A Quantitative Affinity-Profiling System That Reveals Distinct CD4/CCR5 Usage Patterns among Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus Strains

ABSTRACT The affinity of human immunodeficiency virus (HIV) envelope for CD4 and CCR5 appears to be associated with aspects of R5 virus (virus using the CCR5 coreceptor) pathogenicity. However, entry efficiency results from complex interactions between the viral envelope glycoprotein and both CD4 and CCR5, which limits attempts to correlate viral pathogenicity with surrogate measures of envelope CD4 and CCR5 affinities. Here, we present a system that provides a quantitative and comprehensive characterization of viral entry efficiency as a direct interdependent function of both CD4 and CCR5 levels. This receptor affinity profiling system also revealed heretofore unappreciated complexities underlying CD4/CCR5 usage. We first developed a dually inducible cell line in which CD4 and CCR5 could be simultaneously and independently regulated within a physiologic range of surface expression. Infection by multiple HIV type 1 (HIV-1) and simian immunodeficiency virus isolates could be examined simultaneously for up to 48 different combinations of CD4/CCR5 expression levels, resulting in a distinct usage pattern for each virus. Thus, each virus generated a unique three-dimensional surface plot in which viral infectivity varied as a function of both CD4 and CCR5 expression. From this functional form, we obtained a sensitivity vector along with corresponding metrics that quantified an isolate's overall efficiency of CD4/CCR5 usage. When applied to viral isolates with well-characterized sensitivities to entry/fusion inhibitors, the vector metrics were able to encapsulate their known biological phenotypes. The application of the vector metrics also indicated that envelopes derived from elite suppressors had overall-reduced entry efficiencies compared to those of envelopes derived from chronically infected viremic progressors. Our affinity-profiling system may help to refine studies of R5 virus tropism and pathogenesis.

Tissue-Specific Sequence Alterations in the Human Immunodeficiency Virus Type 1 Envelope Favoring CCR5 Usage Contribute to Persistence of Dual-Tropic Virus in the Brain

ABSTRACT Most human immunodeficiency virus type 1 (HIV-1) strains isolated from the brain use CCR5 for entry into macrophages and microglia. Strains that use both CCR5 and CXCR4 for entry (R5X4 strains) have been identified in the brains of some individuals, but mechanisms underlying the persistence of R5X4 viruses compartmentalized between the brain and other tissue reservoirs are unknown. Here, we characterized changes in the HIV-1 envelope (Env) that enhance the tropism of R5X4 variants for brain or lymphoid tissue. R5X4 Envs derived from the brains of two individuals had enhanced CCR5 usage in fusion assays compared to R5X4 Envs derived from matched spleen or blood, which was associated with reduced dependence on specific residues in the CCR5 N terminus and extracellular loop 1 (ECL1) and ECL3 regions. In contrast, spleen/blood-derived Envs had enhanced CXCR4 usage compared to brain-derived Envs, which was associated with reduced dependence on residues in the CXCR4 N terminus and ECL2 region. Consequently, brain-derived Envs had preferential CCR5 usage for HIV-1 entry into the JC53 cell line, could use either CCR5 or CXCR4 for entry into monocyte-derived macrophages (MDM), and could use CCR5 (albeit inefficiently) for entry into peripheral blood mononuclear cells (PBMC), whereas the entry of spleen-derived Envs was CXCR4 dependent in all three cell types. Mutagenesis studies of Env amino acid variants influencing coreceptor usage showed that S306 in the gp120 V3 region of brain-derived Envs reduces dependence on the CCR5 N terminus and enhances CCR5 usage for HIV-1 entry into PBMC and MDM, whereas R306 in spleen-derived Envs reduces dependence on the CXCR4 N terminus and confers the CXCR4 restricted phenotype. These results identify mechanisms underlying R5X4 HIV-1 persistence in different tissue reservoirs. Tissue-specific changes in the gp120 V3 region that increase the efficiency of CCR5 or CXCR4 usage, and thereby influence coreceptor preference, may enhance the tropism of R5X4 strains for CCR5-expressing macrophage lineage cells in the brain and CXCR4-expressing T cells in lymphoid tissues, respectively.