[16] Modification of the CCR5 binding site by mutations in gp120 influence drug susceptibility and viral infectivity in one subject with clinical resistance to vicriviroc

18th International HIV Drug Resistance Workshop

Basic Principles & Clinical Implications

June 9–13 2009, Fort Myers, Florida, USA

MODIFICATION OF THE CCR5 BINDING SITE BY MUTATIONS IN GP120 INFLUENCE DRUG SUSCEPTIBILITY AND VIRAL INFECTIVITY IN ONE SUBJECT WITH CLINICAL RESISTANCE TO VICRIVIROC

Antivir Ther 2009; 14 Suppl 1:A18 (abstract no. 16)

RA Ogert¹, Y Hou¹, L Ba¹, C Buontempo¹, N Murgolo², P Qiu², J Duca³, J Strizki¹, R Ralston¹ and JA Howe¹
¹Virology, Schering–Plough Research Institute, Kenilworth, NJ, USA; ²Molecular Design and Informatics, Schering–Plough Research Institute, Kenilworth, NJ, USA ³3D Drug Design, Schering–Plough Research Institute, Kenilworth, NJ, USA

OBJECTIVES: The mechanisms by which HIV type-1 (HIV-1) may develop resistance to vicriviroc, a small-molecule CCR5 antagonist, are not well understood. We sought to characterize the viral adaptations that confer phenotypic resistance in a clinical isolate.

METHODS: Site-directed mutagenesis was used to introduce reciprocal point mutations into resistant or baseline env genes. Relative viral infectivity and VCV susceptibility studies were performed using a single-cycle pseudoparticle assay with U87-CD4/CCR5 cells or 293T cells transfected with CD4 and wild-type or mutant CCR5.

RESULTS: The HIV-1 env gene from the clade D strain isolated at the time of study discontinuation contained six amino acid changes in the V3 loop and one in the C4 region as compared with the baseline clone. Pseudovirus generated with the chimeric env were completely resistant to VCV, but remained exclusively R5-tropic. Back mutations E315Q, G321R and F317L in the tip and the stem of the V3 loop region from the resistant env restored complete and partial susceptibility to VCV, respectively; back mutation of V3 loop residues N320D, K328E and R429G in C4 significantly reduced pseudovirus infectivity without altering the resistant phenotype. Forward mutagenesis showed that none of the six amino acid residues were sufficient to confer the resistant phenotype to the baseline chimeric gp120. A combination of four specific mutations in V3 was required to restore complete resistance. Structural modeling demonstrated that certain mutations identified in the V3 loop map to the binding domain in gp120 for the N terminus of CCR5. Entry of pseudovirus generated with resistant envelopes was more sensitive than baseline counterparts to point mutations Y10A, D11A, Y14A and Y15A that eliminate important binding determinants in the N terminus of CCR5.

CONCLUSIONS: The amino acid changes we characterized primarily conferred VCV resistance or influenced envelope infectivity. A specific combination of four of the seven mutations was required for the complete VCV resistance phenotype, suggesting that CCR5 antagonists have a high barrier to resistance. Functionally, the combination of mutations resulted in increased dependence on the envelope interaction with the N terminus of CCR5 for viral entry.

2009-06-09
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