relative luciferase activities in transduced cells showed that expression of a3g - d128k or a3g - d128k - p2a potently inhibited lentivector hl [ wt ] transduction to 6 % and 7 %, respectively, compared to the lentivector prepared in the absence of a3g ( figure 1c ), indicating that the presence of the p2a tag did not significantly affect the antiviral activity of a3g - d128k. to test whether the a3 and 3g portions of a3g - d128k retained any antiviral activity, virions produced from eyfpip, a3dr ( dk ), and 3gdr ( dk ) vectors were normalized for p24 capsid protein ( ca ) amounts and used to transduce 293t cells ( figure 1d ). flow cytometry analysis of the transduced cells showed that expression of the a3 ( a3dr ) or 3g ( 3gdr ) truncated proteins of a3g - d128k in the lentivector producer cells resulted in < 2 - fold inhibition of transduction ( ∼50 % and ∼87 % eyfp cells compared to cells transduced with eyfpip, respectively ). expression of a3dr resulted in ∼50 % inhibition of transduction, suggesting that a3dr may be packaged into the virions and suppress lentivector transduction by inhibition of dna synthesis. in future studies, it may be possible to shorten the a3 portion and shift the ∼900 - bp direct repeat to suppress the ∼2 - fold inhibition of lentivector transduction. a3g - d128k transduction efficiency was determined for vectors with ( a3x3g ( dk ) ) and without ( a33g ( dk ) ) stop codons between the direct repeats by transducing 293t cells with serial dilutions of viruses ( figure 1e ). recombination between the repeated regions during transfection of the virus - producing cells could potentially reconstitute a3g - d128k; therefore, viruses were prepared in the presence or absence of hiv - 2 vif ( vif2 ), which can induce degradation of a3g - d128k. the results showed that, in the absence of vif2 co - expression, the transduction efficiency with the a3g ( dk ) vector was nearly undetectable, while the transduction efficiency with the a33g ( dk ) and a3x3g ( dk ) viruses was increased to 28 % and 49 %, respectively ( compare cells transduced with 20 ng p24 ca - containing virus ). co - expression of vif2 increased the transduction efficiencies of the a3g ( dk ), a33g ( dk ), and a3x3g ( dk ) viruses to 8 %, 65 %, and 73 %, respectively. the results indicated that the stop codons between the a3 and 3g direct repeats in the a3x3g ( dk ) vector prevented expression of a a33g - d128k fusion protein that retained antiviral activity and resulted in the highest amounts of infectious lentivectors that expressed eyfp in the target cells. next, we analyzed the expression of a3g - d128k and a33g - d128k in the lentivector producer cells and their incorporation into virions by western blotting ( figure 1f ). a3g - d128k - p2a and a33g - d128k - p2a fusion proteins were readily detected in the absence of vif2 ( lanes 1 – 3 and 7 – 9, respectively ) or the presence of vif2 ( lanes 4 – 6 and 10 – 12, respectively ). a3g - d128k - p2a was also detected at a very low level in cells transfected with a33g ( dk ) in the absence of vif2 ( lanes 7 – 9 ) but not in the presence of vif2 ( lanes 10 – 12 ), confirming that a3g - d128k was reconstituted at a low efficiency by dna recombination during transfection and that the a33g - d128k fusion protein was targeted for degradation by vif2. virion incorporation of a33g - d128k - p2a or the reconstituted a3g - d128k - p2a could not be detected because of low levels of virion incorporation and sensitivity of detection. a3g - d128k - p2a was efficiently packaged into virions, and co - expression of vif2 substantially reduced virion incorporation of a3g - d128k but did not eliminate it, since a faint band was detectable upon overexposure of the blot ( data not shown ). to determine the frequency of a3g - d128k reconstitution during reverse transcription, 4 independent pools of 293t cells transduced with the a3x3g ( dk ) virus were generated, and their genomic dna was used to pcr - amplify proviral dna using primers flanking the a3x3g sequence ( figure 1g ). the results showed that most of the amplified dna ( ∼98 % ) generated a 1, 290 - bp band labeled a3g ( dk ) that was expected from direct repeat deletion, while a 2, 190 - bp band that was expected in the absence of deletion was undetectable. pcr amplification of the a33g ( dk ) vector dna indicated that direct repeat deletion occurred in ∼14 % of the amplified dna fragments, indicating that most of the direct repeat deletion occurred during reverse transcription and not during pcr amplification. to determine the frequency of a3g - d128k gene reconstitution more precisely, 69 single - cell clones of transduced cells that were selected for puromycin resistance were isolated and analyzed by pcr amplification ( figure 1h ). a3g - d128k reconstitution was observed in 61 of the 69 cell clones, providing a reconstitution frequency of 89 %, which was similar to the previously observed > 87 % deletion frequency for a 971 - bp repeated sequence. we determined the extent to which proviral dnas in target cells transduced with the a3g ( dk ), a33g ( dk ), and a3x3g ( dk ) vectors were hyper mutate d by pcr amplification and sequencing of a 982 - bp proviral dna fragment ( figure s2; table 1 ). in the absence of vif2 co - expression, a high frequency of a3g ( dk ) proviral dnas was hyper mutate d ( 52 / 52 clones ), a33g ( dk ) proviral dnas were hyper mutate d to a lesser extent ( 28 / 55 clones ), and very few a3x3g ( dk ) proviral dnas were hyper mutate d ( 3 / 63 clones ). interestingly, the higher frequency of hypermutation observed for the a33g ( dk ) proviral dnas compared to the a3x3g ( dk ) vector suggested that the a33g - d128k fusion protein was expressed, incorporated into virions, and retained cytidine deaminase activity. co - expression of vif2 resulted in lower hypermutation frequencies for all three vectors, although a high frequency of hypermutation was still observed for a3g ( dk ) proviral genomes ( 40 / 60 clones ). proviral genomes derived from the a3x3g ( dk ) vector had ∼65 - fold and ∼164 - fold fewer g - to - a mutations than the proviral genomes from the a3g ( dk ) vector in the absence or presence of vif2, respectively ( table 1 ). overall, the a3x3g ( dk ) self - activating vector, in the presence of vif2 co - expression, efficiently suppressed a3g - d128k expression in the lentivector producer cells and inactivation of the viral vector by hypermutation. table 1frequency of hyper mutate d clones and g - to - a hypermutation profile for a3g - d128k, a33g - d128k, and a3x3g - d128k virus produced in the absence or presence of vif2vector and presence or absence of vif2no. of hyper mutate d clones / total clones sequenced ( % ) average g - to - a mutations / hyper mutate d clonedi nucleot ide context of g - to - a mutationstotal g - to - a mutationstotal g - to - a mutations / total clones sequencedgggagcgta3g ( dk ) −52 / 52 ( 100 % ) 40. 31, 85122747862, 11640. 7 + 40 / 60 ( 66. 7 % ) 18. 56784461073812. 3a33g ( dk ) −28 / 55 ( 50. 9 % ) 13. 131916383466. 3 + 3 / 84 ( 3. 6 % ) 15. 867872841a3x3g ( dk ) −3 / 63 ( 4. 7 % ) 11. 635122400. 63a + 0 / 53 ( 0 % ) 0112040. 075aafold difference in g - to - a mutations for a3g ( dk ) versus a3x3g ( dk ) in the absence of vif2 ( 40. 7 / 0. 63 = 65 - fold ) or presence of vif2 ( 12. 3 / 0. 075 = 164 - fold ). frequency of hyper mutate d clones and g - to - a hypermutation profile for a3g - d128k, a33g - d128k, and a3x3g - d128k virus produced in the absence or presence of vif2 fold difference in g - to - a mutations for a3g ( dk ) versus a3x3g ( dk ) in the absence of vif2 ( 40. 7 / 0. 63 = 65 - fold ) or presence of vif2 ( 12. 3 / 0. 075 = 164 - fold ).