Background
There is a paucity of understanding of how HIV latent reservoirs survive and expand, escape immune clearance, and reactivate upon ART cessation. The integration site (IS) of proviral DNA into the host genome could (i) determine the transcriptional status of the provirus and (ii) influence the expansion of infected cells. In this study, we determined the proviral IS associated with clonal expansion and latency of HIV infected cells in an in vivo model.
Methods
Memory CD4+ T cells isolated from healthy human donors were infected with dual reporter- tagged, replication-defective HIV-1 to generate large populations (millions) of infected cells, each carrying a single transcriptionally active reporter provirus at a distinct IS. Clonal expansion and proviral reporter expression dynamics of infected cells was analyzed post-engraftment in NSG mice. HIV-1 IS were determined by PCR-based amplification of host-viral junction and next-generation sequencing.
Results
Following engraftment of HIV-1 infected CD4+ T cells in mice, the fraction of cells in which the HIV-1 reporter provirus was transcriptionally active decreased over time. HIV-1 IS analysis in long-term persistently infected cell clones led to the identification of 646 unique IS and the majority of IS were found in genic regions (82%) of human chromosomes. Comparison of genic IS in multiple animals led to the identification of recurrent integration in genes including NPLOC4, PACS1, NFATC3, NOSIP, and ATF7IP. Gene length and expression level correlated with increased frequency of integration, both in pre-engraftment cells and in expanded cell clones after engraftment. Sorting of cells into populations with active and latent proviruses at late time points after engraftment revealed that IS harboring transcriptionally latent proviruses were more likely to be found in non-genic, pericentric regions. To study and characterize cells with latent HIV-1 proviruses, we successfully generated and expanded single cell clones from engrafted cells, each carrying distinct proviral IS. Here, we observed that proviral reporter expression in latently infected clones was independent of cellular activation status and maintained during in vitro expansion.
Conclusions
HIV IS affects proviral transcriptional status, and latent proviruses were found more frequently integrated in non-genic regions. Current and future work involves studying the mechanisms underpinning individual IS-driven clonal survival of infected cells and proviral transcriptional status.
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