Background:  The majority of genetic determinants of HIV-1 envelope phenotype have remained largely intractable due to the extreme genetic variability of env. In a novel approach, we demonstrate that the diversity of phenotypes among individual env variants within individual patient virus quasi-species can be exploited to map mutations and polymorphisms that confer important phenotypic properties.

Methods:  Full-length env sequences were amplified from patient plasma samples and were evaluated as populations and individual clones. Env sequences were determined by conventional methods. Co-receptor use and susceptibility to entry inhibitors and antibodies were measured using a single replication cycle pseudovirion assay. Env-mediated infectivity was evaluated using a multi-cycle replication assay. Membrane fusion was assessed using a GFP/luciferase reporter assay.

Results:  Multiple env clones isolated from individual virus populations exhibited a wide range of viral infectivity, membrane fusion, and drug and neutralizing antibody susceptibilities. Subtle changes (1 or 2 amino acid changes) in gp120 constant regions C1, C2, C4, and gp41 are capable of dramatically altering the envelope phenotype. A single L261S substitution in C2 greatly decreased viral infectivity (100-fold) and cell-cell membrane fusion (70-fold), and was accompanied by a decreased sensitivity to sCD4 (>100-fold). Infectivity, membrane fusion and sCD4 sensitivity was partially restored by substitutions T639A and V749A in gp41. This suggests small changes in gp41 can influence the interaction between envelope and the CD4 receptor. A second virus with substitutions K117E in C1 and K421E in C4 exhibited low infectivity (30-fold) and membrane fusion (5-fold) and enhanced sensitivity to sCD4 (>100-fold), monoclonal antibodies that target either gp120 or gp41, and autologous and heterologous patient plasmas. Thus, these two substitutions resulted in a pan neutralization sensitive phenotype.

Conclusions:  Clonal analyses of individual virus populations using high throughput assay systems enables identification of distinct changes in env that confer alterations in phenotype. These observations are consistent with a structurally integrated model of the gp120-gp41 glycoprotein complex. Application of this approach to additional virus populations will be used to extend our appreciation of envelope structure-function relationships that can be applied to the development of entry inhibitors and vaccines.