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

Abstract Background 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.Results 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.Conclusions 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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Deficient uracil base excision repair leads to persistent dUMP in HIV proviruses during infection of monocytes and macrophages

10.1101/2020.03.10.985283 ◽

2020 ◽

Author(s):

Mesfin Meshesha ◽

Alexandre Esadze ◽

Junru Cui ◽

Natela Churgulia ◽

Sushil Kumar Sahu ◽

...

Keyword(s):

Base Excision Repair ◽

Reverse Transcription ◽

Excision Repair ◽

Dna Glycosylase ◽

Target Cells ◽

Viral Dna ◽

Uracil Dna Glycosylase ◽

Monocytes And Macrophages ◽

Base Excision ◽

Uracil Base

AbstractNon-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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Dynamics in Uracil Base Excision Repair

ACS Symposium Series - Structural Biology of DNA Damage and Repair ◽

10.1021/bk-2010-1041.ch004 ◽

2010 ◽

pp. 47-58

Author(s):

Joshua I. Friedman ◽

James T. Stivers

Keyword(s):

Base Excision Repair ◽

Excision Repair ◽

Base Excision ◽

Uracil Base

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Linking uracil base excision repair and 5-fluorouracil toxicity in yeast

Nucleic Acids Research ◽

10.1093/nar/gkj430 ◽

2006 ◽

Vol 34 (1) ◽

pp. 140-151 ◽

Cited By ~ 76

Author(s):

L. Seiple

Keyword(s):

Base Excision Repair ◽

Excision Repair ◽

Base Excision ◽

Fluorouracil Toxicity ◽

Uracil Base

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Ung1p-mediated uracil-base excision repair in mitochondria is responsible for the petite formation in thymidylate deficient yeast

FEBS Letters ◽

10.1016/j.febslet.2009.04.003 ◽

2009 ◽

Vol 583 (9) ◽

pp. 1499-1504 ◽

Cited By ~ 3

Author(s):

Chia-Yi Chien ◽

Chen-Kung Chou ◽

Jin-Yuan Su

Keyword(s):

Base Excision Repair ◽

Excision Repair ◽

Base Excision ◽

Uracil Base

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

eLife ◽

10.7554/elife.18447 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 21

Author(s):

Erik C Hansen ◽

Monica Ransom ◽

Jay R Hesselberth ◽

Nina N Hosmane ◽

Adam A Capoferri ◽

...

Keyword(s):

Base Excision Repair ◽

Defense Mechanism ◽

Excision Repair ◽

Blood Monocytes ◽

Viral Dna ◽

Recent Infection ◽

Base Excision ◽

Viral Genomic Rna ◽

Uracil Base ◽

Hiv 1

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection.

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