Identification and Characterization of PWWP Domain Residues Critical for LEDGF/p75 Chromatin Binding and Human Immunodeficiency Virus Type 1 Infectivity

Abstract

Lens epithelium-derived growth factor (LEDGF)/p75 functions as a bimodal tether during lentiviral DNA integration: its C-terminal integrase-binding domain interacts with the viral preintegration complex, whereas the N-terminal PWWP domain can bind to cellular chromatin. The molecular basis for the integrase-LEDGF/ p75 interaction is understood, while the mechanism of chromatin binding is unknown. The PWWP domain is homologous to other protein interaction modules that together comprise the Tudor clan. Based on primary amino acid sequence and three-dimensional structural similarities, 24 residues of the LEDGF/p75 PWWP domain were mutagenized to garner essential details of its function during human immunodeficiency virus type 1 (HIV-1) infection. Mutating either Trp-21 or Ala-51, which line the inner wall of a hydrophobic cavity that is common to Tudor clan members, disrupts chromatin binding and virus infectivity. Consistent with a role for chromatin-associated LEDGF/p75 in stimulating integrase activity during infection, recombinant W21A protein is preferentially defective for enhancing integration into chromatinized target DNA in vitro. The A51P mutation corresponds to the S270P change in DNA methyltransferase 3B that causes human immunodeficiency, centromeric instability, and facial anomaly syndrome, revealing a critical role for this amino acid position in the chromatin binding functions of varied PWWP domains. Our results furthermore highlight the requirement for a conserved Glu in the hydrophobic core that mediates interactions between other Tudor clan members and their substrates. This initial systematic mutagenesis of a PWWP domain identifies amino acid residues critical for chromatin binding function and the consequences of their changes on HIV-1 integration and infection.Integration, catalyzed by the viral integrase protein, is an essential step in the replication cycles of all retroviruses. Integration into cellular chromatin provides an optimal environment for gene expression and ensures that the viral genetic material is inherited by daughter cells upon division. Integration proceeds via the following steps: (i) integrase binding to the cDNA end regions that are synthesized during reverse transcription; (ii) hydrolysis adjacent to invariant CA sequences near both 3Ј ends (3Ј processing); (iii) transfer of the reactive 3Ј-OH ends to the 5Ј-phosphates of a double-stranded cut in cellular chromatin (DNA strand transfer); and (iv) repair of the resulting DNA recombination intermediate, which is likely accomplished by host cell enzymes. (See reference 61 for a detailed overview of retroviral integration.)Although all retroviruses rely on integrase 3Ј processing and DNA strand transfer activities, significant differences exist in the way the various viral genera select their chromosomal integration sites. These differences manifest themselves at the level of local DNA sequence (20, 68) and genetic structure (reviewed in reference 2). Lentiviruses, for example, favor integration into active transcription uni...

Role of the PWWP Domain of Lens Epithelium-derived Growth Factor (LEDGF)/p75 Cofactor in Lentiviral Integration Targeting

“…HIVbased lentiviral vectors showed a reduced transduction efficiency compared with ⌬N 93 -LEDGF cells (supplemental Fig. S9b; p ϭ 0.0113, two-tailed t test), in line with earlier reports (38,67) …”

“…⌬N 93 -LEDGF AT-locates to the nucleus and does not interact with mitotic chromatin (supplemental Fig. S9a) (36,38). HIVbased lentiviral vectors showed a reduced transduction efficiency compared with ⌬N 93 -LEDGF cells (supplemental Fig.…”

Implication of Serine Residues 271, 273, and 275 in the Human Immunodeficiency Virus Type 1 Cofactor Activity of Lens Epithelium-Derived Growth Factor/p75

“…These data indicated that LEDGF/p75 chromatin binding requires the functional interaction of the PWWP domain and two AT hook motifs (22). More recently, a mutagenesis analysis of evolutionarily conserved residues within the PWWP domain suggested that a hydrophobic cavity in this protein domain has a fundamental role in the chromatin binding of LEDGF/p75 (34).…”

Lens epithelium-derived growth factor fusion proteins redirect HIV-1 DNA integration

“…S1). The PWWP domain is the primary chromatin binding determinant, although the protein or modification to which it binds is currently unknown (15,16). LEDGF promotes the association of the PIC with the host genome, enhancing the integration of HIV-1 DNA (16)(17)(18)(19)(20).…”