Experimental strategy to assess integrase catalytic activities and PIC protein content. Cytoplasmic extract of HIV-1-infected cells was processed in the indicated ways to determine levels of integrase 3′ processing activity that occurred in infected cells, levels of in vitro DNA strand transfer activity, and levels of PIC-associated proteins as detected by MM-PCR footprinting and Western blotting. IN, integrase.

Strategy for detecting 3′ processing activity in infected cell extracts. HaeIII and HindIII cleave HIV-1 cDNA approximately 100 bp from the U3 and U5 ends, respectively. 3′ processing by integrase shortens the U3 minus strand and U5 plus strand by 2 nucleotides (nt). Following cell lysis and cleavage with HaeIII and HindIII, both processed and nonprocessed strands were detected by indirect end labeling using strand-specific riboprobes.

MM-PCR footprints of wild-type, 604del, and revE1 PICs. (A) The U3 end of HIV-1. In lane 1, naked plasmid DNA was used as the target for Mu transposition. Lane 2, deproteinized wild-type cDNA; lane 3, native wild-type PICs; lane 4, deproteinized 604del cDNA; lane 5, native 604del PICs; lane 6, deproteinized revE1 cDNA; lane 7, revE1 PICs. E, region of transpositional enhancements; F, footprinted regions. Although the deproteinized 604del cDNA in lane 4 did not amplify well, results of other experiments showed that this pattern of Mu transposition was indistinguishable from the wild-type and revE1 deproteinized cDNA patterns in lanes 2 and 6, respectively. (B) The U5 end. The samples in lanes 1 to 7 were the same as those in panel A. WT-E, the wild-type region of transpositional enhancements; WT-F, the wild-type footprinted region; 604-E, 604del region of weak transpositional enhancements; rev-E, the revE1 region of transpositional enhancements; rev-F, the revE1 footprinted region. The U5 ends of 604del and revE1 were shorter in panel B due to the 26- and 47-bp deletions, respectively. Due to the polarity of Mu transposition, only the very ends of the nonprocessed U3 and U5 strands can be analyzed by MM-PCR (8, 39).

Two-LTR circle content of cells infected with wild type, 604del, and integrase mutants Q62K and D116N. Lane 1, lysate from cells infected with wild type; lane 2, lysate from 604del-infected cells; lane 3, lysate from Q62K-infected cells; lane 4, lysate from D116N-infected cells; lane 5, lysate from mock-infected cells. 222 and 196 indicate the sizes of PCR products for wild type and 604del, respectively, in base pairs.

In vitro DNA strand transfer activity of wild-type, 604del, and revE1 PICs. The integration reactions contained wild-type PICs (lane 1), 604del PICs (lane 2), and revE1 PICs (lane 3). cDNA, 9.7-kb HIV-1 integration substrate; IP, 15.1-kb integration product.

Structure of wild-type, 604del, and revE1 cDNA ends and detection of in vivo 3′ processing activity. (A) The processed U5 plus strand. Lane 1, DNA from wild-type-infected cells; lane 2, DNA from 604del-infected cells; lane 3, DNA from revE1-infected cells. WT(+)sss, cleavage product of wild-type plus-strand strong-stop DNA; WTun, the unprocessed wild-type strand (105 nucleotides); WTpro, the processed wild-type strand (103 nucleotides); 604(+)sss, cleavage product of 604del plus-strand strong-stop DNA; 604un, the unprocessed 604del strand (79 nucleotides); 604pro, the processed 604del strand (77 nucleotides); REV(+)sss, cleavage product of revE1 plus-strand strong-stop DNA; REVun, the unprocessed revE1 strand (60 nucleotides); REVpro, the processed revE1 strand (58 nucleotides). (B) The processed U3 minus strand. The samples in lanes 1 to 3 were the same as those in panel A. un, the 103-nucleotide unprocessed minus strand; pro, the processed 101-nucleotide product. (C) The nonprocessed U5 minus strand. The samples in lanes 1 to 3 were the same as those in panel A. The wild-type, 604del, and revE1 nonprocessed strands are indicated as WTnon, 604non, and REVnon, respectively. The higher-molecular-weight bands in lanes 1 to 3 display mobilities consistent with the minus-strand strong-stop products of each of the viruses. (D) The nonprocessed U3 plus strand. The samples in lanes 1 to 3 were the same as those in panel A. non, the 103-nucleotide nonprocessed strand. For panels A and C, the DNA levels in lanes 2 and 3 appear lower than the levels in lanes 1 because the 604del and revE1 deletions removed U5 sequences that are present in these wild-type-derived riboprobes. The sizes of the unprocessed, processed, and nonprocessed U5 and U3 strands were confirmed by comparing their migration distances to those of an M13 sequencing ladder (31).

Integrase protein content of gradient-purified wild-type, 604del, and revE1 PICs. Lane 1, 50 ng of recombinant HIV-1 integrase; lane 2, 25 ng of recombinant integrase; lane 3, gradient-purified wild-type PICs; lane 4, 604del PICs; lane 5, revE1 PICs. IN, integrase.