PP 1.30 – 00159 Dating HIV-1 reservoir formation in ARV-suppressed Ugandans

Background: HIV-1 expresses a 2.6-kb antisense transcript (Ast) with protein-coding and noncoding roles.We previously showed that Ast promotes HIV-1 latency by recruiting the PRC2 complex to the 5′LTR leading to deposition of H3K27me3 marks and epigenetic silencing.We sought to identify Ast domains and motifs involved in these effects.We also assessed Ast expression in resting CD4+ T cells from ART-suppressed PLWH.Methods: Functional activity of Ast mutants was assessed via stable transduction in the Jurkat E4 latency model.ChIP, ChIRP and RIP assays were used to assess the interaction between Ast and its binding partners.Expression of Ast in patient-derived samples was assessed by CARD-SGS and by digital PCR.Results: The 5′ end of Ast mapping in the U3 region of the 3′ LTR (U3AST) interacts with the homologous U3 region of the proviral 5′LTR.Nucleotide substitutions in two pyrimidine-rich motifs within U3AST disrupted interaction between Ast and 5′ LTR.Over-expression of the U3AST fragment in latently infected cells reversed latency by displacing endogenous Ast from the 5′ LTR.A G-quadruplex (G4) motif at the center of Ast mediates interaction with PRC2.Mutation of 70 nt comprising the G4 motif abolished the interaction with PRC2 and the latencypromoting function of Ast.Full suppressive activity requires interaction with additional host factors, including the transcriptional silencers YY1 and CTCF, and members of the chromatin remodeling complexes BAF and NuRD.Substitution of a domain at the 3′ of Ast abolished the interaction with these host factors and latency-promoting function of Ast.CARD-SGS analyses detected on average 1 copy of Ast in ∼5% of infected cells from donors on ART, whereas more sensitive digital PCR detected Ast in ∼26% of infected cells.Figure.Ast model Conclusion: The HIV-1 Ast promotes viral latency in vitro via direct recruitment of transcriptional silencers and chromatin remodeling complexes to HIV-1 5′LTR, supporting its use in cure strategies.Expression of Ast is detectable in latently infected CD4+ T cells from ART-suppressed PLWH.

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