Alkylating HIV-1 Nef - a potential way of HIV intervention

作者：Jin, Yong-Jiu^*; Zhang, Xiaoping; Cai, Catherine Yi; Burakoff, Steven J.

来源：AIDS Research and Therapy, 2010, 7: 26.

DOI：10.1186/1742-6405-7-26

• IMMUNODEFICIENCY-VIRUS TYPE-1
• CELL-SURFACE CD4
• PHENYLALANINE CHLOROMETHYL KETONE
• MEDIATED DOWN-REGULATION
• CRYSTAL-STRUCTURE
• PROTEIN-KINASE
• CATALYTIC SUBUNIT
• EXPRESSION
• PHOSPHORYLATION
• BINDING

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 (A(56)W(57)L(58)) 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.

• 出版日期2010