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

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

doi:10.1038/s41598-020-66153-z

Abstract

A key component of antiretroviral therapy (ART) for HIV patients is the nucleoside reverse transcriptase inhibitor (NRTI) is tenofovir. Recent reports of tenofovir toxicity in patients taking ART for HIV cannot be explained solely on the basis of off-target inhibition of mitochondrial DNA polymerase gamma (Polγ). PrimPol was discovered as a primase-polymerase localized to the mitochondria with repriming and translesion synthesis capabilities and, therefore, a potential contributor to mitochondrial toxicity. We established a possible role of PrimPol in tenofovir-induced toxicity in vitro and show that tenofovir-diphosphate incorporation by PrimPol is dependent on the n-1 nucleotide. We identified and characterized a PrimPol mutation, D114N, in an HIV+ patient on tenofovir-based ART with mitochondrial toxicity. This mutant form of PrimPol, targeting a catalytic metal ligand, was unable to synthesize primers, likely due to protein instability and weakened DNA binding. We performed cellular respiration and toxicity assays using PrimPol overexpression and shRNA knockdown strains in renal proximal tubular epithelial cells. The PrimPol-knockdown strain was hypersensitive to tenofovir treatment, indicating that PrimPol protects against tenofovir-induced mitochondrial toxicity. We show that a major cellular role of PrimPol is protecting against toxicity caused by ART and individuals with inactivating mutations may be predisposed to these effects.

Highlights

A key component of antiretroviral therapy (ART) for Human immunodeficiency virus (HIV) patients is the nucleoside reverse transcriptase inhibitor (NRTI) is tenofovir
Prior studies from our lab identified a Polγ R953C mutant in an HIV+ patient, which may predispose the patient to NRTI-induced mitochondrial toxicity by altering the ability of Polγ to discriminate between natural nucleotides and NRTI nucleotides[29]
The current study is focused on defining potential mechanisms of NRTI-mediated nephrotoxicity in HIV+ patients who are taking tenofovir-containing antiretroviral drug regimens

Summary

Introduction

A key component of antiretroviral therapy (ART) for HIV patients is the nucleoside reverse transcriptase inhibitor (NRTI) is tenofovir. Mechanistic studies have shown that Polγ incorporates the natural dATP substrate much more efficiently and selects against www.nature.com/scientificreports the active tenofovir diphosphate (TFV-DP) metabolite leading to a very favorable in vitro discrimination factor, suggesting that the Polγ hypothesis cannot fully explain the proposed mitochondrial toxicity caused by TDF15,16. These discrepancies may be explained by factors such as differences in metabolism, binding affinity and rate of incorporation of the respective NRTIs by Polγ, ineffective exonuclease removal, and the role of additional host cell polymerases[17,18,19]. We surmise that the presence of inactivating mutations in PrimPol such as D114N might contribute to the mitochondrial toxicity associated renal toxicity in some patients on TDF-based ART

Methods

Results

Discussion

Conclusion