11th International AIDS Conference Vancouver, British Columbia — July 7-12, 1996

Int Conf AIDS 1996 Jul 7-12; 11:6 (abstract no. We.A.153)
Paulus CR, Beibetainger M, Wolf H, Wagner R; Institut fur Medizinische Mikrobiologie und Hygiene, Regensburg, Germany. Fax: +49 941 944 6402.

OBJECTIVE: The frameshift protein p6* encoded directly upstream of the protease (PR) in the HIV-1 pol reading frame is supposed to play a role in the intracellular regulation of PR activity. This limitation of cytoplasmic protease activation is necessary to prevent premature processing of Gag and Gag-Pol precursors and unspecific cleavage of cellular proteins.

METHODS: To allow structural and functional characterization of the p6* transframe protein the p6* coding sequence was cloned into the vector pGEX-KG and expressed in E. coli as a fusion protein with glutathione S-transferase (GST). Purification of the bacterially produced protein was performed by affinity chromatography, thrombin cleavage and subsequent gelfiltration of the native p6*. Absorption, fluorescence, circular dichroism and 1H-NMR spectroscopy were applied to structurally characterize the purified protein. The interaction of p6* with the HIV-1 protease was investigated using a kinetic assay as described by Zhand et al. (1991).

RESULTS: The expression and native purification strategy described resulted in milligram amounts of highly pure p6* protein allowing stuctural and functional investigations. Two-dimensional NMR spectra provided essentially complete sequence specific resonance assignments at pH 5.9. Although there is a helix forming tendency in the Nterminus of the protein, the experiments indicate that p6* has no overall stable secondary or tertiary structure with the single tryptophane residue exposed in aqueous solution. To provide extra evidence that the lack of structure is not an artefact of our method of expression and purification, we investigated the interaction of our p6* sample with the HIV-1 protease. From our measurements we conclude for the first time that p6* is predominantly a dimerization inhibitor, the dissociation constant being approximately 1 micromolar.

CONCLUSIONS: The results reported provide the basis for further studies to understand the intrinsic regulation of HIV-1 protease activity in the viral life cycle, which may lead to new ideas for the design of protease inhibitors.

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WeA153

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