Interaction of the HIV-1 nucleocapsid protein with 2-mercaptobenzamide thioesters and [psi]-RNA
Jenkins, Lisa Marie
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The HIV-1 nucleocapsid protein (NCp7) is a protein that plays several crucial roles throughout the retroviral lifecycle. As a nucleic acid-binding protein, NCp7 is essential for reverse transcription, viral integration, and viral assembly. The most important function of NCp7, though, is the dimerization and packaging of the genomic RNA into a new virion. Dimerization and packaging both involve the interaction of NCp7 with the stem-loop sequences in the ¨ site, located at the 5 region of the genomic RNA. NCp7 contains two highly-conserved structural zinc-binding domains, in which three cysteine and one histidine residues are used to coordinate zinc. These zinc-binding domains are required for sequence-specific RNA-binding by NCp7. The highly conserved nature of NCp7 and its importance at various stages of the HIV-1 lifecycle have made it a target for the development of new antiviral therapies. Compounds have been developed that function by ejecting the coordinated zinc from NCp7, such as the 2-mercaptobenzamide thioester compounds. UV/Visible spectroscopy, NMR spectroscopy, mass spectrometry, and gel mobility shift assays have been used to investigate the mechanism of action of the 2-mercaptobenzamide thioesters. We have determined that these thioester compounds specifically target the Cys39 in the carboxyl-terminal zinc-binding domain of NCp7, leading to the ejection of zinc and the loss of nucleic acid binding. When NCp7 is already bound to RNA, however, the 2-mercaptobenzamide thioester compounds are unable to interact with NCp7. The interaction of these compounds with several cellular zinc-binding domains has also been studied. It was found that the 2-mercaptobenzamide thioesters were able to interact with other Cys2HisCys zinc-binding domains, as well as with proteins containing Cys4 zinc-binding domains. Proteins containing classical Cys2His2 and interleaved RING finger-like zinc-binding domains did not interact with the thioester compounds. In addition, NMR spectroscopy and gel mobility shift assay were used to investigate the interaction of NCp7 with the internal loop of the first stem-loop from the ¨ site. Binding of NCp7 to this RNA destabilized two base pairs around the internal loop. The internal loop could function as a nucleation point for the destabilization of the stem during dimerization. NCp7 could facilitate this destabilization as part of its dimerization function.