OLIGOMERIZATION OF THE HYDROPHOBIC HEPTAD REPEAT OF GP41

The transmembrane protein of human immunodeficiency virus type 1 (HN 1 ) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix. To evaluate the po tential of the hydrophobic heptad repeat to induce protein oligomeriza tion, this region of gp41 has been cloned into the bacterial expressio n vector pRIT2T. The resulting plasmid, pRIT3, expresses a fusion prot ein consisting of the Fc binding domain of monomeric protein A, a bact erial protein, and amino acids 538 to 593 of HIV-1 gp41. Gel filtratio n chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that t he chimeric protein formed a higher-order multimer that was greater th an a dimer. Thus, we have identified a region of HIV-1 gp41 which is c apable of directing the oligomerization of a fusion protein containing monomeric protein A. Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been eng ineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography. A point mutation in the heptad repeat, which ch anged:the central isoleucine to an alanine, caused a significant (>60% ) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligome rization, Deletions of one, two, or three amino acids following the fi rst isoleucine also resulted in a profound decrease in oligomerization . The inhibitory effects of the mutations on oligomer formation correl ated with the effects of the same mutations on envelope glycoprotein-m ediated fusion. A possible role of the leucine zipper-like region in t he fusion process and in an oligomerization event distinct from assemb ly of the envelope glycoprotein complex is discussed.