furthermore, the proportion of hyper mutate d viral dnas increased from 0 / 33 to 18 / 34 in cultures in which 75 % of the cells expressed a3g - d128k ( p < 0. 005, fisher ’ s exact test ). thus, inhibition of virus replication was correlated with a3g - d128k expression and its antiviral activity in a subpopulation of target cells. in an effort to select for a3g - d128k - resistant viral variants, we passaged the nl4 - 3 virus for ∼3 months in 25 % cem: 75 % cem / d128k mixed - cell cultures, which supported viral replication with a 5 - day delay in peak virus production ( figure 4a ). six rounds of infections were carried out by passaging the virus for 2 weeks during each round and using the culture supernatant at the peak of virus production to initiate the next round of infection ( figure 4c ). virus from the peak of virus production in the initial 25 % cem: 75 % cem / d128k culture ( figure 4a, round 1 ) was used to infect 6 flasks ( labeled a – f ) containing fresh 25 % cem: 75 % cem / d128k mix and cultured for 2 weeks ( figure 4c, round 2 ). this protocol was repeated for rounds 3 – 6, and the results of infection of 100 % cem, 75 % cem / d128k, and 100 % cem / d128k cells with virus from round 6 are shown in figure 4d. virus replication was not detected in 100 % cem / d128k cultures after infection with virus from rounds 1 – 6, indicating that a3g - d128k - resistant virus did not emerge after > 3 months of culture. in all cases, virus replication with wild - type nl4 - 3 kinetics was detected in 100 % cem cells, and delayed replication kinetics were observed in the 25 % cem: 75 % cem / d128k cell mix, confirming the presence of replicating virus in the cultures. analysis of integrated proviruses at the time point of peak virus production from the 25 % cem: 75 % cem / d128k cell mix infected with round 6 virus showed that > 50 % ( up to 78 % ) of the integrated proviruses were hyper mutate d, with an average of 7 – 14 g - to - a changes per hyper mutate d clone in the 896 - nt vif region ( figure 4e, round 6 ). overall, these results indicated that cd4 t cell lines expressing a3g - d128k were capable of long - term hiv - 1 inhibition without detectable emergence of a3g - d128k - resistant virus. cem / d128k cells express endogenous a3g and a3f, but cemss cells do not. we hypothesized that a3g - d128k - resistant virus might emerge more efficiently in the absence of expression of endogenous a3g and a3f, since vif would not need to retain the ability to induce degradation of the endogenous a3 proteins. to test this hypothesis, we generated cemss cells that stably expressed a3g - d128k by transduction with the a3x3g ( dk ) lentivector and selecting a pool of puromycin - resistant cells ( cemss / d128k ). western blotting analysis showed that the cemss / d128k cell pool expressed a3g - d128k at 37 % of the level observed with cem / d128k ( figure 5a ) and that expression of a3g - d128k did not alter the growth kinetics of the cemss cells ( figure s3c ). we infected the parental cemss and cemss / d128k cells with equivalent p24 ca amounts of nl4 - 3 virus and monitored hiv - 1 replication kinetics ( figure 5b ). the results showed that nl4 - 3 replication was readily detected in the cemss cells, but no virus replication was detected in the cemss / d128k cells for 42 days, indicating that resistance to a3g - d128k did not emerge after prolonged culture, even in the absence of endogenous a3 proteins. similar to the results observed for cem / d128k cells ( figure 3f ), infection of cemss / d128k cells with subtype - b - derived patient isolates did not result in the detectable emergence of a3g - d128k - resistant virus ( figures 5c and 5d ). figure 5absence of selection for nl4 - 3 variants that can replicate in cemss / d28k cells ( a ) cemss cells were transduced with a3x3g ( dk ) virus and selected for puromycin resistance. western blot analysis shows the level of a3g - d128k - p2a expressed in the cemss / d128k cell pool ( 37 % ) compared to the level in the cem / d128k cell pool ( set to 100 % ) and normalized to α - tubulin. blot probed with α - p2a ( green ) and α - tubulin ( red ). ( cemss / d128k and parental cemss cells were infected with nl4 - 3 ( 70 pg p24 ca / 1 × 10 cells ). supernatants were harvested every 2 to 3 days, and p24 ca amounts were determined by elisa. multiple rounds of infection were monitored over a 40 - day period. nl4 - 3 shows wild - type kinetics with peak virus at day 11, with no detectable virus in cemss / d128k cells. averages of two independent infections are indicated. error bars represent sd. ( c and d ) equal amounts of p24 - containing virus from nl4 - 3 and three subtype b patient isolates ( ptb - 1, - 2, and - 3 ) were used to infect ( c ) cemss and ( d ) cemss / d128k cell lines, respectively. supernatants were harvested every 2 to 3 days for 42 days and monitored by elisa for p24 ca expression. averages of replication kinetics of two independent infections are shown. error bars represent sd. ( western analysis of puromycin - resistant cemss / d128k single - cell clones expressing a3g - d128k. two cemss / d128k clones are shown with lower a3g - d128k levels ( 0. 35× and 0. 70× ) compared to endogenous a3g expressed in cem cells ( 1× ). blot probed with α - a3g ( green ) and α - tubulin ( red ). ( 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. absence of selection for nl4 - 3 variants that can replicate in cemss / d28k cells ( a ) cemss cells were transduced with a3x3g ( dk ) virus and selected for puromycin resistance. western blot analysis shows the level of a3g - d128k - p2a expressed in the cemss / d128k cell pool ( 37 % ) compared to the level in the cem / d128k cell pool ( set to 100 % ) and normalized to α - tubulin. blot probed with α - p2a ( green ) and α - tubulin ( red ). ( cemss / d128k and parental cemss cells were infected with nl4 - 3 ( 70 pg p24 ca / 1 × 10 cells ). supernatants were harvested every 2 to 3 days, and p24 ca amounts were determined by elisa. multiple rounds of infection were monitored over a 40 - day period. nl4 - 3 shows wild - type kinetics with peak virus at day 11, with no detectable virus in cemss / d128k cells. averages of two independent infections are indicated. error bars represent sd. ( c and d ) equal amounts of p24 - containing virus from nl4 - 3 and three subtype b patient isolates ( ptb - 1, - 2, and - 3 ) were used to infect ( c ) cemss and ( d ) cemss / d128k cell lines, respectively. supernatants were harvested every 2 to 3 days for 42 days and monitored by elisa for p24 ca expression. averages of replication kinetics of two independent infections are shown. error bars represent sd. ( western analysis of puromycin - resistant cemss / d128k single - cell clones expressing a3g - d128k. two cemss / d128k clones are shown with lower a3g - d128k levels ( 0. 35× and 0. 70× ) compared to endogenous a3g expressed in cem cells ( 1× ). blot probed with α - a3g ( green ) and α - tubulin ( red ). (