Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells

Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R.; Hosmane, Nina N.; Capoferri, Adam A.; Bruner, Katherine M.; Pollack, Ross A.; Zhang, Hao; Drummond, M. Bradley; Siliciano, Janet M.; Siliciano, Robert F.; Stivers, James T.

doi:10.7554/elife.18447

Cited by 38 publications

(149 citation statements)

References 77 publications

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“…Monocytes and dendritic cells had low incision activity on the U:A substrate ( Fig. 2c), resonating with the low level of uracil base excision in monocytes 12 and confirming that myeloid lineages have unique uracil repair phenotypes 13 . However, these differences in uracil repair were not apparent for the U:G substrate, presumably due to redundant activities of SMUG1 14 and MBD4 15 in incising U:G-containing substrates.…”

supporting

confidence: 54%

Simultaneous measurement of biochemical phenotypes and gene expression in single cells

Richer

Riemondy²,

Hardie

et al. 2019

Preprint

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ABSTRACTMethods to measure heterogeneity among cells are rapidly transforming our understanding of biology but are currently limited to molecular abundance measurements. We developed an approach to simultaneously measure biochemical activities and mRNA abundance in single cells to understand the heterogeneity of DNA repair activities across thousands of human lymphocytes, identifying known and novel cell-type-specific DNA repair phenotypes. Our approach provides a general framework for understanding functional heterogeneity among single cells.

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supporting

confidence: 54%

Simultaneous measurement of biochemical phenotypes and gene expression in single cells

Richer

Riemondy²,

Hardie

et al. 2019

Preprint

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“…We also observed that around 50% of GFP Ϫ MDM harbored some amount of intranuclear HIV-1 DNA. A recent study suggested that a subset of MDM may be resistant to HIV-1 infection due to increased dUTP incorporation into HIV-1 RT products and their subsequent degradation (48). It is therefore possible that the fewer EdU puncta within the GFP Ϫ MDM subset may result from the degradation of uracilated HIV-1 RT products.…”

Section: Discussionmentioning

confidence: 99%

Imaging HIV-1 Genomic DNA from Entry through Productive Infection

Stultz

Cenker

McDonald

2017

J Virol

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In order to track the fate of HIV-1 particles from early entry events through productive infection, we developed a method to visualize HIV-1 DNA reverse transcription complexes by the incorporation and fluorescent labeling of the thymidine analog 5-ethynyl-2=-deoxyuridine (EdU) into nascent viral DNA during cellular entry. Monocyte-derived macrophages were chosen as natural targets of HIV-1, as they do not divide and therefore do not incorporate EdU into chromosomal DNA, which would obscure the detection of intranuclear HIV-1 genomes. Using this approach, we observed distinct EdU puncta in the cytoplasm of infected cells within 12 h postinfection and subsequent accumulation of puncta in the nucleus, which remained stable through 5 days. The depletion of the restriction factor SAMHD1 resulted in a markedly increased number of EdU puncta, allowing efficient quantification of HIV-1 reverse transcription events. Analysis of HIV-1 isolates bearing defined mutations in the capsid protein revealed differences in their cytoplasmic and nuclear accumulation, and data from quantitative PCR analysis closely recapitulated the EdU results. RNA fluorescence in situ hybridization identified actively transcribing, EdUlabeled HIV-1 genomes in productively infected cells, and immunofluorescence analysis confirmed that CDK9, phosphorylated at serine 175, was recruited to RNApositive HIV-1 DNA, providing a means to directly observe transcriptionally active HIV-1 genomes in productively infected cells. Overall, this system allows stable labeling and monitoring of HIV genomic DNA within infected cells during cytoplasmic transit, nuclear import, and mRNA synthesis. IMPORTANCEThe fates of HIV-1 reverse transcription products within infected cells are not well understood. Although previous imaging approaches identified HIV-1 intermediates during early stages of infection, few have connected these events with the later stages that ultimately lead to proviral transcription and the production of progeny virus. Here we developed a technique to label HIV-1 genomes using modified nucleosides, allowing subsequent imaging of cytoplasmic and nuclear HIV-1 DNA in infected monocyte-derived macrophages. We used this technique to track the efficiency of nuclear entry as well as the fates of HIV-1 genomes in productively and nonproductively infected macrophages. We visualized transcriptionally active HIV-1 DNA, revealing that transcription occurs in a subset of HIV-1 genomes in productively infected cells. Collectively, this approach provides new insights into the nature of transcribing HIV-1 genomes and allows us to track the entire course of infection in macrophages, a key target of HIV-1 in infected individuals.KEYWORDS fluorescent-image analysis, human immunodeficiency virus, macrophages H uman immunodeficiency virus type 1 (HIV-1) infection can be viewed as occurring in early (entry) stages, including reverse transcription (RT), nuclear import, and the integration of the viral genome, and late (productive) stages, including viral transcr...

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“…These contexts arise from two innate immune responses that result in the introduction of dUMP into viral DNA [1–5]. These responses involve either enzymatic cytosine deamination of the viral (-) strand cDNA by APOBEC DNA deaminases [1,2] or the incorporation of dUMP opposite to adenine on either strand of viral DNA during reverse transcription [3,4,6]. The incorporation pathway arises specifically in quiescent cells because [dUTP] is typically higher in such non-dividing cells and most DNA polymerases readily utilize dUTP as a substrate in competition with dTTP [7–9].…”

Section: Introductionmentioning

confidence: 99%

“…The two uracilation pathways are quite unique because cytosine deamination leads to G⍰A transition mutations at specific trinucleotide sequences on (+) strand genomic RNA [10], while dUMP incorporation opposite to adenine occurs on both strands and is not intrinsically mutagenic [3]. Previous studies by us and others indicate that the majority of the dUMP that is present with in vitro infected monocyte-derived-macrophages (MDM) arises from dUMP incorporation by reverse transcriptase [4,6]. Once the viral DNA products enter the nuclear compartment, both types of lesions are substrates for uracil excision by the enzyme nuclear uracil DNA glycosylase (hUNG2)[3], the first enzyme in the UBER pathway.…”

Section: Introductionmentioning

confidence: 99%

Deficient uracil base excision repair leads to persistent dUMP in HIV proviruses during infection of monocytes and macrophages

Meshesha

Esadze

Cui

et al. 2020

Preprint

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Non-dividing cells of the myeloid lineage such as monocytes and macrophages are target cells of HIV that have low dNTP pool concentrations and elevated levels of dUTP, which leads to frequent incorporation of dUMP opposite to A during reverse transcription (“uracilation”). One factor determining the fate of dUMP in proviral DNA is the host cell uracil base excision repair (UBER) system. Here we explore the relative UBER capacity of monocytes (MC) and monocyte-derived macrophages (MDM) and the fate of integrated uracilated viruses in both cell types to understand the implications of viral dUMP on HIV diversification and infectivity. We find that monocytes are almost completely devoid of functional UBER, while macrophages are mainly deficient in the initial enzyme uracil DNA glycosylase (hUNG2). Accordingly, dUMP persists in viral DNA during the lifetime of a MC and can only be removed after differentiation of MC into MDM. Overexpression of human uracil DNA glycosylase in MDM prior to infection resulted in rapid removal of dUMP from HIV cDNA and near complete depletion of dUMP-containing viral copies. This finding establishes that the low hUNG2 expression level in these cells limits UBER but that hUNG2 is restrictive against uracilated viruses. In contrast, overexpression of hUNG2 after viral integration did not accelerate the excision of uracils, suggesting that they may poorly accessible in the context of chromatin. We found that viral DNA molecules with incorporated dUMP contained unique (+) strand transversion mutations that were not observed when dUMP was absent (G→T, T→A, T→G, A→C). These observations and other considerations suggest that dUMP introduces errors predominantly during (-) strand synthesis when the template is RNA. These mutations may arise from the increased mispairing and duplex destabilizing effects of dUMP relative to dTMP during reverse transcription. Overall, the likelihood of producing a functional virus from in vitro infection of MC is about 50-fold and 300-fold reduced as compared to MDM and activated T cells. The results implicate viral dUMP incorporation in MC and MDM as a potential viral diversification and restriction pathway during human HIV infection.

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Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells

Cited by 38 publications

References 77 publications

Simultaneous measurement of biochemical phenotypes and gene expression in single cells

Simultaneous measurement of biochemical phenotypes and gene expression in single cells

Imaging HIV-1 Genomic DNA from Entry through Productive Infection

Deficient uracil base excision repair leads to persistent dUMP in HIV proviruses during infection of monocytes and macrophages

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