The Structure-Specific Recognition Protein 1 Associates with Lens Epithelium-Derived Growth Factor Proteins and Modulates HIV-1 Replication

Abstract

The lens epithelium-derived growth factor p75 (LEDGF/p75) is a chromatin-bound protein essential for efficient lentiviral integration. Genome-wide studies have located LEDGF/p75 inside actively transcribed genes where it mediates lentiviral integration. Although its role in HIV-1 integration is clearly established, the role of LEDGF/p75-associated proteins in HIV-1 infection remains unexplored. Using protein-protein interaction assays, we demonstrated that LEDGF/p75 complexes with a chromatin remodeling complex, Facilitates Chromatin Transcription (FACT), a heterodimer of the structure specific recognition protein 1 (SSRP1) and the human homolog of suppressor of Ty 16 (hSpt16). Detailed analysis of the interaction of LEDGF/p75 with the FACT complex indicates that LEDGF/p75 interacts with SSRP1 in an hSpt16-independent manner that requires the PWWP domain of LEDGF proteins and the HMG domain of SSRP1. Functional characterizations demonstrate a LEDGF/p75-independent role of SSRP1 in the regulation of HIV-1 replication. shRNA-mediated partial knockdown of SSRP1 reduces HIV-1, but not Murine Leukemia Virus, infection in human CD4+ T cells. Similarly, SSRP1 knockdown affects infection by HIV-1-derived viruses that express genes from the viral LTR but not from an internal immediate-early CMV promoter, suggesting a role of SSRP1 in LTR-driven gene expression but not viral DNA integration. Together, our data demonstrate for the first time the association of LEDGF proteins with the FACT complex and give further support to a role of SSRP1 in HIV-1 infection.

Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration

“…As shown in Fig. 1 b, no interaction was detected between IN/IBD/FACT by co-immunoprecipitation suggesting that the formation of the IN/LEDGF/FACT complex requires the previously reported physical association between FACT and LEDGF/p75 mediated by SSRP1 and PWWP domains [ 21 ].…”

“…1 a). The close proximity found between SSRP1 and LEDGF/p75 protein in cells strongly supports the enrichment of FACT in the chromatin regions targeted by HIV-1 integration complexes [ 21 ]. Consequently, in view of (i) the histone chaperone activity of FACT, (ii) the previously reported links between this complex and HIV-1 replication and (iii) the importance of chromatin remodeling in regulating HIV-1 integration we further analyzed its potential function in modulating the insertion of viral DNA into chromatin.…”

“…1 b and Additional file 3 : Figure S3). To determine whether the IN/LEDGF/FACT interaction occurs via the LEDGF/SSRP1 interaction we used a 326–471 amino-acid LEDGF/p75-GST construct lacking the PWWP SSRP1 interacting domain (as determined previously, [ 21 ]) but carrying the integrase binding domain (IBD). We first confirmed that the IBD interacts with HIV-1 IN but not FACT by GST pull down (Additional file 3 : Figure S3).…”

“…This chromatin remodeling complex is tightly associated with the PolII transcription machinery [ 17 – 19 ] and was previously shown to participate in the regulation of HIV-1 LTR-driven transcription [ 20 ]. Moreover, a recent study reported that the SSRP1 FACT component interacts with LEDGF/p75 via their HMG and PWWP domains [ 21 ] (a summary of the previously identified interactions is shown in Fig. 1 a).…”

Twenty years of research on the DFS70/LEDGF autoantibody-autoantigen system: many lessons learned but still many questions

“…Subsequent studies revealed that DFS70/ LEDGF critically associates with the MLL/Menin transcription complex to drive the expression of Hox genes and oncogenic transformation in leukemias caused by the MLL gene [75]. More recently, El Ashkar et al [76] PWWP [156] demonstrated that conditional knockout of the Psip1 gene (which encodes DFS70/LEDGF) from blood cells in a mouse model was dispensable for normal hematopoiesis but critical for MLL-mediated leukemogenesis. Consistent with this, leukemia cells expressing MLL and overexpressing DFS70/LEDGF IBD mutants were defective for MLL interactions and displayed decreased clonogenic growth [77].…”