474   Correlation of gp41 Binding and Antiviral Potencies of the T-20 Fusion Inhibitor Using Clinical Trial Isolate-Derived Sequences.

M. A. Mink*, S. Janumpalli, D. K. Davison, S. M. Mosier, J. B. Erickson, R. A. Picking, P. Sista, D. M. Lambert, and T. J. Matthews.
Trimeris, Durham, NC.

Background:The HIV envelope glycoprotein gp41 contains two heptad repeat sequences, HR1 and HR2. A synthetic HR2-derived T-20 peptide is thought to target the HR1 domain and results in HIV fusion inhibition. Consistent with this mechanism, T-20 resistance mutations derived by in vitro passage of virus mapped to the HR1 region (Rimsky et al. , J. Virol. 72:986-993, 1998). Sequence analysis of virus isolates derived from patients participating in T-20 clinical trials have revealed additional mutations in the HR1 region. These mutations were incorporated into E. coli -expressed polypeptides. We studied the interaction of T-20 with these polypeptides to derive information related to the mechanism of action of T-20. We also incorporated these substitutions into HIV genomes and tested their T-20 phenotype.

Methods:The ectodomain of HIV gp41 lacking the HR2 domain was expressed in E. coli as a maltose binding protein chimera. Site-directed mutagenesis of this recombinant plasmid was used to derive the various proteins containing the substitutions indicated. Protein was affinity purified by amylose column chromatography. T-20 peptide was synthesized containing flourescein at the amino-terminus. We determined equilibrium saturation binding parameters using a constant amount of probe mixed with increasing amounts of the target protein. Binding was measured by fluorescence polarization. HIV-1 DNA clone pNL4-3 was used to rescue mutated HIV. Each substitution was rescued, and sensitivity to T-20 was determined by a Magi cell assay. The concentration required to inhibit 50% of virus was determined.

Results:Changes in T-20 binding affinity to each substituted gp41 polypeptide directly correlated with the T-20 sensitivity phenotype of virus containing the identical substitution.

Conclusion:HR2 binding affinity to HR1 domains containing mutations associated with T-20 resistance correlate to T-20 phenotypic sensitivity.