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. 1998 Dec 22;95(26):15270-4.
doi: 10.1073/pnas.95.26.15270.

The barrier-to-autointegration protein is a host factor for HIV type 1 integration

H Chen  1 , A Engelman
Affiliations

The barrier-to-autointegration protein is a host factor for HIV type 1 integration

H Chen et al. Proc Natl Acad Sci U S A. .

Abstract

In vivo, retroviral integration is mediated by a large nucleoprotein complex, termed the preintegration complex (PIC). PICs isolated from infected cells display in vitro integration activity. Here, we analyze the roles of different host cell factors in the structure and function of HIV type 1 (HIV-1) PICs. PICs purified by size exclusion after treatment with high salt lost their integration activity, and adding back an extract from uninfected cells restored this activity. In parallel, the native protein-DNA intasome structure detected at the ends of HIV-1 by Mu-mediated PCR footprinting was abolished by high salt and restored by the crude cell extract. Various purified proteins previously implicated in retroviral PIC function then were analyzed for their effects on the structure and function of salt-treated HIV-1 PICs. Whereas relatively low amounts (5-20 nM) of human barrier-to-autointegration factor (BAF) protein restored integration activity, substantially more (5-10 microM) human host factor HMG I(Y) was required. Similarly high levels (3-8 microM) of bovine RNase A, a DNA-binding protein used as a nonspecific control, also restored activity. Mu-mediated PCR footprinting revealed that of these three purified proteins, only BAF restored the native structure of the HIV-1 protein-DNA intasome. We suggest that BAF is a natural host cofactor for HIV-1 integration.

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Figures

Figure 1
Figure 1
Functional reconstitution of salt-stripped HIV-1 PICs with uninfected cell extract. Integration reactions were deproteinized and analyzed by Southern blotting. Lane 1, integration activity of untreated, gradient-purified PICs. Lane 2, HIV-1 PICs treated with 1.2 M KCl and then purified by spin column chromatography and gradient centrifugation before the integration assay. Lane 3, BSA included in the reconstitution buffer. Lanes 4–6 included 5, 10, and 15 μl, of crude cell extract, corresponding to approximately 35, 70, and 105 μg of total protein, respectively. cDNA, 9.7-kb HIV-1 linear DNA substrate; IP, 15.1-kb integration product. Approximately 3-fold more material was loaded in lanes 2–6, accounting for the increased level of cDNA.
Figure 2
Figure 2
Structural reconstitution of the HIV-1 intasome with uninfected cell extract. Mu transposition reactions were deproteinized and analyzed by denaturing sequencing gels after two rounds of PCR. The U3 end of HIV-1 DNA was analyzed in A, and the U5 end was analyzed in B. Lanes 1, the pattern of Mu transposition into deproteinized cDNA. Lanes 2, the native structure of the HIV-1 intasome. Note the regions of strong transpositional enhancements (E) and DNA footprints (F) relative to the deproteinized control. Lanes 3, salt-stripped PICs. Note the similarity in patterns between the salt-stripped and deproteinized PICs. Lanes 4, reconstitution buffer control. Lanes 5, salt-stripped PICs reconstituted with 5 μl of uninfected cell extract. The reaction products were run alongside DNA-sequencing ladders to determine the distance from the ends of HIV-1 (nucleotide position 1 in U3 and position 9,718 in U5). The U3 and U5 footprints extended approximately 250 and 200 bp, respectively, in the native structure.
Figure 3
Figure 3
Functional reconstitution of salt-stripped HIV-1 PICs by using purified proteins. Integration reactions were deproteinized and analyzed by Southern blotting. Lane 1, activity of PICs partially purified under native (150 mM KCl) conditions. Lane 2, activity of salt-stripped PICs. Lanes 3–7 included 6.25, 12.5, 25, 50, and 100 ng of BAF per reaction. Lanes 8–12 included 500, 2,000, 8,000, 16,000, and 32,000 ng of HMG I(Y) per reaction. Lanes 13–16 contained 500, 2,000, 8,000, and 16,000 ng of RNase A per reaction. Neither HMG I(Y) nor RNase A was tested at lower protein concentrations. Other labeling is the same as in Fig. 1.
Figure 4
Figure 4
Structural reconstitution of the HIV-1 intasome with purified BAF. Salt-stripped PICs were reconstituted with the indicated proteins and analyzed by MM-PCR footprinting. A and B present the results for the U3 and U5 ends, respectively. Lanes 1–4, as indicated. Lanes 5–7 included 12.5, 25, and 50 ng of BAF per reaction. The integration activities of these reconstitution mixtures were undetectable, and about 8% and 35% of the activity of the untreated native sample was detected, respectively (data not shown). Lanes 8–10 included 2,000, 8,000, and 16,000 ng of HMG I(Y) per reaction. These mixtures displayed undetectable, and about 7% and 11% of the activity of the untreated sample, respectively (data not shown). Lanes 11–13 contained 2,000, 8,000, and 16,000 ng of RNase A per reaction. The mixture containing 2 μg of RNase A displayed about 20% of the activity of the untreated sample; the mixture with 16 μg displayed about 35% activity. Other labeling is the same as in Fig. 2.
Figure 5
Figure 5
BAF and HMG I(Y) inhibit each other’s activity. Reconstituted salt-stripped PICs were tested for integration activity. Lane 1, activity of the preparation before salt stripping (approximately 25% of substrate converted to product). Lane 2 contained 50 ng of BAF (about 8% of substrate converted to product). Lanes 3–5 contained 1, 5, and 25 μg of HMG I(Y), respectively. About 5% integration activity was recovered in lane 5. In lanes 6–8, 50 ng of BAF and 1, 5, and 25 μg of HMG I(Y), respectively, were added simultaneously to the reconstitution reactions. The approximate levels of activity in lanes 6–8 were 9, 5, and 3%, respectively. In lane 9, 50 ng of BAF was preincubated with the stripped PICs for 30 min, and then 25 μg of HMG I(Y) was added (about 2% activity recovered); lane 10, the order of addition in lane 9 was reversed (about 4% activity recovered). In lane 11, 50 ng of BAF was preincubated, followed by 5 μg of HMG I(Y) (about 8% activity recovered); lane 12, the order of addition in lane 11 was reversed (about 8% activity recovered). Other labeling is the same as in Fig. 1.
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