Background:  HIV-1 evolves from R5 strains that use CCR5 for entry to R5X4 strains that use both CCR5 and CXCR4, and in some cases to X4 strains that use only CXCR4. We recently showed that R5X4 isolates use CXCR4 preferentially on primary lymphocytes, although they use both CCR5 and CXCR4 on macrophages. It is not known whether R5X4 strains use the 2 pathways equally well on primary macrophages. It also is unclear whether CCR5 and CXCR4 differ in the efficiency with which they support entry in the context of a primary target cell that expresses both co-receptors.

Methods:  We used quantitative polymerase chain reaction (PCR) to measure entry by 2 prototype and 3 primary R5X4 isolates in the presence or absence of CCR5 and CXCR4 blockers in primary macrophages and lymphocytes. To address the efficiency with which R5X4 isolates use each co-receptor on macrophages, we compared sensitivity to the fusion inhibitor T20 when entry was restricted to CCR5 or CXCR4 by single co-receptor inhibitors.

Results:  In lymphocytes CXCR4 blocking completely abrogated R5X4 strain entry but CCR5 blocking had no effect, confirming use of CXCR4 only. In macrophages, blocking each co-receptor independently reduced entry by ~20 to 80%, while blocking both pathways completely prevented entry, indicating that both CCR5 and CXCR4 were used. Of the 5 isolates, 2 utilized CCR5 and CXCR4 approximately equally, 2 used CCR5 more efficiently than CXCR4, and 1 entered more efficiently through CXCR4. For all 5 strains, the sensitivity to T20 did not differ based on whether entry was restricted to CCR5 or CXCR4. There was also no systematic difference between T20 sensitivity for R5X4 entry into macrophages through CCR5 compared with the R5 strain BAL, nor for entry through CXCR4 compared with the X4 strain Tybe.

Conclusions:  R5X4 isolates are functionally X4 on lymphocytes but R5X4 on macrophages, suggesting that viral evolution involves an expansion of co-receptor use in macrophages, but a switch from R5 to X4 in lymphocytes. However, R5X4 strains differ quantitatively in relative utilization of each entry pathway in macrophages. Surprisingly, blocking 1 entry pathway on macrophages does not result in a compensatory increase in entry through the other pathway, even though it is likely that virus, and not co-receptor, availability is limiting. Finally, based on T20 sensitivity assay, R5X4 isolates use macrophage CCR5 and CXCR4 with comparable efficiency, and with similar efficiency to that of the R5 and X4 reference strains BAL and Tybe.