title: Alkylating HIV-1 Nef - a potential way of HIV intervention.
abstract: BACKGROUND
  Nef is a 27 KDa HIV-1 accessory protein. It downregulates CD4 from infected cell surface, a mechanism critical for efficient viral replication and pathogenicity. Agents that antagonize the Nef-mediated CD4 downregulation may offer a new class of drug to combat HIV infection and disease. TPCK (N-alpha-p-tosyl-L-phenylalanine chloromethyl ketone) and TLCK (N-alpha-p-tosyl-L-lysine chloromethyl ketone) are alkylation reagents that chemically modify the side chain of His or Cys residues in a protein. In search of chemicals that inhibit Nef function, we discovered that TPCK and TLCK alkylated HIV Nef.
  
  
  METHODS
  Nef modification by TPCK was demonstrated on reducing SDS-PAGE. The specific cysteine residues modified were determined by site-directed mutagenesis and mass spectrometry (MS). The effect of TPCK modification on Nef-CD4 interaction was studied using fluorescence titration of a synthetic CD4 tail peptide with recombinant Nef-His protein. The conformational change of Nef-His protein upon TPCK-modification was monitored using CD spectrometry
  
  
  RESULTS
  Incubation of Nef-transfected T cells, or recombinant Nef-His protein, with TPCK resulted in mobility shift of Nef on SDS-PAGE. Mutagenesis analysis indicated that the modification occurred at Cys55 and Cys206 in Nef. Mass spectrometry demonstrated that the modification was a covalent attachment (alkylation) of TPCK at Cys55 and Cys206. Cys55 is next to the CD4 binding motif (A56W57L58) in Nef required for Nef-mediated CD4 downregulation and for AIDS development. This implies that the addition of a bulky TPCK molecule to Nef at Cys55 would impair Nef function and reduce HIV pathogenicity. As expected, Cys55 modification reduced the strength of the interaction between Nef-His and CD4 tail peptide by 50%.
  
  
  CONCLUSIONS
  Our data suggest that this Cys55-specific alkylation mechanism may be exploited to develop a new class of anti HIV drugs.
authors.affiliation: Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY 10029, USA. Yong-Jiu.Jin@mssm.edu.
authors.forename: Yong-Jiu
authors.initials: YJ
authors.lastname: Jin
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Zhang
authors.forename: Catherine Yi
authors.initials: CY
authors.lastname: Cai
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Burakoff