Background: HIV-1 entry is mediated by CD4 and one of two co-receptors, CCR5 or CXCR4. The ability of an HIV-1 strain to use differing levels of CD4/CCR5 reflects its affinity for CD4 and co-receptor, and is one factor associated with the overall viral infectivity and pathogenicity. We have designed a system to quantitatively describe the efficiency of CD4/CCR5 usage, and used it to examine SIV, HIV-1 strains, and primary isolates from infants with rapid- and slow-progressing (RP vs SP) disease.

Methods: An inducible cell line was created where CD4 and CCR5 expression can be simultaneously and independently controlled with tetracycline and ponasteroneA, respectively. CD4 and CCR5 levels can be induced and controlled within the range of expression found on primary cell targets of HIV-1. Infection was quantified by intracellular p24 staining, or GFP/luciferase activity when using pseudotyped reporter viruses.

Results: Infection of the cells with SIV-316 pseudotyped virus showed dependence on CCR5 but not CD4 concentration, consistent with the known CD4 independence of the virus. Infection with R5 viruses, Bal or YU2, increased with increasing concentrations of both CD4 and CCR5. Infection with 89.6 showed marked dependence on CD4 levels, regardless of the amount of CCR5 present. Infection with primary Clade B and A strains with previously defined high and low affinity for CCR5 and CD4, showed that each virus generates a distinct 3-D surface curve that represents their efficiency of CD4 and CCR5 usage. Mathematical modeling provided a scalar metric that clusters each isolate distinctly on a polar plot, and quantitatively describes their CD4/CCR5 usage pattern.  Finally, at maximal CD4/CCR5 induction, infection with isolates from 3 RP and 4 SP resulted in a significantly higher percentage of infected cells from the RP isolates (0.38-9.1 %  vs 12.11-18.99 % p24+ cells for SP vs RP isolates, respectively; p<0.01).

Conclusions: Various SIV and HIV-1 strains exhibit different patterns of CD4 and CCR5 usage, as shown by distinct 3-D surface curves, which can be clustered distinctly in a scalar polar plot. Also, viruses from RP are more efficient in using CD4 and CCR5 than those from SP. This assay can directly quantify CD4/CCR5 usage by primary HIV isolates, and can be used to study the correlation between CD4/CCR5 usage and viral pathogenicity, and potentially, to monitor the changes in CD4/CCR5 affinity that may occur with the clinical use of CCR5 inhibitors.