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

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-toautointegration factor (BAF) protein restored integration activity, substantially more (5-10 M) human host factor HMG I(Y) was required. Similarly high levels (3-8 M) 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.

Barrier to Autointegration Factor Interacts with the Cone-Rod Homeobox and Represses Its Transactivation Function

“…These results suggest that Baf and Crx interact in vivo. Baf was originally identified as a host cellular factor that plays a role in preventing retroviral DNA from autointegration (integrating into the virus' own genome) and in the formation of preintegration complexes (PICs) during retroviral infection (42,50,51). Baf is evolutionarily conserved from Caenorhabditis elegans to mammals (52) (Fig.…”

Both the Structure and DNA Binding Function of the Barrier-to-Autointegration Factor Contribute to Reconstitution of HIV Type 1 Integration in Vitro

“…Two different proteins, HMG I(Y) (4,5) and the barrier-to-autointegration factor (BAF) (6), were identified by purifying host cell extracts and assaying for reconstitution of salt-disrupted PIC integration activity in vitro. Both HMG I(Y) and BAF display reconstitution activity following their expression and purification from Escherichia coli (3)(4)(5)(6)(7). For both MMLV and HIV-1, recombinant BAF was about 500-fold more active than recombinant HMG I(Y) at reconstituting the integration activity of saltwashed PICs (3,5,7).…”

“…Moloney murine leukemia virus (MMLV) and human immunodeficiency virus type 1 (HIV-1) PICs treated with elevated concentrations of salt and subsequently purified by size exclusion chromatography and nonionic density gradient centrifugation lost their normal intermolecular integration activity (2,3). Adding back protein extracts of uninfected host cells to such salt-depleted PICs restored normal integration activity, showing that host proteins play essential roles in retroviral PIC function in vitro (2)(3)(4)(5). Two different proteins, HMG I(Y) (4,5) and the barrier-to-autointegration factor (BAF) (6), were identified by purifying host cell extracts and assaying for reconstitution of salt-disrupted PIC integration activity in vitro.…”

“…Both HMG I(Y) and BAF display reconstitution activity following their expression and purification from Escherichia coli (3)(4)(5)(6)(7). For both MMLV and HIV-1, recombinant BAF was about 500-fold more active than recombinant HMG I(Y) at reconstituting the integration activity of saltwashed PICs (3,5,7). BAF was not detected in MMLV virions (2,6), leading to models wherein it associates with retroviruses during the processes of reverse transcription and PIC formation in infected cells (3,6).…”

Interaction of the HIV-1 Intasome with Transportin 3 Protein (TNPO3 or TRN-SR2)

“…Inspection of the resulting model revealed the need for manual rebuilding of the loop 841-855; the rebuilt loop was subsequently minimized in vacuo. The overall quality of this minimized model was evaluated using the MOLPROBITY server (55). 98.6% of the residues resided in the allowable regions of a Ramachandran plot and showed generally good stereochemical properties.…”