Summary
Latent replication-competent HIV-1 persists in individuals on long-term antiretroviral therapy (ART). We developed the Full-Length Individual Proviral Sequencing (FLIPS) assay to determine the distribution of latent replication-competent HIV-1 within memory CD4+ T cell subsets in six individuals on long-term ART. FLIPS is an efficient, high-throughput assay that amplifies and sequences near full-length (~9 kb) HIV-1 proviral genomes and determines potential replication-competency through genetic characterization. FLIPS provides a genome-scale perspective which addresses the limitations of other methods that also genetically characterize the latent reservoir. Using FLIPS, we identified 5% proviruses as intact and potentially replication-competent. Intact proviruses were unequally distributed between T cell subsets, with effector memory cells containing the largest proportion of genetically intact HIV-1 proviruses. We identified multiple identical intact proviruses suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir.
Significance
Identifying the source and dynamics of persistent HIV-1 during combinational antiretroviral therapy (cART) is crucial for understanding the barriers to curing HIV infection. Through genetic characterization of HIV-1 DNA in infected cells from peripheral blood and gut-associated lymphoid tissue from patients after long-term suppressive cART, our study reveals that the primary barrier to a cure is a remarkably stable pool of infected memory CD4
+
T cells. Through in-depth phylogenetic analyses, we determined that the HIV-1 reservoir in these cells from eight patients is kept stable during long-term cART and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes.
Memory T cells maintained a relatively constant HIV-1 DNA integrant pool that was genetically stable during long-term effective ART. These integrants appear to be maintained by cellular proliferation and longevity of infected cells, rather than by ongoing viral replication.
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