Probing the topography of HIV-1 nucleocapsid protein with the alkylating agent N-ethylmaleimide

Retroviral nucleocapsid (NC) proteins contain one or two zinc fingers (ZFs) consisting of a CCHC peptide motif that coordinates Zn(II), Mutational and biochemical analyses have shown that NC ZFs an directly involved in multip le stages of viral replication, including genomic RNA encapsidation, virus maturation, and the early infection process. The multiple roles of the cons erved retroviral ZFs make them attractive targets for antiviral agents. We have previously shown that a variety of chemical compounds can inactivate t he whole virus by attacking NC ZFs, For the enhancement of the specificity of antiviral reagents, it is desirable to have a detailed knowledge of the spatial organization of reactive sites on the NC protein in its free and ol igonucleotide-bound states. A method has been developed using chemical prob es to assess the reactivity of specific Cys residues in the NC protein, and is being used to investigate the topography of ZFs in different contexts. In this study we focus on the reaction mechanism of N-ethylmaleimide (NEM) with free HIV-1 NCp7 protein. Our results show that the conformation of fre e NCp7 restricts the initial site of attack to Cys-49 (the most distal Cys residue in the second ZF) and that the reactivity of thiols in full-length protein differs from that of the isolated ZF peptides. A moderate to near c omplete reduction in reaction rate was observed when NCp7 was complexed wit h different oligonucleotides. These findings provide a set of experimentall y determined parameters that can serve to guide computational modeling of t he NC protein and will be useful for the rational design of drugs directed against retroviral ZFs.