Identifying the role of PrimPol in TDF-induced toxicity and implications of its loss of function mutation in an HIV+ patient

Duong, Vincent N.; Zhou, Lei; Martínez‐Jiménez, María I.; He, Linh; Cosme, Moises; Blanco, Luis; Paintsil, Elijah; Anderson, Karen S.

doi:10.1038/s41598-020-66153-z

Cited by 20 publications

(19 citation statements)

References 70 publications

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“…In agreement with the localization of PrimPol in mitochondria, PrimPol-deficient cells were not able to produce these specific replication intermediates, allowing us to conclude that PrimPol helps mtDNA replication by reinitiating forks stalled by ddC-primer incorporation, and also by re-priming from nonconventional origins [11]. In concordance, a recent work reported a patient with mitochondrial toxicity associated with antiviral therapy against HIV who presented an inactivating mutation at the catalytic centre of PrimPol [14]. Therefore, PrimPol has a protective role in mitochondrial toxicity induced by NRTI antiviral therapy [14].…”

Section: Introductionsupporting

confidence: 85%

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Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

Carvalho

Díaz-Talavera

Calvo

et al. 2021

Genes

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PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3′nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg291 residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3′-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg291, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3′elongation site, thus avoiding synthesis of abortive primers.

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Section: Introductionsupporting

confidence: 85%

“…In concordance, a recent work reported a patient with mitochondrial toxicity associated with antiviral therapy against HIV who presented an inactivating mutation at the catalytic centre of PrimPol [14]. Therefore, PrimPol has a protective role in mitochondrial toxicity induced by NRTI antiviral therapy [14].…”

Section: Introductionsupporting

confidence: 60%

Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

Carvalho

Díaz-Talavera

Calvo

et al. 2021

Genes

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“…Similarly, GO enrichment analysis, many up and down-regulated genes may play a very key role in the progression of SARS-CoV-2 infection. Enriched genes such as IFNL4 [117], ZFP36 [118], CD274 [119], MIR21 [120], CYP1A1 [121], CMPK2 [122], C4A [123], SERPINE1 [124], CCRL2 [125], CD69 [126], FAH (fumarylacetoacetate hydrolase) [127], ERFE (erythroferrone) [128], SERINC5 [129], ADI1 [130], AP2M1 [131], PRIMPOL (primase and DNA directed polymerase) [132], BIRC5 [133] and TF (transferrin) [134] were responsible for the advancement of various viral infections, but these genes may be involved in the progression of SARS-CoV-2 infection. Enriched genes such as APOBEC3G [135], NLRC5 [136], PTX3 [137], FAP ( broblast activation protein alpha) [138] and CAT (catalase) [139] were linked with the development of in uenza viral infection, but these genes may be associated with progression of SARS-CoV-2 infection as well.…”

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confidence: 99%

Identification of Differentially Expressed Genes and Enriched Pathways in SARS-CoV-2/ COVID-19 using Bioinformatics Analysis

Alshabi

Shaikh

Vastrad

et al. 2020

Preprint

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BackgroundThe exact molecular mechanisms of the progression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain unclear. The current investigation strived to understand and functionally analyze the differentially expressed genes (DEGs) between SARS-CoV-2 infection and mock samples applying extensive bioinformatics analyses.MethodsGSE148729 dataset was downloaded from the Gene Expression Omnibus (GEO) and investigated utilising the limma package in R software to identify DEGs. Pathway and gene ontology (GO) enrichment analysis of the up and down-regulated genes were performed in ToppGene. The HIPPIE database was applied to estimate the interactions of up and down-regulated genes and to construct a protein-protein interaction (PPI) network using Cytoscape software. Receiver operating characteristic (ROC) was utilized for validation.ResultsA total of 928 DEGs (461 up-regulated genes and 467 down-regulated genes) were identified between SARS-CoV-2 infection and mock samples. The up and down-regulated genes were significantly enriched in cytokine-cytokine receptor interaction, and ascorbate and aldarate metabolism. Several significant GO terms, including the response to biotic stimulus and oxoacid metabolic process, were identified. The top hub genes and target genes included JUN, FBXO6, PCLAF, CFTR, TXNIP, PMAIP1, BRI3BP, FAHD1, PROX1, CXCL11, SERHL2 and CFI. ROC curve analysis showed that messenger RNA levels of these ten genes (DDX58, IFITM2, IRF1, PML, SAMHD1, ACSS1, CYP2U1, DDC, PNMT and UGT2A3) exhibited better diagnostic efficiency between SARS-CoV-2 infection and mock.ConclusionsThe current investigation distinguished vital genes and pathways that may be implicated in the progression of SARS-CoV-2 infection, providing a new understanding of the underlying molecular mechanisms of SARS-CoV-2 infection.

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“…Further complicating mitochondrial toxicity, genetic mutations encoding variants of DNA polymerase subunits, which localize to the mitochondrion, could predispose patients to mitochondrial toxicity, e.g. , p140 (R964C, R953C, and E1143D/G) and PrimPol D114N ( 72 , 73 , 74 , 75 , 76 , 77 ). Although we have characterized the differential effects of a single NRTI on proliferating and differentiated HepaRG in this work, other antivirals, drugs, or environmental mitochondrial toxicants should be investigated in the future.…”

Section: Discussionmentioning

confidence: 99%

The antiretroviral 2′,3′-dideoxycytidine causes mitochondrial dysfunction in proliferating and differentiated HepaRG human cell cultures

Young

Wheeler

Rahman

et al. 2021

Journal of Biological Chemistry

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Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus infection, and their use can cause mitochondrial toxicity, including mitochondrial DNA (mtDNA) depletion in several cases. The first-generation NRTIs, including 2′,3′-dideoxycytidine (ddC), were originally and are still pursued as anticancer agents. NRTI-sensitive DNA polymerases localizing to mitochondria allow for the opportunity to poison proliferating cancer cell mtDNA replication as certain cancers rely heavily on mitochondrial functions. However, mtDNA replication is independent of the cell cycle creating a significant concern that toxicants such as ddC impair mtDNA maintenance in both proliferating and nonproliferating cells. To examine this possibility, we tested the utility of the HepaRG cell line to study ddC-induced toxicity in isogenic proliferating (undifferentiated) and nonproliferating (differentiated) cells. Following ddC exposures, we measured cell viability, mtDNA copy number, and mitochondrial bioenergetics utilizing trypan blue, Southern blotting, and extracellular flux analysis, respectively. After 13 days of 1 μM ddC exposure, proliferating and differentiated HepaRG harbored mtDNA levels of 0.9% and 17.9% compared with control cells, respectively. Cells exposed to 12 μM ddC contained even less mtDNA. By day 13, differentiated cell viability was maintained but declined for proliferating cells. Proliferating HepaRG bioenergetic parameters were severely impaired by day 8, with 1 and 12 μM ddC, whereas differentiated cells displayed defects of spare and maximal respiratory capacities (day 8) and proton-leak linked respiration (day 14) with 12 μM ddC. These results indicate HepaRG is a useful model to study proliferating and differentiated cell mitochondrial toxicant exposures.

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Identifying the role of PrimPol in TDF-induced toxicity and implications of its loss of function mutation in an HIV+ patient

Cited by 20 publications

References 70 publications

Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

Identification of Differentially Expressed Genes and Enriched Pathways in SARS-CoV-2/ COVID-19 using Bioinformatics Analysis

The antiretroviral 2′,3′-dideoxycytidine causes mitochondrial dysfunction in proliferating and differentiated HepaRG human cell cultures

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