Phenotypic and Genotypic Features of Macrophage Tropism in HIV-1

HIV-1 preferentially attaches and enters specific cell types by recognizing cell-specific receptors. HIV-1 typically targets memory CD4+ T cells by restricting attachment to cells expressing high CD4 densities and by using the CCR5 coreceptor. HIV-1 can adapt to enter CD4+ naïve T cells by evolving to use CXCR4 in addition to CCR5. Alternatively, HIV-1 can adapt infecting macrophages by enhancing the entry using low CD4 densities. Early classification confusion and the lack of a reproducible, high-throughput method to assess cellular tropism has hindered robust identification and characterization of macrophage tropism. We developed cellular tropism assays using a novel cell line to perform both high-throughput identification screens and detailed analyses of receptor and coreceptor interactions. Clarified definitions of cellular tropism enabled us to probe the mechanism and consequences of evolving macrophage tropism through genotypic and phenotypic characterizations of subject-matched T-tropic and M-tropic env genes. M-tropic Env proteins are mostly indistinguishable from T-tropic Env proteins, except for subtle differences in the CD4-binding site (CD4bs) revealed by differences in interactions with CD4, competitive inhibition by soluble CD4, anti-CD4bs antibody sensitivity, and the ability of CD4-binding proximal residues to affect CD4 usage. CD4 usage is highly correlated with entry of monocyte-derived macrophages over a continuous range, making CD4 usage a useful proxy for macrophage tropism. Other stages of binding and entry are not major factors in the evolution of macrophage tropism and M-tropic Env proteins are not generally sensitive to antibodies. Nor does the antibody environment appear as a major selective pressure on the evolution of macrophage tropism. The genetic determinants are complex; evolving macrophage tropism requires unique constellations of amino acid substitutions at distal locations in the Env protein. However, substitutions of bulkier, more hydrophobic amino acid residues tend to accumulate in the V5 region of the Env protein. The true genetic determinants of macrophage tropism are still unknown, but we have begun to identify features to help identify new M-tropic viruses, which is an essential part of unraveling the greater mysteries of macrophage tropism.Doctor of Philosoph