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

<p>Abstract</p> <p>Background</p> <p>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-α-p-tosyl-L-phenylalanine chloromethyl ketone) and TLCK (N-α-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.</p> <p>Methods</p> <p>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</p> <p>Results</p> <p>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%.</p> <p>Conclusions</p> <p>Our data suggest that this Cys55-specific alkylation mechanism may be exploited to develop a new class of anti HIV drugs.</p

Genetic control of cytolytic T-lymphocyte responses. II. The role of the host genotype in parental --* F1 radiation chimeras in the control of the specificity of cytolytic T-lymphocyte responses to trinitrophenyl-modified syngeneic cells.,]. Exp. Med

Products of the major histocompatibility complex (MHC) 1 play a key role in the functional specificity of cytolytic T lymphocytes (CTL). Allogeneically stimulated CTL have specificity primarily for products of the H-2K and H-2D loci of the mouse histocompatibility 2 (H-2) complex (1). A high proportion of CTL precursors appear to be committed to react with these allogeneic MHC gene products (2-4). The role of MHC products as target specificities for CTL was further elucidated by the experiments of Doherty et al. (5). These investigators showed that CTL specific for viral antigens required the presence of the stimulating H-2 product on the target cell, as well as the inducing viral determinants, to effect cytolysis. This observation also applied to syngeneic systems in which CTL were induced to chemically modified syngeneic cells (6), non-H-2-1inked histocompatibility determinants (7), and tumor-specific transplantation antigens (8, 9). Shearer (10) originally demonstrated that CTL could be induced in vitro by trinitrophenyl (TNP)-modified syngeneic cells and showed that these CTL onl

Genetic control of cytolytic T-lymphocyte responses. I. Ir gene control of the specificity of cytolytic Tlymphocyte responses to trinitrophenyl-modified syngeneic cells

Several instances of Ir gene control of specific cytolytic T lymphocyte (CTL) 1 responses have been reported. Simpson and Gordon (1) and von Boehmer et al. (2) described two complementing Ir genes that control the CTL response to the HY antigen and map in the I region of the H-2 complex. Schmitt-Verhulst and Shearer (3) observed that spleen cells of mice bearing the H-2 k haplotype develop CTL to trinitrophenyl (TNP)-conjugated syngeneic cells that are specific only for modifications of the H-2K but not H-2D region gene products. This response of H-2 k mice is under the dominant control of H-2-1inked Ir genes. In the preceding report (4), we have investigated another genetic defect in the CTL primary response of H-2 k mouse spleen cells to TNP-conjugated cells in vitro. H-2 k spleen cells, in contrast to cells of other haplotypes, develop primary CTL that react solely with TNP-modified syngeneic cells and do not crossreactively lyse TNP-modified allogeneic spleen target cells. The ability to produce cross-reactive CTL in response to TNP-conjugated H-2 k cells is dominant in spleen cells from (H-T x H-2b)F1 hybrid mice and does not reflect th