10th Conference on Retroviruses and Opportunistic Infections Boston, MA USA - February 10 -14, 2003

Conf Retroviruses Opportunistic Infect 2003 Feb 10-14;10th: abstract no. 23
J. Reeves¹ , S. Gallo², N. Ahmad¹, J. Miamidian¹, P. Harvey¹, M. Sharron¹, S. Pöhlmann¹, T. Pierson¹, M. Biscone¹, J. Sfakianos³, C. Derdeyn³, G. Tomaras⁴, R. Blumenthal², E. Hunter³, R. Doms^1
1Univ of Pennsylvania, Philadelphia; ²Natl Cancer Inst, Frederick, MD; ³Univ of Alabama at Birmingham; and ⁴Duke Univ, Durham, NC

BACKGROUND: Each subunit of the trimeric envelope protein (Env) of HIV is composed of an outer gp120 glycoprotein and a transmembrane gp41 subunit. gp120 binds CD4 and a co-receptor (usually CCR5 or CXCR4) triggering conformational rearrangements in both gp120 and gp41. The extracellular domain of gp41 contains 2 helical regions (HR1 and HR2) that form a 6-helix bundle following receptor engagement to trigger fusion of viral and cellular membranes. HIV entry inhibitors include co-receptor antagonists and fusion inhibitors such as T-20. T-20 binds HR1 and prevents conformational changes required for membrane fusion. HR1 appears to become accessible to T-20 after Env binds CD4, while co-receptor binding likely induces the final conformational changes that lead to membrane fusion. Thus, T-20 binds to a structural intermediate of the fusion process. Mutations in HR1 can impact T-20 sensitivity. Additionally, V3 in gp120 has been shown to impact T-20 sensitivity, the mechanism for which was unclear.

METHODS: Cell-cell fusion and infection assays were utilized to determine the sensitivity of HIV Envs to entry inhibitors. Dye transfer assays were used to determine membrane fusion kinetics. Western blot and FACS assays were used to compare Env:receptor affinity.

RESULTS: Primary viruses can exhibit a wide range of sensitivities to T-20 and this can be independent of HR1 "resistance mutations." We studied chimeric Env proteins containing different V3 loop sequences and found that gp120:co-receptor affinity correlated with T-20 and TAK-779 (CCR5 ligand) sensitivity, with greater affinity resulting in increased resistance to both classes of entry inhibitors. Enhanced affinity conferred more rapid fusion kinetics, reducing the time during which Env is sensitive to T-20. Reduced co-receptor expression levels delayed fusion kinetics and enhanced virus sensitivity to T-20, while increased co-receptor levels had the opposite effect. Additionally, a single amino acid change in the bridging sheet region of the primary virus strain YU2 reduced affinity for CCR5 and markedly increased T-20 sensitivity.

CONCLUSIONS: Co-receptor density and Env mutations that affect receptor engagement and membrane fusion rates can alter entry inhibitor sensitivity. Since co-receptor expression levels are often limiting for virus in vivo, individuals expressing lower levels may respond more favorably to entry inhibitors such as T-20, whose effectiveness we show is dependent in part on fusion kinetics.

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