Mutations in gp41 and gp120 of HIV-1 isolates resistant to hexa-arginine neomycin B conjugate Gadi Borkow, 1 Humberto Herman Lara, 2 and Aviva Lapidot * Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel Received 5 November 2003 Abstract Aminoglycoside–arginine conjugates (AACs) inhibit HIV-1 replication and act as Tat antagonists. AACs compete with monoclonal antibody binding to CXCR4, compete with SDF-1a and HIV-1 gp120 cellular uptake, indicating that they interfere with initial steps of HIV-1 infection. We here present the selection of HIV-1 isolates resistant to hexa-arginine neomycin B conjugate (NeoR6), the most potent anti-HIV-1 AAC. We found in the NeoR6-resistant isolates the following mutations in gp120: I339T in the C3 region, S372L in the V4 region, and Q395K in the C4 region; and in gp41: S668R and F672Y in the ‘heptad repeat’ 2 (HR2) region. These findings strongly suggest that NeoR6 obstructs HIV-1 replication by interfering with the fusion step, dependent on both conformational changes in gp120 following CD4 and CXCR4 interaction, as well as by conformational changes in gp41 in- duced by HR1 and HR2 interaction. The AACs may thus represent a novel family of fusion inhibitors. Ó 2003 Elsevier Inc. All rights reserved. Keywords: HIV-1; Aminoglycoside–arginine conjugates; Viral entry; Gp120; Gp41; Mutations; Heptad repeat For human immunodeficiency virus type 1(HIV-1) to enter a cell, its envelope-protein (Env) must sequentially engage CD4 and a chemokine coreceptor, triggering conformational changes in Env that ultimately lead to fusion between the viral and host cell membranes. Each step of the virus entry pathway is a potential target for novel antiviral agents termed entry inhibitors. The en- velope is composed of two subunits, glycoprotein 120 (gp120) and glycoprotein 41 (gp41). These glycoproteins are proteolytically cleaved from the gp160 Env precursor and associate with each other through noncovalent in- teractions. The Env glycoproteins form trimers on the virion surface, with each monomer consisting of two subunits, gp120 and gp41. During the course of viral infection, the gp120 Env component binds to CD4 on target cells [1] and undergoes conformational changes [2] that allow gp120 to interact with a chemokine receptor, mainly CXCR4 [T cell-tropic (X4) HIV strains] and CCR5 [macrophage-tropic (R5) HIV strains] (reviewed in [3–7]). The third variable region (V3) loop of gp120 has been implicated in chemokine receptor binding as it contains determinants of coreceptor specificity and tro- pism [8–14]. Other domains, such as V1/V2, of gp120 were also found to play a role in the interaction of gp120 with CXCR4 [15] and with CCR5 [8,16,17]. Formation of the tri-molecular complexes (gp120–CD4–chemokine receptor) stabilizes virus binding and triggers a series of conformational changes in gp41. These include exposure of the fusion peptide, which is first displaced toward the cell membrane and inserts into it [18–21]. Following, two conserved regions of gp41, referred to as ‘heptad repeats’ (HR1 and HR2), associate with each other in an anti- parallel fashion to form a-helical trimers of heterodimers (a six-helix bundle). As a result of this transition state, the fusion peptide and transmembrane domain of gp41 are brought into close proximity allowing for lipid mix- ing and cellular and viral membrane fusion, resulting in entry of the viral core into the cell (reviewed in [22]). The discovery of virus entry and the consequent un- derstanding of the receptor-induced conformational changes in the Env protein and virus-cell fusion led to * Corresponding author. Fax: +972-8-934-4142. E-mail address: aviva.lapidot@weizmann.ac.il (A. Lapidot). 1 Present address: Animal Sciences, Faculty of Agriculture, He- brew University, Rehovot, Israel. 2 Present address: Laboratory of Immunology and Virology, University of Nuevo Leon, Mexico. 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.011 Biochemical and Biophysical Research Communications 312 (2003) 1047–1052 BBRC www.elsevier.com/locate/ybbrc ARTICLE IN PRESS ARTICLE IN PRESS