we sought to determine whether a3g - d128k - resistant variants can be selected in culture from a genetically diverse population of viruses derived from patients. three patient - derived subtype - b isolates that were previously characterized to be cxcr4 - tropic were obtained, and their ability to replicate in parental cem cells and cem / d128k cells was determined ( figure 3f ). although all three patient isolates replicated in the parental cem cells with kinetics similar to that of the nl4 - 3 virus, with p24 ca levels peaking around day 7 ( ptb - 2 ) or days 9 – 11 ( ptb - 1 and ptb - 3 ), no viral replication was detected in cem / d128k cells for 42 days ( figure 3f ). the results indicated that a3g - d128k expression was able to restrict the replication of genetically diverse populations of hiv - 1 isolated from subtype - b - infected patients. to confirm that the lack of hiv - 1 replication and spread in cem / d128k and pm1 / d128k cell pools was not due to the inadvertent selection of cell variants that cannot support viral replication, cem / d128k and pm1 / d128k cell lines were infected with normalized inputs of wild - type hiv - 2 or sivmac239 virus, which express vif proteins that can degrade a3g - d128k. sivmac239 and hiv - 2 viruses replicated in the cem / d128k and pm1 / d128k cell lines ( figure s4 ), indicating that both cell lines can support the replication and spread of sivmac239 and hiv - 2 but not hiv - 1. to determine how the proportion of target cells that express a3g - d128k affect inhibition of virus replication, we generated mixtures of cem and cem / d128k cells at different ratios, ranging from 0 % to 100 % cem / d128k cells, and monitored the kinetics of the replication of nl4 - 3 in the cell mixtures for 21 days ( figure 4a ). nl4 - 3 replication peaked on day 7 in cultures that had 0 % or 10 % cem / d128k cells and peaked on days 7 – 9, 9 – 12, and 12 – 14 in cultures that had 25 %, 50 %, and 75 % cem / d128k cells, respectively. virus replication was not detected in cultures that had 90 % or 100 % cem / d128k cells. thus, a3g - d128k expression in a subpopulation of target cells delayed viral replication kinetics in a dose - dependent manner. figure 4absence of selection for nl4 - 3 variants that can replicate in cem / d128k cells ( a ) nl4 - 3 virus was used to infect different ratios of cem: cem / d128k cells. 100 %, 90 %, 75 %, 50 %, 25 %, 10 %, and 0 % cem cells were mixed with 0 %, 10 %, 25 %, 50 %, 75 %, and 100 % cem / d128k cells, respectively. supernatants were collected every 2 to 3 days for 21 days and measured by p24 ca elisa to determine replication kinetics. average of two independent infections per ratio is indicated. error bars represent sd. ( viruses from peak production, normalized for p24 ca, from round 1 cultures 100 % cem ( day 7; indicated by a blue star in a ), 50 % cem: 50 % cem / d128k ( day 9; indicated by a red star in a ), and 25 % cem: 75 % cem / d128k ( day 12; indicated by a green star in a ) were used to infect either 100 % cem or 100 % cem / d128k cells. supernatants were collected every 2 to 3 days for up to 30 days, and p24 ca amounts were quantified by elisa to determine replication kinetics. infection of the parental cem cells showed wild - type nl4 - 3 peak virus at day 7 with no detectable virus measured in 100 % cem / d128k cells. averages of two independent infections per cell ratio are indicated. error bars represent sd. ( protocol to select for hiv - 1 resistance to a3g - d128k. ( viruses from round 6 ( independent flasks a – f ) were used to infect 100 % cem, 25 % cem: 75 % cem / d128k, or 100 % cem / d128k cells, and viral supernatants were collected every 2 to 3 days for up to 28 days. elisa was used to quantify p24 ca to determine replication kinetics. viruses from flasks a – f displayed wild - type kinetics in parental cem cells peaking at days 5 – 7 and showed delayed kinetics, peaking at days 9 – 12, in the 25 % cem: 75 % cem / d128k cell mix as was seen in round 1, as indicated in ( a ). no detectable virus was measured in 100 % cem / d128k cells. ( proviral dna from cell cultures in which virus replication peaked in round 1 and round 6 were analyzed for g - to - a hypermutation by sequencing an 896 - nt vif region. for round 1, proviral dnas from peak virus day 7 for 0 %, 10 %, and 25 % cem / d128k; day 9 for 50 % cem / d128k; and day 12 for 75 % cem / d128k were analyzed. number of clones is combined from 2 independent infections; an 896 - nt vif region was sequenced. for round 6, proviral dna from cell cultures in which virus replication peaked ( day 9 ) was analyzed for 75 % cem / d128k from 6 independent flasks a – f. error bars represent sd. absence of selection for nl4 - 3 variants that can replicate in cem / d128k cells ( a ) nl4 - 3 virus was used to infect different ratios of cem: cem / d128k cells. 100 %, 90 %, 75 %, 50 %, 25 %, 10 %, and 0 % cem cells were mixed with 0 %, 10 %, 25 %, 50 %, 75 %, and 100 % cem / d128k cells, respectively. supernatants were collected every 2 to 3 days for 21 days and measured by p24 ca elisa to determine replication kinetics. average of two independent infections per ratio is indicated. error bars represent sd. ( viruses from peak production, normalized for p24 ca, from round 1 cultures 100 % cem ( day 7; indicated by a blue star in a ), 50 % cem: 50 % cem / d128k ( day 9; indicated by a red star in a ), and 25 % cem: 75 % cem / d128k ( day 12; indicated by a green star in a ) were used to infect either 100 % cem or 100 % cem / d128k cells. supernatants were collected every 2 to 3 days for up to 30 days, and p24 ca amounts were quantified by elisa to determine replication kinetics. infection of the parental cem cells showed wild - type nl4 - 3 peak virus at day 7 with no detectable virus measured in 100 % cem / d128k cells. averages of two independent infections per cell ratio are indicated. error bars represent sd. ( protocol to select for hiv - 1 resistance to a3g - d128k. ( viruses from round 6 ( independent flasks a – f ) were used to infect 100 % cem, 25 % cem: 75 % cem / d128k, or 100 % cem / d128k cells, and viral supernatants were collected every 2 to 3 days for up to 28 days. elisa was used to quantify p24 ca to determine replication kinetics. viruses from flasks a – f displayed wild - type kinetics in parental cem cells peaking at days 5 – 7 and showed delayed kinetics, peaking at days 9 – 12, in the 25 % cem: 75 % cem / d128k cell mix as was seen in round 1, as indicated in ( a ). no detectable virus was measured in 100 % cem / d128k cells. ( proviral dna from cell cultures in which virus replication peaked in round 1 and round 6 were analyzed for g - to - a hypermutation by sequencing an 896 - nt vif region. for round 1, proviral dnas from peak virus day 7 for 0 %, 10 %, and 25 % cem / d128k; day 9 for 50 % cem / d128k; and day 12 for 75 % cem / d128k were analyzed. number of clones is combined from 2 independent infections; an 896 - nt vif region was sequenced. for round 6, proviral dna from cell cultures in which virus replication peaked ( day 9 ) was analyzed for 75 % cem / d128k from 6 independent flasks a – f. error bars represent sd. next, viruses harvested on days of peak viremia from cultures containing 0 %, 50 %, and 75 % cem / d128k cells were used to infect either 100 % cem cells or 100 % cem / d128k cells ( figure 4b ). in all cases, the viruses replicated with wild - type kinetics in cem cells, indicating the presence of infectious virions, but no virus replication was detected in cem / d128k cells, indicating that a3g - d128k - resistant viral variants did not emerge. pcr amplification and sequencing of genomic dna from cells on days of peak virus production revealed extensive g - to - a hypermutation s from cell cultures in which > 25 % of the cells expressed a3g - d128k ( figure 4e, round 1 ).