Characterizing HIV-1 reservoirs in the central nervous system

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Abstract A considerable number of clinical, neuropathological, immunohistochemical and immunological studies suggest that human brain cells, in particular those of myeloid origin, are susceptible to HIV-1 infection; infection of these cells may contribute to neurocognitive dysfunction and viral long-term persistence despite ART. However, among all anatomical tissue locations in the human body, the central nervous system (CNS) arguably represents the most difficult one to access and to evaluate for viral infection and persistence. Recently, we and others have made significant progress in defining viral reservoirs at a single-cell and single-molecule resolution, using a platform of novel next-generation sequencing assays allowing to simultaneously analyzing near full-length proviral sequences, the corresponding chromosomal integration sites and the respective HIV-1 RNA expression profile from individual viral reservoir cells. Using such technologies, we observed evidence for immune-mediated selection mechanisms that enable long-term persistence of viral reservoir cells with features of deep latency, while reservoir cells with higher susceptibility to reactivation signals seemed to be actively selected against; in rare cases of individuals with “elite control”, such selection mechanism resulted in a highly-restricted viral reservoir configuration consisting of intact proviruses located in heterochromatin positions not permissive to viral transcription. Here we will propose to use this established and fully-operational technology pipeline for a detailed analysis of the frequency, clonality and replication competence of proviruses isolated from sorted CNS cells, in particular from myeloid parenchymal microglia, from myeloid perivascular macrophages and astrocytes (Specific Aim 1). In addition, we will utilize novel next-generation sequencing assays for characterizing the chromosomal locations of intact and defective proviruses, and their associated epigenetic chromatin microenvironment, using ATAC-Seq, ChIP-Seq and Methylation-Seq assays (Specific Aim 2). For a functional evaluation of proviruses residing in the CNS, we will subsequently conduct reactivation assays with single patient-derived virally-infected cells from the CNS, allowing us to determine how chromosomal positioning and epigenetic features affect the transcriptional activity of proviruses and their susceptibility to latency-reversing agents (Specific Aim 3). Together, these investigations will provide high-resolution insight into the dynamics of HIV-1 persistence in myeloid CNS cell subsets and may be highly informative for targeted HIV-1 cure interventions.