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Journal of Virology, December 2008, p. 11555-11567, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01561-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Ming-Chieh Shun,1 Yaïr Botbol,2 Xiang Li,1 Francesca Di Nunzio,1 Janet E. Daigle,1 Nan Yan,3 Judy Lieberman,3 Marc Lavigne,2 and Alan Engelman1*

Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Division of AIDS, Harvard Medical School, Boston, Massachusetts 02155,1 Department of Virology, Unit of Structural Biology, Pasteur Institute, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France,2 Immune Disease Institute and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 021153

Received 23 July 2008/ Accepted 11 September 2008

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.

* Corresponding author. Mailing address: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. Phone: (617) 632-4361. Fax: (617) 632-3113. E-mail: alan_engelman{at}dfci.harvard.edu

{triangledown} Published ahead of print on 17 September 2008.

Journal of Virology, December 2008, p. 11555-11567, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01561-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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