The discovery that chemokine receptors serve as co-receptors for HIV-1 fusion & entry has advanced our understanding of viral biology, yet has raised critical questions in the pathogenesis of HIV encephalopathy (HIVE). These questions include the role of CXCR4, which is widely expressed in CNS on microglia, macrophages & neurons and can mediate not just entry (in conjunction with CD4) but functional & neurotoxic effects of Env; secondary co-receptors including CCR3, molecules such as CX3CR1 & APJ that are highly expressed in brain, and the cytomegalovirus receptor US28; and whether there is a role for CD4- independent co-receptor utilization as occurs in neuropathogenic neuroadapted SIV strains. Current knowledge of viral species in HIVE are based mainly on sequences, clones or isolates from CSF or brain tissues. However, CSF variants may not reflect those in parenchyma, and species isolated from tissue may not reflect minority variants or distinguish those from contaminating blood cells Also, cultured virus would not reflect variants adapted to brain cells that replicate poorly in blood cells in vitro. These limitations are an important gap in our understanding of how viruses in vivo interact with chemokine receptor co-receptors and how these interactions, suggested by in vitro of non-human animal models, contribute to pathogenesis. Our hypothesis is that HIV-co-receptor interactions are important in vital compartmentalization & pathogenesis in HIVE, and that an important role is played by viral variants that are minority species present in specific cells in brain or that replicate poorly in culture. In our previous studies we have made significant progress in understanding HIV-1 entry co-receptors macrophage infection, and the biology, genetics & entry co-receptor use of primary isolates. We propose to extend these studies exploiting a novel approach that enables us to clone viral sequences from individual infected cells in brain using single- cell PCR. The goals are to define at the individual cell level chemokine receptor interactions of HIV-1 species in brain in HIVE. We will: (1) Amplify HIV-1 env from individual cells in HIVE autopsy brain & construct functional clones for cell-specific viral analysis; (2) Define the use of major, minor & CD4-independent co-receptors by these functional envs; (3) Determine their target cell tropism & replication characteristics using pseudotype & recombinant infectious viruses, and; (4) Use single cell-derived cDNAs to define cell-specific patterns of altered cellular gene expression in infected versus uninfected microglial in brain tissue from HIVE.
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