The barrier-to-autointegration factor is a component of functional human immunodeficiency virus type 1 preintegration complexes

Abstract

Retroviral integration in vivo is mediated by preintegration complexes (PICs) derived from infectious virions. In addition to the integrase enzyme and cDNA substrate, PICs contain a variety of viral and host cell proteins. Whereas two different cell proteins, high-mobility group protein A1 (HMGA1) and the barrier-to-autointegration factor (BAF), were identified as integration cofactors based on activities in in vitro PIC assays, only HMGA1 was previously identified as a PIC component. By using antibodies against known viral and cellular PIC components, we demonstrate here functional coimmunoprecipitation of endogenous BAF protein with human immunodeficiency virus type 1 (HIV-1) PICs. Since integrase protein and integration activity were also coimmunoprecipitated by anti-BAF antibodies, we conclude that BAF is a component of HIV-1 PICs. These data are consistent with the model that BAF functions as an integration cofactor in vivo.

FIG. 1.

Functional coimmunoprecipitation strategy. Cytoplasmic extracts (fraction I) of acutely infected T cells were subjected to a variety of purification steps to investigate the association of human BAF with HIV-1 PICs. In arm A of the scheme, fraction I was directly immunoprecipitated. Extracts purified by either ultrafiltration or gel filtration chromatography (fraction II) were immunoprecipitated in arm B of the scheme. Whereas integration activity was quantified following Southern blotting as previously described (7, 8), levels of integrase and BAF proteins in fraction PB were detected by Western blotting. PA and SA, pellet and supernatant fractions following immunoprecipitation in arm A of the scheme, respectively; PB and SB, pellet and supernatant fractions after immunoprecipitation of fraction II, respectively.

FIG. 2.

Analysis of PIC activity and integrase and BAF proteins following immunoprecipitation with MAb 3H7. (A) Levels of HIV-1 cDNA synthesis in fraction I (lane 1) and integration activity of various fractions analyzed by Southern blotting. Target DNA was added to the in vitro integration assays shown in lanes 2 and 4 to 7 as indicated. The migration positions of the 9.7-kb HIV-1 substrate and the 15.1-kb integration product are marked cDNA and IP, respectively. Percentage of total cDNA recovered in pellet fractions following immunoprecipitation is indicated beneath lanes 3, 4, and 6. Levels of fraction I (lane 2), PA (lane 4), SA (lane 5), PB (lane 6), and SB (lane 7) integration activities are also indicated beneath the blot. (B) Western blot analysis of integrase and BAF proteins. Whereas integrase was detected by using MAb 8E5 (31) as previously described (8), BAF was detected by using affinity-purified rabbit antiserum at 1:1,000 dilution. The identities of the integrase and BAF proteins in the lane marked 3H7 were confirmed by comigration with purified recombinant proteins. fI, fraction I; Ab, antibody; IN, integrase; n.a., not applicable. Other labeling is the same as that defined in the legend to Fig. 1.

FIG. 3.

Coimmunoprecipitation of integration activity and integrase and BAF proteins by using anti-Ku and anti-Vpr antibodies. (A) Southern blot analysis of HIV-1 cDNA synthesis (lane 1) and PIC activity. Target DNA was included in the in vitro integration reactions shown in lanes 2, 4, and 7 to 12. Whereas fraction I was analyzed in lanes 1 and 2, the fraction II samples shown in lanes 3 to 12 were purified by spin column chromatography. We note that the faint integration product in lane 11 was not revealed by this level of exposure. (B) Western blot analysis of integrase and BAF proteins. fII, fraction II; rIgG, normal rabbit IgG. Other labeling is the same as that described in the legend to Fig. 2.

FIG. 4.

Coimmunoprecipitation of integration activity and integrase protein by anti-BAF_4-20 antibodies. (A) Southern blot analysis. Target DNA was included in the integration reactions depicted in lanes 2, 4, 6, and 7. Whereas fraction I was analyzed in lanes 1 and 2, the fraction II samples in lanes 3 to 7 were purified by spin column chromatography. (B) Western blot analysis. Labeling is the same as that described in the legends to Fig. 2 and 3.

FIG. 5.

Competition of anti-BAF_4-20-mediated PIC immunoprecipitation by recombinant BAF proteins. (A) Western blot analysis of integrase coimmunoprecipitation in the absence of added BAF (lane 1) or in the presence of 10 nM (lane 2) and 100 nM (lane 3) recombinant human BAF. The excess recombinant BAF in the reaction shown in lane 3 contributed to the increased BAF signal. (B) Differential effects of BAF DNA binding mutants on PIC immunoprecipitation. For lane 1, normal rabbit IgG was used in the immunoprecipitation reaction; lanes 2 to 5, anti-BAF_4-20 was used in the immunoprecipitation reaction. Whereas recombinant BAF protein was omitted from the reactions shown in lanes 1 and 2; lanes 3 to 5 contained 100 nM wild-type, K6A, and K18A BAF, respectively. The longer exposure of the anti-BAF Western shown in the lower boxed panel highlights the remaining endogenous BAF protein in lane 5. WT, wild-type; rBAF, recombinant BAF. Other labeling is the same as that described in the legends to Fig. 2 and 3.