Abstract
Although CD4+ memory T cells are considered the primary latent reservoir for HIV-1, replication competent HIV has been detected in tissue macrophages in both animal and human studies. During in vitro HIV infection, the depleted nucleotide pool and high dUTP levels in monocyte derived macrophages (MDM) leads to proviruses with high levels of dUMP, which has been implicated in viral restriction or reduced transcription depending on the uracil base excision repair (UBER) competence of the macrophage. Incorporated dUMP has also been detected in viral DNA from circulating monocytes (MC) and alveolar macrophages (AM) of HIV infected patients on antiretroviral therapy (ART), establishing the biological relevance of this phenotype but not the replicative capacity of dUMP-containing proviruses. As compared to in vitro differentiated MDM, AM from normal donors had 6-fold lower levels of dTTP and a 6-fold increased dUTP/dTTP, indicating a highly restrictive dNTP pool for reverse transcription. Expression of uracil DNA glycosylase (UNG) was 8-fold lower in AM compared to the already low levels in MDM. Accordingly, ∼80% of HIV proviruses contained dUMP, which persisted for at least 14-days due to low UNG excision activity. Unlike MDM, AM expression levels of UNG and SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) increased over 14 days post-HIV infection, while dUTP nucleotidohydrolase expression decreased. These AM-specific effects suggest a restriction response centered on excising uracil from viral DNA copies and increasing relative dUTP levels. Despite the restrictive nucleotide pools, we detected rare replication competent HIV in AM, peripheral MC, and CD4+ T cells from ART-treated donors. These findings indicate that the potential integration block of incorporated dUMP is not realized during in vivo infection of AM and MC due to the near absence of UBER activity. In addition, the increased expression of UNG and SAMHD1 in AM post-infection is too slow to prevent integration. Accordingly, dUMP persists in integrated viruses, which based on in vitro studies, can lead to transcriptional silencing. This possible silencing outcome of persistent dUMP could promote viral latency until the repressive effects of viral dUMP are reversed.
Competing Interest Statement
The authors have declared no competing interest.
ABREVIATIONS
- AM
- alveolar macrophage
- APE1
- Apurinic/apyrimidinic endonuclease 1
- A3A
- Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A
- A3G
- Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G
- ART
- antiretroviral therapy
- BAL
- Bronchoalveolar lavage
- BLT
- bone marrow-liver-thymus
- cDNA
- Complementary DNA
- CNS
- central nervous system
- DAPI
- 4’,6-diamidino-2-phenylindole
- ddPCR
- Droplet Digital PCR
- dNTP
- Deoxynucleoside triphosphate
- dsDNA
- double stranded DNA
- dU
- deoxyuridine
- dUMP
- deoxyuridine monophosphate
- DUT
- DUTP pyrophosphohydrolase
- dUTP
- deoxyuridine triphosphate
- dT
- Thymidine
- dTTP
- Thymidine triphosphate
- Env
- viral envelope protein
- ERT
- early reverse transcripts
- HAP1
- a near-haploid human cell line
- HIV
- human immunodeficiency virus
- hUNG
- human uracil DNA glycosylase
- IDT
- Integrated DNA Technologies
- IUPM
- infectious units per million
- Lig III
- DNA Ligase 3
- LRT
- late reverse transcripts
- LTR
- long terminal repeat
- MC
- monocyte
- M-CSF
- Macrophage colony-stimulating factor
- MDM
- Monocyte-derived macrophage
- NEB
- New England Biolabs
- P24
- a component of the HIV particle capsid
- PBMC
- peripheral blood mononuclear cell
- PHA
- Phytohemagglutinin
- Pol β
- DNA polymerase beta
- QVOA
- Quantitative Viral Outgrowth Assay
- RPP30
- Ribonuclease P/MRP Subunit P30
- RT-PCR
- Reverse transcription polymerase chain reaction
- SAMHD1
- SAM domain and HD domain-containing protein 1
- SIV
- simian immunodeficiency virus
- TCR β
- T-cell receptor chain beta
- TMP
- Thymidine monophosphate
- TTP
- Thymidine triphosphate
- UBER
- uracil base excision repair
- UNG
- uracil DNA glycosylase