Mitochondrial (mt) DNA replication in disease:
HIV/AIDS, congestive heart failure, hepatitis, renal failure

William Lewis, MD

Primary Laboratory Research:

- Mitochondrial DNA replication in disease: AIDS, congestive heart failure, hepatitis, renal failure
One classic biological teaching is that mitochondria are the "powerhouses of the cell", particularly in tissues that require significant energy, like heart, liver and muscle. The focus of the Lewis lab is the diverse effects of toxins on mitochondria structure and function. The primary focus is the untoward mitochondrial effects of a class of drugs called nucleoside reverse transcriptase inhibitors (NRTI) used to treat HIV infection, that are known to inhibit mitochondrial (mt-) DNA replication in vitro. These drugs are toxic to various tissues including heart, skeletal muscle, liver and kidney. Our overall goals are to (1) define the molecular mechanisms of how NRTIs change the abundance of precursors for mtDNA replication in vivo (2) define the effects of active NRTI triphosphates on the mitochondrial replicon in vivo (3) define the effects of specific polymerase mutants on the replication of mtDNA in vivo and (4) define the effects of the mtDNA template itself on its replication in vivo. The approach taken employs tissue specific targeting of genes involved in the processes and evaluation of structural and functional defects that result using state of the art biochemical, molecular, physiological, and pathological approaches. Conditional, tissue specific targeting also is addressed. The direct clinical benefits of these studies are twofold (1) the toxicity of important compounds is defined so that better therapeutics can be designed and administered and (2) mechanisms of organ specific changes in HIV disease and related illnesses are elucidated.

Proposed mechanism of nucleoside analog toxicity to mtDNA

Proposed mechanism of nucleoside analog toxicity to mtDNA (small double circle), with attached DNA pol (in block) in which analog triphosphates serve as inhibitors of mtDNA replication (panel A, B). Details of interaction of mtDNA with DNA pol : Fialuridine (FIAU) -TP serves as an alternate substrate for dTTP with DNA pol and is incorporated (inserted into mtDNA as F) into the nascent chain because it possesses a 3'-OH. Panel C, D: In contrast, analog triphosphates (like AZT-TP) that lack 3'-OH compete with dTTP as substrates of DNA pol . Additionally, the latter analog monophosphates terminate mtDNA synthesis (inserted into DNA as Z) because nascent chain cannot be extended unless AZT is recognized as erroneous. Oxidative stress and mtDNA mutation are included in the "mitochondrial dysfunction hypothesis" 8,9,56

Active Grants:

·	2R01HL 059798-06 (Lewis) 07/01/1997-06/30/2010 NHLBI/NIH Cardiomyopathy in AIDS. At present, there is an amended application under review. This application's initial goal was to define TG transgenic effects of selected HIV gene products and mammalian DNA-pol-? in AIDS CM.
·	1R01 HL079867-01 (Lewis) 09/01/06-08/31/11 Acquired mtDNA Depletion and Nucleoside Reverse Transcriptase Inhibitors Goals: This project dissects the roles of enzymes involved in the second phosphorylation of NRTIs to diphosphates. Using transgenic mice with conditional, targeted expression, overexpression of native and mutant TMPK is evaluated in NRTI treatment protocols

Recent Funding:

·	1R01 HL 072707 (Lewis) 9/01/02-08/31/07 NHLBI/NIH Nucleoside Analogs, Mitochondria, and AIDS Cardiomyopathy Goals: To define mechanisms of mitochondrial defects of NRTIs in cardiac mitochondria. This model examines effects of NRTIs on HIV Tat, Vpr, Vpu, Nef and cardiac myocyte apoptosis in HIV. Competitive continuation submitted.
·	CFAR03 Small Grant Award (Kohler) 09/01/06-08/31/07 AIDS Nephropathy and Antiretroviral (Tenofovir) Renal Toxicity Goals: This project evaluates mechanisms of renal tubular epithelial mitochondrial damage associated with tenofovir disoproxil fumare (TDF, Viread) treatment.

Laboratory Members:

James J. Kohler, PhD
Instructor, Pathology & Laboratory Medicine
Rm 7126 WMRB
404-712-9013

Seyed H. Hosseini, PhD
Research Specialist Supervisor
Rm 7120 WMRB
404-727-1258

Ioan Cucoranu, MD
Post-Doctoral Fellow
Rm 7231 WMRB

Earl Fields, MS
Research Specialist
Lab 7205 WMRB

Elgin Green, MS
Research Specialist Lead
Lab 7207 WMRB

Chad Haase, BS
Research Specialist Lead
Lab 7127 WMRB

Amy Hoying Brandt, MS
Research Specialist Lead
Lab 7207 WMRB

David Johnson, BS
Research Specialist
Lab 7207 WMRB

Rodney Russ
Animal Technician III
Lab 7127 WMRB

Bree Wittich, MS
Research Specialist Lead
Laba 7205 WMRB

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