Defining a macrophage-tropic phenotype for HIV-1 to assess a role in pathogenesis is complicated by the fact that HIV-1 isolates vary continuously in their ability to enter monocyte-derived macrophages (MDMs) in vitro, and MDMs vary in their ability to support HIV-1 entry. To overcome these limitations, we identified consistent differences in entry phenotypes between five paired blood-derived, T cell-tropic HIV-1 env genes, four of which are CCR5-using (R5) and one of which is CXCR4-using (X4),

more »

... and cerebrospinal fluid (CSF)-derived, R5 macrophage-tropic env genes. We performed entry assays using the CD4- and CCR5-inducible Affinofile cell line, expressing a range of CD4 levels that approximates the range from MDMs to CD4+ T cells. The macrophage-tropic viruses were significantly better at infecting cells expressing low levels of CD4 than the T cell-tropic viruses from the same subjects, with the titration of CD4 providing a distinctive and quantitative phenotype. This difference in CD4 utilization was not due to macrophage-tropic viruses being CD4 independent. Furthermore, macrophage-tropic viruses did not differ from paired T cell-tropic viruses in their ability to use low levels of CCR5 (tpaired = −1.39; P = 0.24) or their use of an alternative conformation of CCR5. We also infected MDMs with a panel of viruses and observed that infectivity of each virus differed across four donors and between three preparations from a single donor. We concluded that the evolutionary transition from replication in T cells to that in macrophages involves a phenotypic transition to acquire the ability to infect cells expressing low levels of CD4 and that this phenotype is more reliably measured in Affinofile cells than in macrophages.