Human Mitochondrial Dna Polymerase With Anti-hiv Nucleotides

National Institute Of Environmental Health Sciences

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Abstract

The mode and effect of antiviral nucleotide analogs, by AZT, ddI, 3TC, D4T and others on the inhibition and fidelity of the mitochondrial DNA polymerase and mitochondrial DNA replication have been documented and characterized in my laboratory. We now know what structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to mitochondrial toxicity. We previously compared the inhibition, insertion, and exonucleolytic removal of five currently approved antiviral nucleotide analogs on the purified human recombinant DNA polymerase gamma. The apparent Km and kcat values were determined for the incorporation of TTP, dCTP, dGTP, 2-3-dideoxy-TTP (ddTTP), 3-azido-TTP (AZT-TP), 2-3-dideoxy-CTP (ddCTP), 2-3didehydro-TTP (D4T-TP), (-)-2,3-dideoxy-3-thiacytidine (3TC-TP), and carbocyclic 2,3-didehydro-dGTP (CBV-TP). Kinetic studies indicate that the apparent in vitro hierarchy of mitochondrial toxicity for the approved NRTIs is: ddC(zalcitabine) = ddI(didanosine) = D4T(stavudine) > >3TC(lamivudine) >PMPA(tenofovir)> AZT(zidovudine) > CBV(abacavir). The human pol gamma utilized dideoxynucleotides and D4T-TP in vitro as efficiently as the natural deoxynucleoside triphosphates, whereas AZT-TP, 3TC-TP and CBV-TP were moderate inhibitors of chain elongation. We have also identified genetic variants of the mitochondrial DNA polymerase that increases the susceptibility of these NRTI to cause mitochondrial toxicity and identified critical amino acids in the mitochondrial DNA polymerase that allow for insertion of these NRTIs into mitochondrial DNA. In collaboration with Miriam Poirier at the NCI, will are evaluating mitochondrial DNA for mutations and deletions from patas monkeys that have been exposed in utero to NRTIs. Pregnant patas monkeys were exposed with human equivalent doses of AZT, 3TC, abacavir and nevirapine. Tissues were collected at birth, 1 and 3 years of age and will be analyzed by next generation sequencing for point mutations and deletions in mitochondrial DNA. This analysis will help us to understand the long term consequences of NRTI treatment on children exposed in utero to antiretroviral therapy. To aid in the identification of mitochondrial DNA alterations, we have developed a highly sensitive method, called LostArc, to decipher and interrogate mitochondrial deletions from patient and animal samples. This method can detect 1 deletion event per million mtDNA genomes and is being used on the patas monkey samples to identify and quantitate the level of mtDNA deletions in patas monkeys exposed to anti-HIV analogs as compared to untreated patas monkey samples. The interplay between HIV and COVID-19 infection is noteworthy. Beyond the obvious role of mitochondria in the innate immune response to SARS-CoV-2 infection, the off-target effects of antiviral therapy on mitochondria must also be considered. Patients infected with HIV and SARS-CoV-2 while on antiviral therapy show a prolonged duration of COVID-19 infection that can exceed 6 months. This extended period of infection facilitates viral evolution and permits development of more complex (and dangerous) variants, which is believed to be one basis for the origin of the Omicron variant. Understanding the interplay between chronic HIV infection, antiviral drugs, SARS-CoV-2 infection and viral evolution is critical to controlling the spread of new variants. In 2001 we showed for the first time that monophosphate metabolites of NRTIs, namely AZT-monophosphate (AZT-MP), could inhibit the exonuclease proofreading function of Pol gamma, raising the possibility of altering the fidelity of mtDNA replication. In vivo, the metabolic pathway for phosphorylation of AZT encounters a bottleneck at production of AZT-DP, resulting in accumulation of a high level (200 uM) of AZT-MP in mitochondria. Such concentrations of AZT-MP can inhibit both Pol exonuclease, as well as thymidylate kinase 2 (TK2). So far, the most effective antiviral for COVID-19 is remdesivir. Like AZT, remdesivir is administered as a pro-drug that gets metabolized to remdesivirTP. To address the off target affect of remisdivir, we investigated the core replication complex of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Among the proteins required for faithful replication of the SARS-CoV-2 genome are nonstructural protein 14 (NSP14), a bifunctional enzyme with an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase, and its accessory protein, NSP10. The difficulty in producing pure and high quantities of the NSP10/14 complex has hampered the biochemical and structural study of these important proteins. We developed a straightforward protocol for the expression and purification of both NSP10 and NSP14 from Escherichia coli and for the in vitro assembly and purification of a stoichiometric NSP10/14 complex with high yields. Using these methods, we observe that NSP10 provides a 260-fold increase in kcat/Km in the exoribonucleolytic activity of NSP14 and enhances protein stability. We also probed the effect of two small molecules on NSP10/14 activity, remdesivir monophosphate and the methyltransferase inhibitor S-adenosylhomocysteine. Our analysis highlights two important factors for drug development: first, unlike other exonucleases, the monophosphate nucleoside analog intermediate of remdesivir does not inhibit NSP14 activity; and second, S-adenosylhomocysteine modestly activates NSP14 exonuclease activity. In total, our analysis provides insights for future structure-function studies of SARS-CoV-2 replication fidelity for the treatment of coronavirus disease 2019.