Host proteins interacting with the Moloney murine leukemia virus integrase: multiple transcriptional regulators and chromatin binding factors

Abstract

Background: A critical step for retroviral replication is the stable integration of the provirus into the genome of its host. The viral integrase protein is key in this essential step of the retroviral life cycle. Although the basic mechanism of integration by mammalian retroviruses has been well characterized, the factors determining how viral integration events are targeted to particular regions of the genome or to regions of a particular DNA structure remain poorly defined. Significant questions remain regarding the influence of host proteins on the selection of target sites, on the repair of integration intermediates, and on the efficiency of integration.

Results: We describe the results of a yeast two-hybrid screen using Moloney murine leukemia virus integrase as bait to screen murine cDNA libraries for host proteins that interact with the integrase. We identified 27 proteins that interacted with different integrase fusion proteins. The identified proteins include chromatin remodeling, DNA repair and transcription factors (13 proteins); translational regulation factors, helicases, splicing factors and other RNA binding proteins (10 proteins); and transporters or miscellaneous factors (4 proteins). We confirmed the interaction of these proteins with integrase by testing them in the context of other yeast strains with GAL4-DNA binding domain-integrase fusions, and by in vitro binding assays between recombinant proteins. Subsequent analyses revealed that a number of the proteins identified as Mo-MLV integrase interactors also interact with HIV-1 integrase both in yeast and in vitro.

Conclusion: We identify several proteins interacting directly with both MoMLV and HIV-1 integrases that may be common to the integration reaction pathways of both viruses. Many of the proteins identified in the screen are logical interaction partners for integrase, and the validity of a number of the interactions are supported by other studies. In addition, we observe that some of the proteins have documented interactions with other viruses, raising the intriguing possibility that there may be common host proteins used by different viruses. We undertook this screen to identify host factors that might affect integration target site selection, and find that our screens have generated a wealth of putative interacting proteins that merit further investigation.

Figure 1

Expression of DNA binding domain-IN plasmids and control plasmids used in the yeast two-hybrid screens. (A) Lysates from strain CTY10-5d were electrophoresed on 10% SDS-PAGE gels, transferred to PVDF membrane and probed with anti-lexA. Lane 1, pSH2-1 empty vector; lane 2, pSH2-MoMLV IN; lane 3, pSH2-MoMLV IN with 5'six-glycine linker; lane 4, pSH2-HIV-1 IN; lane 5, pSH2-mouse LEDGF; lane 6, pNlexA empty vector; lane 7, MoMLV IN-pNlexA. (B) Lysates from strain SFY526 were electrophoresed on 10% SDS-PAGE gels, transferred to PVDF and probed with anti-GAL4-DB. Lane 1, strain without vector; lane 2, pGBKT7 empty vector; lane 3, pGBKT7-MLV Gag; lane 4, pGBKT7-MoMLV IN; lane 5, pGBKT7-HIV-1 IN; lane 6, pGBKT7-mLEDGF.

Figure 2

Construction and expression of MoMLV IN deletion plasmids in CTY10-5d. (A)Schematic of pSH2-1 MLV IN truncation constructs. 1–408, full-length mIN; 1–124, mIN-Zn; 1–296, mIN-ZnDDE; 97–225, mIN-DDE; 107–408, mIN-DDECOOH; 220–408, mIN-COOH. (B) Lysates from strain CTY10-5d were electrophoresed on 12% SDS-PAGE gels, transferred to PVDF membranes and probed with anti-LexA. The indicated lysates are shown left to right.

Figure 3

Expression and binding tests of maltose binding and glutathione-S transferase fusion proteins. (A) MBP lysates were bound to amylose resin, eluted with 15 mM maltose, electrophoresed on 10% SDS-PAGE gels, and stained with Coomassie brilliant blue. Lanes 2–4, expression of pmalc2 (empty vector), pmalc2-mIN, and pmalc2-hIN in TB1 cells. For the GST fusions, the lysates were bound to glutathione sepharose, eluted with 10 mM reduced glutathione, electrophoresed on 10% SDS-PAGE gels and stained with Coomassie brilliant blue. Lanes 5–13, representative loads of GST-yeast two hybrid clones: pGEX2TPL, mLEDGF, Fen-1, Enx-1, TFIIE-β, Ku70, ABT1, PRC, and Brd2. (B) Lanes 2–12, GST-yeast-two hybrid clones: AF9, Baz2b, B-ATF, Ankrd49, Znfp38, SF3a3, U2AF²⁶, U5snRNP, KIF3A, Radixin, and Ran bp10. Lane 1 in A and B: Molecular weight marker.

Figure 4

In vitrobinding interactions between MoMLV and HIV-1 integrases and selected proteins identified in the yeast two-hybrid screen. In vitro binding assays between the pmalc2 empty vector (MBP), full-length pmalc2-MoMLV IN (mIN) or full-length pmalc2-HIV-1 IN (hIN) and seventeen of the clones isolated in the screen, plus mLEDGF expressed as GST fusions. The MBP fusion lysates were incubated with amylose resin, washed extensively, resuspended in equal volumes of buffer, and then aliquoted to separate tubes. These tubes were incubated with the GST fusion lysates, washed and eluted with 15 mM maltose. 25 μl of each eluate was electrophoresed on 10 or 12% SDS-PSGE gels, transferred to PVDF membranes, and the same Western was probed with anti-GST, stripped, and then probed with anti-MBP. All Westerns are loaded from left to right: MBP, mIN, and hIN fusion reactions. All upper panels, anti-MBP. All lower panels, anti-GST. (A) Maltose binding protein fusions with empty GST vector; MBP fusions with Brd2, AF9, and Ankrd49. (B) MBP fusions with mLEDGF, Fen-1, Enx-1, and TFIIE-β. (C) MBP fusions with Ku70, PRC, Baz2b, and ABT1. (D) MBP fusions with SF3a3, U5snRNP, KIF3A, and Radixin. (E) MBP fusions with Znfp38, U2AF²⁶, and Ran bp10.

Figure 5

In vitro binding interactions between MoMLV and HIV-1 integrases and selected proteins after treatment of the lysates with nucleases to eliminate nucleic acid bridges between the proteins. In vitro binding assays between the empty vector (MBP), full-length pmalc2-MoMLV IN (mIN) or full-length pmalc2-HIV-1 IN (hIN) and a subset of the clones isolated in the screen. All Westerns are loaded from left to right: MBP, mIN, hIN and the indicated GST fusion reactions. Upper panels, anti-MBP. Lower panels, anti-GST. (A) Left, maltose binding protein fusions with empty GST vector; right, MBP fusions with Ku70 and Brd2. (B) MBP fusions with U2AF²⁶, TFIIE-β, and Ankr49. (C) MBP fusions with the indicated proteins AF9, PRC, Fen-1, Baz2b, and Enx-1.