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

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.

A) Statistical significance of the estimated rates for TFV-DP incorporation in Fig. 1C and 1D using the one-way ANOVA test, **** = p<0.0001. B) Denaturing PAGE showing the preceding nucleotide preference for TFV-DP incorporation by PrimPol is present for two independent primer/template sequences, D20A/D45 (left two gels) and D21/D36 (right two gels). In both cases, PreA TFV-DP incorporation is favored over PreC. C) The preceding nucleotide preference also occurs with the isolated polymerase domain of PrimPol (amino acids 1-354) using the D20A/D45 primer/template. D) The preceding nucleotide preference was tested using different NRTIs, d4T (dT analog, D22T/D45, top), (-)-3TC (dC analog, D23C/D45, middle), and (-)-FTC (dC analog, D23C/D45, bottom). In all cases there was not a strong preceding nucleotide preference, demonstrating that the effect observed with TFV-DP is unique in the NRTIs tested. Figure S2. Determining the kinetic parameters of TFV-DP and dATP incorporation by WT PrimPol dependent on the preceding nucleotide in the primer strand (see Table 1).
In the case of TFV-DP incorporation by PrimPol, the amplitude of product formation varied with TFV-DP concentration. A-D) The kinetic parameters of TFV-DP incorporation by PrimPol were calculated by plotting the amplitude of single turnover experiments with PrimPol and TFV-DP against the concentrations of TFV-DP used. The Kd was determined by the concentration at half of the amplitude max. E) The similar rates across TFV-DP concentrations were averaged to determine the kpol. F) The kpol and Kd of dATP incorporation by PrimPol was determined by plotting the rates of each single turnover experiment against the concentration of TFV-DP used in each experiment. Table S1. Comparison of incorporation efficiencies of NRTIs by PrimPol (see Table 1).
Values for TFV-DdP incorporation by PrimPol under optimal conditions (preceding dT in the primer) are compared to the incorporation efficiency values of other NRTIs by PrimPol. The errors represent the standard error values of the parameters that corresponds to a confidence level of 68.3%, or to one standard deviation. a Values obtained from Ref. 28. Figure S3. TFV-DP is able to reduce the product length of primers during elongation but unable to inhibit dimer formation (see Fig. 1).
A) Dimer formation during primer initiation by PrimPol was monitored in various concentrations of TFV-DP. TFV-DP did not compete with ATP even at excess concentrations. B) The interference of TFV-DP in primer elongation by PrimPol was monitored. Using a 3'-GTCT-5' and a 3'-GCTC-5' substrate, TFV-DP was added at different concentrations in competition with dATP or ATP. Figure S4. Thermal shift assay of WT and D114N PrimPol dependent on the presence of Mg 2+ or Mn 2+ and DNA (see Fig. 4G).
WT or D114N PrimPol (5 µM) was mixed with either MgCl2 or MnCl2 (10 µM) and dsDNA (5 µM) and the unfolding of the protein was monitored through fluorescence by SYPRO orange. The derivatives of the relative fluorescence units were plotted against temperature, the local minima identified, and the corresponding temperatures were recorded. Numbers are shown as the mean ± SD. For experiment 1A and 1B, n=6, and for experiment 2, n=3. A) Western blotting of PrimPol of the scrambled, wild-type PrimPol overexpression, and shRNA knockdown renal proximal tubule epithelial cell lines. B) Immunoblotting of TRAP1 in RPTECs after treatment of 30 µM TDF for 3 days. The lanes for each treatment represent a technical replicate, n=3. C) Quantified relative amounts of TRAP1 levels across 3 biological replicates. The normalized levels of TRAP1 to GAPDH were compared for each cell strain to the untreated control. The shRNA and scrambled cell lines show a downward, but statistically nonsignificant (one-way ANOVA), trend of TRAP1 levels compared to the PrimPol overexpression strain. D) Seahorse XF Analyzer MitoStress assay schematic to measure parameters related to mitochondrial respiration. A) Cells were seeded in 6-well plate at a low density and then counted after 2 and 4 days, n=3. B) The PrimPol RPTEC strains were treated with abacavir over 5 days and cellular proliferation was monitored using the MTT assay, n=3. C) As a control, the cells were treated with efavirenz, an NNRTI, over 5 days and the MTT assay was used to assess the toxicity, n=3. Significance was determined by one-way ANOVA, * = p<0.05, ** = p<0.01, and *** = p<0.001. Figure S7. Standard curves for qPCR quantification of mtDNA.
The target DNA, A) mtDNA or B) nucDNA, was serial diluted and the cycle threshold was plotted against the concentration of DNA. Relative mtDNA content of RPTECs treated with TDF was calculated through qPCR. CT values were obtained and relative mtDNA was calculated by subtracting the CT of mtDNA from nucDNA. The final value was then determined by the equation = 2 * 2 ∆ . Those values were then normalized to the no treatment control. For each biological replicate, three technical replicates were conducted. For Western blots, polyacrylamide gels were cut before membrane transfer. A) Fig. 1C. B) Fig. S5A, Samples were run on the same gel and corresponding molecular weights were cut out before transfer. C) Fig. S5B Samples were run on the same gel and corresponding molecular weights were cut out before transfer, as denoted by the solid line. Different gels are separated by the dashed line. All membranes were exposed at the same time. D, E) Fig. 5I, Samples were run on the same gel and corresponding molecular weights were cut out before transfer.