Background:  HIV-1 co-receptor switching involves envelope mutations that usually result in R5 to R5X4 to X4 transitions. We have examined the functional consequences of envelope mutations in 4 distinct switching pathways resulting in viruses with co-receptor preference ranging from R5 >X4, R5 = X4, X4 >R5, to pure X4 to determine common and distinct features of each pathway.

Methods:  Site-directed mutagenesis was used to introduce most combinations of mutations in envelope previously shown to generate R5X4 or X4 variants of BaL or ADA isolates. For all combinations of mutations preserving infectivity on either CCR5- or CXCR4-expressing target cells, we determined the sensitivity of the resulting envelope mutant to co-receptor inhibitors, soluble CD4, and the broadly neutralizing antibodies b12-IgG and 4E10.

Results:  Common features of Env mutations involved in co-receptor switching were decreased CCR5 binding, increased sensitivity to CCR5 inhibitors, and increased resistance to soluble CD4 that was confirmed by direct binding assays. Increased binding to CD4 preceded measurable increases in CXCR4 binding and increased resistance to CXCR4 inhibitors. Unique features of different co-receptor switch intermediates included varying sensitivity to antibody neutralization and the fraction of mutated envelopes with loss of entry function. Infection of CXCR4-expressing target cells was more sensitive to antibody neutralization than infection of CCR5-expressing target cells.

Conclusions:  Increased CD4 binding and decreased CCR5 binding are common features of co-receptor switch intermediates, even when the final virus utilizes CCR5 more efficiently than the parental R5 isolate. Increased CD4 binding may provide enough fitness advantage to allow survival of intermediates with poor binding to both CCR5 and CXCR4. Neutralizing antibody may slow the emergence of R5X4 or X4 variants.