A poorly understood molecular player in the intrinsic immune response against HIV is the simple nucleotide deoxyuridine triphosphate (dUTP). In non-replicating immune cells, dUTP levels are typically high, and upon HIV infection, viral reverse transcriptase incorporates dUTP into the nascent viral genome to generate U/A base pairs instead of the normal Watson-Crick T/A pairs. In the absence of nuclear uracil base excision repair (UBER) heavily uracilated viral DNA is fully competent for integration, but silences viral gene expression. Because UBER is virtually undetectable in resting cells, these findings indicate that uracilated proviruses will persist until a resting cell is activated. These considerations lead us to propose that persistent U/A base pairs play a previously unrecognized role in HIV-1 latency and reactivation. To characterize this aspect of HIV infection we will use a novel next-generation DNA sequencing method (BE-SEQ) to map the spatial distribution and temporal fate of "invisible" U/A base pairs in unintegrated and integrated HIV-1 DNA in a UBER-deficient cell line. High-resolution uracil mapping will provide valuable mechanistic information t understand how dUTP is incorporated into minus and plus strand DNA products by reverse transcriptase (RTase), and how its spatial distribution, persistence and possible error-prone removal may impact viral persistence in UBER-deficient immune cells. We will then determine the extent and persistence of uracil generated from dUTP incorporation or enzymatic cytosine deamination in proviral DNA obtained from resting CD4+ T cells of HIV-1 infected patients. These resting T cells will then be activated in vitro, and the fate of proviral uracils will be evaluated using a new trans-well viral outgrowth assay. Invisible uracils that silence viral gene transcription in the resting state could be repaired after reactivation, or instead, lead to lethal genetic alterations by error prone repair mechanisms. Overall, these studies will determine the distribution, persistence and fate of U/A pairs in proviral DNA sequences and their potential effect on viral latency and infection.
This proposal will elucidate how the simple base uracil in DNA can impact HIV-1 infectivity and persistence in human immune cells. This work has the potential to uncover new routes for pharmacological intervention in HIV infection and long term AIDS eradication.
|Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R et al. (2016) Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells. Elife 5:|