37°c and 5 % co2 for 5 h, and the cells were washed and placed in 3 ml media. at 2 - or 3 - day intervals, the virus - cell suspension was mixed, and 1 ml culture was removed and centrifuged at 16, 000 × g for 10 min in a heraeus biofuge fresco centrifuge. the resulting virus supernatant was removed and stored at −80°c, while the cell pellet was frozen and subsequently used for genomic dna extractions. of the remaining 2 ml culture, 1 ml culture was discarded, and the remaining 1 ml was resuspended with 2 ml fresh media. the sample was then incubated for another 2 – 3 days, and the process was repeated for the duration of the experiment. to assay for replication kinetics, the amount of hiv - 1 virus in culture supernatants was determined by quantifying the amount of p24 ca by elisa. the amounts of hiv - 2 and sivmac239 virus in culture supernatants were determined by infection of tzm - bl cells and quantifying the luciferase activity 48 – 72 h after infection. total genomic dna from transduced or infected cells was isolated using the qiaamp dna blood mini kit ( qiagen, hilden, germany ), and an 896 - nt vif region from integrated proviruses was amplified by pcr using primers nl43 - seq - 4921f ( 5 ′ - gagatccagtttggaaaggac - 3 ′ ) and nl43 - seq - 5816r ( 5 ′ −ctgtcgagtaacgcctattc - 3 ′ ). alternatively, a 982 - nt puro - wpre ( woodchuck hepatitis virus post - transcriptional regulatory element ) region was amplified using primers purofor ( 5 ′ - cgcgcagcaacagatggaag - 3 ′ ) and nefrev ( 5 ′ - cttgtgcttctagccaggca - 3 ′ ). the pcr products were resolved on a 1 % agarose gel, the 896 - bp band was extracted from the gel using the qiaquick gel extraction kit ( qiagen ), and the dna was cloned into a topo ta plasmid ( invitrogen ). the plasmid dna was extracted from the bacterial colonies using the nucleospin 8 plasmid kit ( clontech ). individual clones were sequenced ( macrogen ) and evaluated for nucleot ide and amino acid changes and further analyzed for the presence of hypermutation using hypermut ( https: / / www. hiv. lanl. gov / content / sequence / hypermut / hypermut. html ). sequenced clones containing single g - to - a changes were not considered hyper mutate d, as single mutations may have resulted from errors during pcr or sequencing. pbmcs were grown in rpmi medium supplemented with 10 % fetal calf serum ( hyclone ) and 1 % penicillin - streptomycin ( final concentration: penicillin, 50 u / ml; and streptomycin, 50 μg / ml; gibco ). pbmcs were stimulated for 3 days at 37°c with il - 2 ( 40 u / ml; sigma ) and pha - p ( 5 μg / ml; sigma ) or dynabeads human t - activator cd3 / cd28 ( invitrogen ). cells were washed to remove pha - p, and cd4 t cells were isolated using the dynal cd4 positive isolation kit ( invitrogen ). flow cytometry analysis was performed to determine the percent activated cd4 t cells in the isolated population using the following antibod ies ( bd pharmingen, san jose, ca, usa ): fluorescein - iso thiocyanate ( fitc ) - conjugated mouse anti-human cd4, fitc - conjugated mouse immunoglobulin ( ig ) g κ isotype control, phycoerythrin ( pe ) - conjugated mouse anti-human cd25, and pe - conjugated mouse igg1 κ isotype control. activated cd4 t cells were spin - inoculate d with virus at an moi of 10 ( 1, 200 × g, 1 h, room temperature ) in the presence of 4 μg / ml polybrene. cell / virus mix was incubated for 20 h at 37°c. subsequently, the cells were washed, and flow cytometry analysis was performed 72 h post - transduction to determine the proportion of eyfp cells. culturing and infection of cd34 hspcs were performed as previously described. umbilical cord blood cd34 hspcs were pre - stimulated for 24 h in iscove ’ s modified dulbecco ’ s medium ( imdm ) supplemented with 20 % bit 9500 ( stemcell technologies, cambridge, ma, usa ), 50 ng / ml each of human interleukin-6 and thrombopoietin, 150 ng / ml human stem cell factor, and 100 ng / ml flt - 3 ligand ( peprotech, rocky hills, nj, usa ).