importantly, none of the 52 proviral dnas from cells transduced with the a3x3g ( dk ) virus produced in the presence of vif2 were hyper mutate d, indicating that the a3x3g ( dk ) vectors were very effective in suppressing a3g - d128k expression in the virus - producing cells. our observed high efficiency of homologous recombination ( 89 % – 98 % ) for the ∼900 - bp direct repeat sequence of a3g is consistent with previous observations by us and others that a 701 - bp direct repeat and a 971 - bp direct repeat were deleted at frequencies of 91 % and 81 %, respectively. intriguingly, we were unable to select for a3g - d128k - resistant viral variants in multiple viral cultures for > 3. 5 months, suggesting that a3g - d128k has a high genetic barrier to development of resistance. one possible reason for the high genetic barrier is that the g - to - a hypermutation induced by a3g is a lethal “ all - or - nothing ” phenomenon. hyper mutate d proviruses in infected patients contain, on average, 231 g - to - a substitutions, of which ∼47 result in stop codons; thus, the probability of a virus escaping a3g - mediated inactivation by sublethal mutagenesis is < 10. we postulate that multiple amino acid substitutions in vif must appear simultaneously to acquire the ability to induce a3g - d128k degradation. if the appearance of one or two of the amino acid changes does not confer complete resistance to a3g - d128k, the all - or - nothing nature of hypermutation predicts that these intermediate substitution mutants would be quickly eliminated from the viral population. the probability of acquiring three specific nucleot ide substitutions in a single viral genome during one replication cycle is the product of the rate for each substitution, which is 3 × 10 / nucleot ide / replication cycle: thus, the probability of acquiring three specific substitutions is ( 3 × 10 ) × ( 3 × 10 ) × ( 3 × 10 ) or 2. 7 × 10. thus, because of the relatively small size of the viral populations in the cell culture assays and the limited number of replication cycles ( ∼100, assuming one replication cycle per day ), it is unlikely that a mutant with three simultaneous amino acid changes will appear in our selection experiments. a3g - d128k - resistant variants did not emerge in cemss / d128k cells, which did not express other a3 proteins, suggesting that the high genetic barrier to resistance is intrinsic to the all - or - nothing nature of lethal hypermutation. consistent with our results, a computational study predicted that the most potent therapeutic anti-hiv - 1 a3g - based strategy is to use a vif - binding site mutant. previous efforts to select for a3g - resistant vif - defective viral variants have yielded negative results or mutations that were unable to confer the ability to replicate in a3g - expressing t cell lines. 66, 67, 68 in a recent study, vif null viral variants with env substitutions were selected, but these mutations did not confer a3f resistance, suggesting that the mechanism of resistance may be specific to the level of a3g expression. potential benefits of gene therapy with vif - resistant a3g - d128k are that ccr5 - tropic as well as cxcr4 - tropic viruses will be inhibited, and that it is possible to express more than one vif - resistant a3 protein to inhibit viral replication and spread. distinc t vif - a3g and vif - a3f interactions are necessary for inducing degradation of the a3 proteins, indicating that multiple vif mutations may be required to overcome different vif - resistant a3 proteins. a3g - d128k differs from wild - type human a3g by a single amino acid, which should minimize elimination of the genetically engineered cells by the host immune system. in vivo patient studies of elite controllers have been shown to not only attenuate vif proteins but also increase a3g mrna and protein levels, leading to lower hiv provirus burden, emphasizing the importance of vif - apobec3 interactions in controlling viral loads. although our results indicate that efficient lentivector delivery of vif - resistant a3g to hiv - 1 target cells is a promising anti-hiv gene therapy, additional technical developments and testing in a humanized mouse model system will be needed before its implementation in the clinic. first, it will be necessary to demonstrate that a sufficiently high proportion of primary cd4 + t cells, the target of hiv - 1 infection, can be engineered to express a3g - d128k to effectively inhibit hiv - 1 replication and spread. our cell - mixing experiments in which parental cem and cem / d128k cells were mixed at different ratios suggest that a3g - d128k expression in > 75 % of the target cells may be needed to significantly inhibit viral replication and spread. our efficiency of transduction of the activated cd4 + t cells was lower than the efficiencies reported in recent studies but could be improved by adapting recently improved transduction methods. in future studies, it may be possible to achieve high levels of lentiviral transduction as well as enable pharmacologic selection of the transduced cells. second, the a3g - d128k - expressing cells will be susceptible to infection by hiv - 1, resulting in their elimination from the patients. it may be possible to provide the a3g - d128k - expressing cells with a selection advantage over non - expressing cells by co - expression of anti - ccr5 shrnas or a fusion peptide that inhibits hiv - 1 entry. successful combination of a3g - d128k expression with elements that provide a selection advantage to the transduced cells, as well as achieving a high efficiency of lentivector transduction, may establish the feasibility of hiv - 1 gene therapy with vif - resistant a3g. in summary, we constructed and characterized self - activating lentiviral vectors that can efficiently deliver vif - resistant a3g - d128k to a high proportion of cd4 t cells and cd34 hspcs. additional improvements in lentivector transduction efficiency, providing the a3g - d128k - expressing cells with a selective advantage, and testing the efficacy of this anti-hiv gene therapy in an animal model system will help determine whether gene therapy with vif - resistant a3g can provide a viable anti-hiv treatment and functional cure strategy. plasmids used in this study are designated with a “ p, ” while the names of viruses and proviruses generated from these plasmids are not. pc - help is an hiv - 1 helper construct that lacks a packaging signal and primer - binding site; the helper construct expresses all the viral proteins except nef and envelope, and phcmv - g expresses the g glyco protein of vesicular stomatitis virus ( vsv - g ). pvif2 - ha expresses codon - optimized hemagglutinin ( ha ) - tagged vif from hiv - 2. pcdna - apo3gd128k expresses a3g mutant d128k and was used to pcr - amplify the a3g - d128k sequences used for construction of other vectors. phl [ wt ] is an hiv - 1 luciferase - encoding vector that expresses all the hiv - 1 proteins except nef and envelope. pnl4 - 3 expresses a replication - competent subtype b hiv - 1. pmj4 is a subtype c hiv - 1 infectious molecular clone, obtained through the nih aids reagent program, division of aids, national institute of allergy and infectious diseases ( niaid ), nih ( from drs. thumbi ndung ’ u, boris renjifo, and max essex; catalog # 6439 ). p90cf402. 1 is an intersubtype recombinant ae hiv - 1 infectious molecular clone, obtained through the nih aids reagent program, division of aids, niaid, nih ( from drs. feng gao and beatrice hahn; catalog # 3284 ). prod12 is a full - length infectious molecular clone of hiv - 2 ( a gift from keith peden ). psivmac239 is an infectious clone of simian immunode ficiency virus ( siv ), obtained through the nih aids reagent program, division of aids, niaid, nih ( from dr. ronald c. desrosiers; catalog # 12249 ).