we verified that endogenous a3g and a3f expression in cd4 t cell lines 174xcem ( hereinafter referred to as cem ) and pm1 was similar to that in h9 t cells ( figure 2a ); a3g and a3f expression in h9 cells was previously determined to be comparable to that in primary cd4 t cells. 46, 47, 48 the cem cells can be infected with cxcr4 - tropic hiv - 1 virions, while the pm1 cells can be infected with cxcr4 - tropic as well as ccr5 - tropic virions. either cem and pm1 cells were transduced with the a33g ( dk ) or a3x3g ( dk ) lentivectors and placed directly on puromycin selection, or eyfp cells were first sorted using flow cytometry and then placed on puromycin selection ( figure 2b ). western blotting analysis of puromycin - resistant pm1 ( pm1 / d128k ) or cem ( cem / d128k ) cell pools indicated that a3g - d128k reconstitution was efficient and that the a3g - d128k - p2a fusion protein was expressed ( figure 2c ). the undeleted a33g ( dk ) - p2a protein could be detected in 293t cells transfected with this vector using an anti - p2a or an anti - a3g antibod y but not in the pm1 / d128k and cem / d128k cell pools. the intensities of the endogenous a3g and the slightly larger a3g ( dk ) - p2a bands detected using an anti - a3g antibod y indicated that the a3g ( dk ) - p2a protein levels were slightly higher than that for the endogenous a3g protein in pm1 cells ( 1. 4 - to 1. 8 - fold ) and were at 20 % – 30 % of the endogenous a3g protein in the cem cells. stable a3g - d128k expression in the cem and pm1 cell lines did not have a detectable effect on their viability or growth kinetics ( figures s3a and s3b ). figure 2cem / d128k and pm1 / d128k cell lines ( a ) western blot analysis of endogenous expression levels of a3g and a3f in cem, pm1, and h9 t cell lines. ( protocol used to create a3g - d128k - expressing pm1 / d128k and cem / d128k cell lines. ( western blot analysis of puromycin - resistant cem / d128k and pm1 / d128k cell lines ( n = 5 ). upper blot probed with α - p2a antibod y ( green bands ) and α - tubulin antibod y ( red bands ). lower blot probed with α - a3g antibod y ( green bands ) and α - tubulin antibod y ( red bands ). two representative puromycin - resistant a3g - d128k - expressing cem and pm1 cell pools ( derived from a33g ( dk ) or a3x3g ( dk ) vector virus ) are indicated. lanes labeled a3g - d128k - p2a and a33g - d128k - p2a show deleted ~ 46 - kda and undeleted ~ 82 - kda bands from transfected 293t cells, respectively. cem / d128k and pm1 / d128k cell lines ( a ) western blot analysis of endogenous expression levels of a3g and a3f in cem, pm1, and h9 t cell lines. ( protocol used to create a3g - d128k - expressing pm1 / d128k and cem / d128k cell lines. ( western blot analysis of puromycin - resistant cem / d128k and pm1 / d128k cell lines ( n = 5 ). upper blot probed with α - p2a antibod y ( green bands ) and α - tubulin antibod y ( red bands ). lower blot probed with α - a3g antibod y ( green bands ) and α - tubulin antibod y ( red bands ). two representative puromycin - resistant a3g - d128k - expressing cem and pm1 cell pools ( derived from a33g ( dk ) or a3x3g ( dk ) vector virus ) are indicated. lanes labeled a3g - d128k - p2a and a33g - d128k - p2a show deleted ~ 46 - kda and undeleted ~ 82 - kda bands from transfected 293t cells, respectively. to determine whether stable expression of a3g - d128k inhibited hiv - 1 replication and spread, 4 cem / d128k pools and three pm1 / d128k pools ( generated from independent transductions with a3x3g ( dk ) virus ) were infected with equivalent p24 ca amounts of nl4 - 3 virus, and virus replication and spread were monitored by determining the amount of p24 ca in the culture supernatant every 2 to 3 days for 27 days ( figures 3a and 3b ). nl4 - 3 virus replicated in the parental cem and pm1 cells, and p24 ca amounts peaked at 7 days post - infection. however, no nl4 - 3 replication was detected in the cem / d128k and pm1 / d128k cells for 27 days, indicating that a3g - d128k expression potently inhibited nl4 - 3 replication. furthermore, no a3g - d128k - resistant viral variants emerged in 7 independent cultures for 27 days. similar inhibition of replication of an cxcr4 - tropic subtype ae recombinant ( 90cf402. 1. 8 ) was observed in the cem / d128k and pm1 / d128k cell pools for 24 – 30 days ( figures 3c and 3d, respectively ). inhibition of replication of ccr5 - tropic subtype c virus from molecular clone mj4 in the pm1 / d128k cells was also observed for 26 days ( figure 3e ), indicating that a3g - d128k expression in t cells can potently inhibit the replication and spread of multiple subtypes, including highly prevalent subtypes b, c, and ae recombinant. figure 3replication kinetics of group m viruses in cem / d128k and pm1 / d128k cell linesnormalized virus from subtype b ( nl4 - 3 ), recombinant subtype ae, and subtype c ( mj4 ) virus and patient - derived subtype b isolates were used to infect parental and a3g - d128k - expressing t cell lines. target cells were infected with virus ( ~ 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 up to 42 days post - infection. ( a and b ) replication kinetics of nl4 - 3 in parental ( a ) cem and ( b ) pm1 cells, with no detectable virus 27 days post - infection in 4 and three independent cem / d128k and pm1 / d128k cell lines, respectively. the averages of replication kinetics from duplicate flasks are indicated. error bars represent sd. ( c and d ) replication kinetics of recombinant subtype ae in parental ( c ) cem and ( d ) pm1 cells, with no detectable virus up to 30 days post - infection in cem / d128k and pm1 / d128k cell lines. replication kinetics from duplicate flasks are indicated for each cell line. ( replication kinetics of ccr5 - tropic subtype c clone mj4 in parental pm1 cells, with no detectable virus in pm1 / d128k cells 26 days post - infection. replication kinetics from duplicate flasks are indicated. ( replication kinetics of three subtype b patient isolates ( ptb - 1, - 2, and - 3 ) in cem cells. no detectable virus was measured in the cem / d128k cell line 42 days post - infection. the averages of replication kinetics from duplicate flasks are indicated. replication kinetics of group m viruses in cem / d128k and pm1 / d128k cell lines normalized virus from subtype b ( nl4 - 3 ), recombinant subtype ae, and subtype c ( mj4 ) virus and patient - derived subtype b isolates were used to infect parental and a3g - d128k - expressing t cell lines. target cells were infected with virus ( ~ 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 up to 42 days post - infection. ( a and b ) replication kinetics of nl4 - 3 in parental ( a ) cem and ( b ) pm1 cells, with no detectable virus 27 days post - infection in 4 and three independent cem / d128k and pm1 / d128k cell lines, respectively. the averages of replication kinetics from duplicate flasks are indicated. error bars represent sd. ( c and d ) replication kinetics of recombinant subtype ae in parental ( c ) cem and ( d ) pm1 cells, with no detectable virus up to 30 days post - infection in cem / d128k and pm1 / d128k cell lines. replication kinetics from duplicate flasks are indicated for each cell line. ( replication kinetics of ccr5 - tropic subtype c clone mj4 in parental pm1 cells, with no detectable virus in pm1 / d128k cells 26 days post - infection. replication kinetics from duplicate flasks are indicated. ( replication kinetics of three subtype b patient isolates ( ptb - 1, - 2, and - 3 ) in cem cells. no detectable virus was measured in the cem / d128k cell line 42 days post - infection. the averages of replication kinetics from duplicate flasks are indicated.