replication kinetics of nl4 - 3 in cemss / d128k clones ( 100 % ) or a mix of 20 % cemss: 80 % cemss / d128k low ( blue ) or medium ( red ) clones. viral supernatants were collected every 2 to 3 days, and p24 ca was measured by elisa up to 28 days post - infection. no nl4 - 3 virus was detected in the 100 % cemss / d128k cells ( top ). severely delayed kinetics were observed in the 20 % cemss: 80 % cemss / d128k clone mix 28 days post - infection ( bottom ). ( normalized virus from day 28, round 1, 20 % cemss: 80 % cemss / d128k cells ( low and medium, indicated by blue and red stars, as also shown in the lower panel of f ) was used to infect 100 % cemss / d128k or 100 % cemss cells. viral supernatants were collected up to 42 days post - infection and analyzed for p24 ca by elisa. no nl4 - 3 replication was detected in 100 % cemss / d128k cell lines, and near - wild - type kinetics ( days 11 – 13 ) were observed in 100 % cemss cells. to determine whether resistant virus would emerge in cemss / d128k cells expressing lower amounts of a3g - d128k, single - cell clones were selected that expressed a3g - d128k - p2a at a 35 % ( low ) or a 70 % ( medium ) level compared to endogenous a3g expressed in cem cells ( figure 5e ). both cemss / d128k cell clones were infected with nl4 - 3 and passaged for 28 days. as seen with cem / d128k cells, the cemss / d128k cell clones also inhibited productive viral replication ( figure 5f, top ). however, nl4 - 3 infection of a 20 % cemss: 80 % cemss / d128k cell mix for each cell clone showed severely delayed kinetics, and virus production did not become detectable for 20 days after infection ( figure 5f, bottom ). viruses from the 20 % cemss: 80 % cemss / d128k ( low or medium ) cultures were used to infect 100 % cemss / d128k cell clones and monitored for viral replication and spread ( figure 5g, top ). no virus replication was detected after 42 days, indicating that a3g - d128k - resistant virus did not emerge, even though replication - competent virus was detected upon infection of the parental cemss cells ( figure 5g, bottom ). overall, expression of a3g - d128k in the absence of endogenous a3 proteins did not lead to the emergence of a3g - d128k - resistant viral variants. it was previously reported that substitution of hiv - 1 vif amino acids drmr with african green monkey amino acids se ( r / m ) q conferred the ability to induce degradation of a3g - d128k. however, the hiv - 1 vif semq variant is unable to induce degradation of human a3f in single replication cycle assays. to determine whether the hiv - 1 vif semq mutant can replicate in a3g - d128k expressing cells, we constructed an nl4 - 3 mutant in which the vif amino acids drmr were replaced with semq ( nl4 - 3 [ semq ] ), which also resulted in the substitution of integrase ( in ) c - terminal 5 amino acids from rqdad to krdad. we generated nl4 - 3 [ semq ] virus and infected the parental cem and cemss as well as cem / d128k and cemss / d128k cells ( figure 6 ). in the parental cem and cemss cells, the nl4 - 3 [ semq ] peak virus production was delayed by by 2 – 3 days compared to wild - type nl4 - 3, suggesting that the semq mutant may have a lower fitness than wild - type nl4 - 3 ( figures 6a and 6c ). in cem / d128k or cemss / d128k cells, wild - type nl4 - 3 replication was not detected for 42 days, whereas nl4 - 3 [ semq ] replicated with severely delayed kinetics ( figures 6b and 6d ). these results suggest that the nl4 - 3 [ semq ] mutant can overcome the antiviral activity of a3g - d128k, but it either is less efficient at inducing a3g - d128k degradation or has a lower fitness than wild - type nl4 - 3. figure 6replication kinetics of the nl4 - 3 vif semq mutant in cem / d128k and cemss / d128k cell lines ( a – d ) normalized virus from nl4 - 3 and nl4 - 3 [ semq ] was used to infect ( a ) cem, ( b ) cem / d128k, ( c ) cemss, and ( d ) cemss / d128k cell lines. supernatants were harvested every 2 to 3 days for up to 42 days and monitored by elisa for p24 ca expression. averages of replication kinetics from two independent infections are indicated. replication kinetics of the nl4 - 3 vif semq mutant in cem / d128k and cemss / d128k cell lines ( a – d ) normalized virus from nl4 - 3 and nl4 - 3 [ semq ] was used to infect ( a ) cem, ( b ) cem / d128k, ( c ) cemss, and ( d ) cemss / d128k cell lines. supernatants were harvested every 2 to 3 days for up to 42 days and monitored by elisa for p24 ca expression. averages of replication kinetics from two independent infections are indicated. it is desirable to transduce activated cd4 t cells, the natural targets of hiv - 1 infection, and cd34 hspcs, which can differentiate to form new cd4 t cells. cd4 t cells from 5 different donors ( figure 7a ) were transduced with the a3x3g ( dk ) lentivector ( moi = 10 ), resulting in the expression of eyfp in 9 % – 15 % of the cd4 t cells. transduction of cd34 hspcs from three different donors with the a3x3g ( dk ) lentivector ( moi = 20 ) resulted in eyfp expression in 32 % of hspcs, and transduction with the lv - gfp lentivector resulted in gfp expression in ∼40 % of the hspcs ( figure 7b ); no apparent cytotoxicity was observed ( data not shown ). these results confirmed that the a3x3g ( dk ) lentivector can efficiently transduce activated cd4 t cells and cd34 hspcs. figure 7efficient transduction of cd4 t cells and cd34 hspcs with the a3x3g - d128k - expressing vector ( a ) cd4 t cells from 5 donors were transduced with a3x3g ( dk ) vector virus at an moi of 10, and the proportion of eyfp cells was determined by facs 72 h post - infection. ( cd34 hspcs from 3 donors were transduced with a3x3g ( dk ) vector virus or a control gfp lentiviral vector ( lv - gfp ) at an moi of 20, and the proportion of fluorescent cells was determined by facs 6 days post - transduction. average from two independent infections is indicated. efficient transduction of cd4 t cells and cd34 hspcs with the a3x3g - d128k - expressing vector ( a ) cd4 t cells from 5 donors were transduced with a3x3g ( dk ) vector virus at an moi of 10, and the proportion of eyfp cells was determined by facs 72 h post - infection. ( cd34 hspcs from 3 donors were transduced with a3x3g ( dk ) vector virus or a control gfp lentiviral vector ( lv - gfp ) at an moi of 20, and the proportion of fluorescent cells was determined by facs 6 days post - transduction. average from two independent infections is indicated. two patients have remained free of hiv - 1 in the absence of therapy for > 18 months to several years after hspc transplantation of donor cells with a δ32 deletion in the ccr5 coreceptor, suggesting that inactivating the ccr5 coreceptor using gene therapy might achieve a cure. however, inactivation of the ccr5 does not prevent infection with cxcr4 - tropic and dual - tropic viruses and is likely to select for variants that can utilize the cxcr4 coreceptor, suggesting a need for strategies to suppress infection with ccr5 -, cxcr4 -, and dual - tropic viruses. other gene therapy strategies that are being explored to achieve a functional cure that can potentially inhibit replication of all viruses, regardless of the coreceptor they utilize, include expression of a fusion peptide, genetically modified chimeric antigen receptor ( car ) - expressing t cells, deletion of hiv - 1 proviruses by gene editing, and harnessing the antiviral activity of host restriction factors. 59, 60, 61 in the studies reported here, we developed self - activating lentiviral vectors that suppress a3g - d128k expression in the virus - producing cells and efficiently reconstitute a functional a3g - d128k by homologous recombination during reverse transcription. consequently, > 70 % of the target 293t cells were transduced with the a3x3g ( dk ) vector, compared to only 8 % transduction efficiency with the control a3g ( dk ) vector.