Background
Cure strategies that seek to permanently block reversal of HIV-1 latency remain largely unexplored. We previously showed that the HIV-1 antisense transcript AST induces a closed chromatin state at the HIV-1 5’LTR that blocks transcription and promotes latency in cell line models. Here, we assessed the ability of AST to prevent latency reversal in CD4+ T cells from PLWH, and its potential application in “block and lock” cure strategies. We also elucidated the underlying molecular mechanisms.
Methods
AST or a control RNA were expressed by nucleofection in CD4+ cells from PLWH under suppressive ART. Cells were then treated with SAHA, Panobinostat, or anti-CD3/CD28, and cell-associated HIV-1 gag RNA levels were evaluated by RT-qPCR. Identification of AST structural domains and sequence motifs was performed in the Jurkat E4 latency model. RIP and ChIRP assays were used to study the interaction of AST with chromatin factors and the 5’LTR, respectively. ChIP assays were used to assess epigenetic modifications at the 5’LTR.
Results
We found that ectopic expression of AST in CD4+ T cells from ART-suppressed PLWH blocks latency reversal in response to the HDAC inhibitors, SAHA and Panobinostat, and to TCR stimulation, enforcing HIV-1 transcriptional silencing. The ability of AST to promote HIV-1 latency requires a quadruplex forming G-rich motif of AST that interacts with and recruits the suppressive epigenetic complex, PRC2 to the 5’LTR. Replacement of this motif with an unrelated sequence disrupts AST binding to PRC2 and reversed latency. Two pyrimidine-rich motifs in the 5’ terminus of AST facilitate its interaction with the 5’ LTR via sequence homology. Targeted nucleotide substitutions in these two pyrimidine-rich motifs abolish the interaction of AST with the 5’ LTR and its latency-promoting activity. An unbiased proteomics screen of AST interactors revealed an array of previously known and potential new HIV-1-suppressive factors, including the SWI/SNF complex, TDP-43, PTBP1, MATR3, and hnRNPK.
Conclusions
Ectopic expression of AST inhibits HIV-1 transcription in PLWH-derived CD4+ T cells and locks proviruses in a state of permanent latency. AST functions as a scaffold for the recruitment and assembly of a biomolecular complex at the 5’ LTR leading to epigenetic silencing of HIV-1 transcription. Overall, our studies identify AST as a first-in-class biological molecule capable of enforcing latency in PLWH-derived cells, showing that AST has potential applications in cure strategies.
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