Figure 3.
Two distinct IN complexes are detected following cell infection. SupT1 cells were either not infected (A) or infected with HIV-1IN-HA, and WCE were prepared at 2 h (B) and 6 h p.i. (C). WCE were subjected to gel filtration on a Superdex 200HR 10/300 column. Fractions were collected and analyzed by Western blotting using antibodies against LEDGF/p75, TNPO3, HA, MA, and CA. Inputs represent 3.5& of WCE load. Two complexes containing IN were detected, a high molecular weight complex (IN complex I, >1.3 MDa), and a low molecular weight complex (IN complex II, ~440KDa). To detect IN-HA at 2h p.i. in (B), the film was exposed for 5 minutes, whereas detection of IN-HA at 6h p.i. in (C) required film exposure of 30 minutes. (D) Viral DNA co-elutes with IN complex I. Same as (A) and (B) except DNA was extracted from each fraction and late reverse transcription product (LRT) content was quantified by real time PCR. Non-infected (NI) samples were used as controls (not shown). Results shown are representative of two independent experiments. Dividing lines indicate the grouping of parts of the different gels with identical times of film exposure.