J Virol. 2008 Dec;82(23):11555-67. Epub 2008 Sep 17.

Identification and characterization of PWWP domain residues critical for LEDGF/p75 chromatin binding and human immunodeficiency virus type 1 infectivity.

Shun MC, Botbol Y, Li X, Di Nunzio F, Daigle JE, Yan N, Lieberman J, Lavigne M, Engelman A.

Source

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Division of AIDS, Harvard Medical School, Boston, MA 02115, USA.

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.

PMID:: 18799576; [PubMed - indexed for MEDLINE]
PMCID:: PMC2583668

Free PMC Article

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FIG. 1.

LEDGF/p75 schematic and PWWP domain sequence alignments. (A) LEDGF/p75 domain organization and previously described mutants used in the present study. Binding of recombinant protein to DNA in vitro is predominantly mediated by the NLS and dual copy of the AT-hook (ATh) DNA-binding motif, with the PWWP domain (gray shade) contributing a minor role (1, 56, 62). Chromatin binding is largely mediated by the PWWP domain and AT-hooks, with charged regions (CRs) 1 to 3 making secondary contributions (29, 62). MutL1 harbors 12 missense mutations: 6 in the NLS and 3 in each copy of the AT-hook (1, 62). (B) Amino acid alignment of the N-terminal PWWP domains from select human HRP family members. Secondary structural elements elucidated from the solution structure of the HDGF domain (31) are shown above the alignment. Residues targeted for mutagenesis are noted below by triangles; numbers refer to LEDGF/p75 positions. The coloring scheme denotes the following levels of sequence conservation: black, amino acid identity at three or more positions; dark gray, amino acid similarity at all four positions; light gray, chemically similar side chains at three of four positions. (C) Alignment of DNMT3B and LEDGF/p75 PWWP domains. Secondary elements elucidated from the X-ray crystal structure of the murine DNMT3B domain (47) are shown above the corresponding human sequence, whereas HDGF structural elements are drawn below the LEDGF/p75 sequence. The substitution of Pro for Ser-270 (boxed) in DNMT3B is associated with ICF syndrome (53). Black denotes identical amino acids, whereas gray indicates homologous side chains as defined by the following chemical groupings (51): Gly, Ala, Ser, Thr, and Pro; Met, Val, Leu, and Ile; Phe, Tyr, and Trp; Asp, Glu, Asn, and Gln; Lys, Arg, and His; Cys. PR, proline-rich.

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

J Virol. J Virol;82(23):11555-11567.