subsequently, the reverse transcriptase and the growing point of the nascent dna dissociate from the 3 ′ direct repeat and anneal to the 5 ′ direct repeat, resulting in the deletion of one copy of the direct repeat and any intervening sequences. because the a3 and 3g portions of a3g do not express a catalytically active a3g, functional a3g - d128k is not expressed in the virus - producing cells and, therefore, cannot inhibit virion infectivity, but a reconstituted a3g - d128k is expressed in the target cells, leading to inhibition of subsequent rounds of hiv - 1 replication. figure 1a3g - d128k direct repeat vectors, transduction, and frequency of a3g reconstitution ( a ) lentiviral vectors pa3x3g ( dk ) and pa33g ( dk ) contain an overlapping ~ 900 - bp homologous region of a3g - d128k ( “ 3, ” indicated by black arrows ). a3 and 3g are the 5 ′ and 3 ′ fragments of a3g - d128k, respectively, separated by three stop codons ( + stop; “ x ” ) or no stop codons ( - stop ), fused in frame with the self - cleaving porcine teschovirus - 1 2a peptide ( p2a ) and enhanced yellow fluorescent protein ( eyfp ). an internal promoter from cytomegalovirus ( cmv ), an internal ribosomal entry site ( ires ), and a puromycin ( puro ) - selectable marker are present, followed by the woodchuck hepatitis virus post - transcriptional regulatory element ( wpre ). these vectors contain intact long terminal repeats ( ltrs ). indicated below the vector are the protein products that are expected following viral infection, direct repeat deletion during reverse transcription, translation, and p2a cleavage. rre, rev response element; ψ, hiv - 1 rna packaging signal; cppt, central polypurine tract. ( vector peyfpip expresses eyfp, and vector pa3g ( dk ) expresses the intact a3g - d128k fused in frame with p2a and eyfp. vectors pa3dr ( dk ) and p3gdr ( dk ) express the a3 and 3g portions of the a3g - d128k direct repeat, respectively. ( normalized p24 capsid protein ( ca ) from hl [ wt ] virus made in the presence of cmv - driven vectors expressing a3g - d128k or a3g - d128k - p2a - eyfp was used to transduce 293t target cells. indicated are the average luciferase light units normalized to hl [ wt ] in the absence of a3g - d128k ( 100 % ) ( n = 3 ). ( normalized p24 ca from each vector virus was used to transduce 293t target cells; indicated is the percentage of eyfp cells 48 h post - transduction normalized to eyfpip ( 100 % ) ( n = 3 ). ( normalized p24 ca from vector virus pa3g ( dk ), pa33g ( dk ), and pa3x3g ( dk ) produced in the presence or absence of vif2 was used to transduce 293t target cells, and the proportion of eyfp cells 48 - h post - transduction was determined. ( western blot analysis of cell lysates and virions showing expression and encapsidation of a3g ( dk ) p2a and / or a33g ( dk ) p2a. ( puromycin - resistant pools of 293t cells transduced with a3x3g ( dk ) virus were analyzed for the deletion frequency of the a3g - d128k ~ 900 - bp direct repeat. primers flanking a3g ( dk ) were used for pcr amplification, with positive plasmid controls indicated for a3g ( dk ) ( 2, 190 bp ) and undeleted a33g ( dk ) ( 1, 290 bp ). band density analysis indicates an average 98 % direct repeat deletion frequency for 4 independent pools ( a – d ). low levels of deletion were detected with the plasmid a33g ( dk ) control due to direct repeat deletion during pcr ( 14 % ). ( puro selected 293t cell clones transduced with a33g ( dk ) virus. primers flanking a3g ( dk ) were used for pcr amplification with plasmid controls shown for a3g ( dk ) ( 1, 864 bp ) and undeleted a33g ( dk ) ( 964 bp ). pcr analysis shows an average 89 % direct repeat deletion frequency from three independent transductions ( 69 total clones ). a3g - d128k direct repeat vectors, transduction, and frequency of a3g reconstitution ( a ) lentiviral vectors pa3x3g ( dk ) and pa33g ( dk ) contain an overlapping ~ 900 - bp homologous region of a3g - d128k ( “ 3, ” indicated by black arrows ). a3 and 3g are the 5 ′ and 3 ′ fragments of a3g - d128k, respectively, separated by three stop codons ( + stop; “ x ” ) or no stop codons ( - stop ), fused in frame with the self - cleaving porcine teschovirus - 1 2a peptide ( p2a ) and enhanced yellow fluorescent protein ( eyfp ). an internal promoter from cytomegalovirus ( cmv ), an internal ribosomal entry site ( ires ), and a puromycin ( puro ) - selectable marker are present, followed by the woodchuck hepatitis virus post - transcriptional regulatory element ( wpre ). these vectors contain intact long terminal repeats ( ltrs ). indicated below the vector are the protein products that are expected following viral infection, direct repeat deletion during reverse transcription, translation, and p2a cleavage. rre, rev response element; ψ, hiv - 1 rna packaging signal; cppt, central polypurine tract. ( vector peyfpip expresses eyfp, and vector pa3g ( dk ) expresses the intact a3g - d128k fused in frame with p2a and eyfp. vectors pa3dr ( dk ) and p3gdr ( dk ) express the a3 and 3g portions of the a3g - d128k direct repeat, respectively. ( normalized p24 capsid protein ( ca ) from hl [ wt ] virus made in the presence of cmv - driven vectors expressing a3g - d128k or a3g - d128k - p2a - eyfp was used to transduce 293t target cells. indicated are the average luciferase light units normalized to hl [ wt ] in the absence of a3g - d128k ( 100 % ) ( n = 3 ). ( normalized p24 ca from each vector virus was used to transduce 293t target cells; indicated is the percentage of eyfp cells 48 h post - transduction normalized to eyfpip ( 100 % ) ( n = 3 ). ( normalized p24 ca from vector virus pa3g ( dk ), pa33g ( dk ), and pa3x3g ( dk ) produced in the presence or absence of vif2 was used to transduce 293t target cells, and the proportion of eyfp cells 48 - h post - transduction was determined. ( western blot analysis of cell lysates and virions showing expression and encapsidation of a3g ( dk ) p2a and / or a33g ( dk ) p2a. ( puromycin - resistant pools of 293t cells transduced with a3x3g ( dk ) virus were analyzed for the deletion frequency of the a3g - d128k ~ 900 - bp direct repeat. primers flanking a3g ( dk ) were used for pcr amplification, with positive plasmid controls indicated for a3g ( dk ) ( 2, 190 bp ) and undeleted a33g ( dk ) ( 1, 290 bp ). band density analysis indicates an average 98 % direct repeat deletion frequency for 4 independent pools ( a – d ). low levels of deletion were detected with the plasmid a33g ( dk ) control due to direct repeat deletion during pcr ( 14 % ). ( puro selected 293t cell clones transduced with a33g ( dk ) virus. primers flanking a3g ( dk ) were used for pcr amplification with plasmid controls shown for a3g ( dk ) ( 1, 864 bp ) and undeleted a33g ( dk ) ( 964 bp ). pcr analysis shows an average 89 % direct repeat deletion frequency from three independent transductions ( 69 total clones ). vectors pa33g ( dk ) and pa3x3g ( dk ) were constructed with and without stop codons between a3 and 3g and fused with a self - cleaving porcine teschovirus - 1 2a peptide ( p2a ) and enhanced yellow fluorescent protein ( eyfp ); the vectors also expressed the puromycin - resistance gene ( puro ) from an internal ribosomal entry site ( ires ) ( figure 1a ). control vectors were also constructed that expressed eyfp ( peyfpip ), a3g - d128k ( pa3g ( dk ) ), and the a3 ( pa3dr ( dk ) ) or the 3g portions ( p3gdr ( dk ) ) of a3g - d128k ( figure 1b ). control vector pcmv - a3g ( dk ) expressed an untagged a3g - d128k from a cytomegalovirus ( cmv ) promoter, whereas the pcmv - a3g ( dk ) - p2a - eyfp vector expressed a3g - d128k - p2a fusion protein and eyfp after cleavage between p2a and eyfp ( figure 1c ). to compare the antiviral activity of a3g - d128k - p2a with a3g - d128k, virus was prepared from cells that expressed a3g - d128k, a3g - d128k - p2a fusion protein, or no a3g in the presence of the hiv - 1 luciferase vector, phl [ wt ].