Background:  Co-receptor utilization by HIV-1 strains is generally defined in vitro in cell lines expressing

CCR5 or CXCR4, although primary lymphocytes and macrophages are the principal targets in vivo. R5 strains dominate early in infection, but CXCR4-using strains emerge later in a substantial minority. Many late-stage CXCR4-using strains can use both CXCR4 and CCR5 (R5X4 strains) in cell lines, but the specific pathways used by these late-stage strains to infect primary cells and in vivo are unknown. Methods:  We examined 2 R5X4 prototypes (89.6 and DH12) and 3 R5X4 primary isolates and measured productive replication and/or polymerase chain reaction (PCR)-based entry assay in primary cells in the presence or absence of CCR5 or/and CXCR4 antagonist.

Results:  HIV-1 89.6 and 3 primary strains were completely restricted to CXCR4-mediated entry in primary lymphocytes, even though lymphocytes are permissive for CCR5-mediated entry by R5 strains (BaL). Prototype DH12 was largely (>90%) dependent on CXCR4 for entry and infection of lymphocytes. In contrast, in primary macrophages these R5X4 isolates used both CCR5 and CXCR4. The R5X4 strains were also more sensitive than R5 strains to a CCR5 antagonist in U87/CCR5 cell lines, suggesting less efficient interactions between R5X4 Env and CCR5.

Conclusions:  These results indicate that co-receptor phenotype in transformed cells does not necessarily predict utilization in primary cells, and many or most strains characterized as R5X4 use CXCR4 only to infect primary lymphocytes. Less efficient interaction between R5X4 strains and CCR5 may be responsible for the inability to use CCR5 on lymphocytes, which express relative low CCR5 levels. These results have implications for the evolution from R5 to R5X4 during disease progression, and for the use of chemokine receptor antagonists as antiretroviral therapeutics. Furthermore, since isolates that acquire CXCR4 utilization retain the ability to use CCR5 on macrophages despite their inability to use CCR5 on lymphocytes, these results also raise the possibility that a CCR5-mediated macrophage reservoir is required for sustained infection in vivo.

Keywords: coreceptor; primary lymphocytes; macrophage reservoir