Restoration of the antiviral activity of 3'-azido-3'-deoxythymidine (AZT) against AZT-resistant human immunodeficiency virus by delivery of engineered thymidylate kinase to T cells.
Emergence of antiviral drug resistance is a major challenge to human immunodeficiency virus (HIV) therapy. The archetypal example of this problem is loss of antiviral activity of the nucleoside analogue 3'-azido-3'-deoxythymidine (AZT), caused by mutations in reverse transcriptase (RT), the viral polymerase. AZT resistance results from an imbalance between rates of AZT-induced proviral DNA chain termination and RT-induced excision of the chain-terminating nucleotide. Conversion of the AZT prodrug from its monophosphorylated to diphosphorylated form by human thymidylate kinase (TMPK) is inefficient, resulting in accumulation of the monophosphorylated AZT metabolite (AZT-MP) and a low concentration of the active triphosphorylated metabolite (AZT-TP). We reasoned that introduction of an engineered, highly active TMPK into T cells would overcome this functional bottleneck in AZT activation and thereby shift the balance of AZT activity sufficiently to block replication of formerly AZT-resistant HIV. Molecular engineering was used to link highly active, engineered TMPKs to the protein transduction domain of Tat for direct cell delivery. Combined treatment of HIV-infected T cells with AZT and these cell-permeable, engineered TMPKs restored AZT-induced repression of viral production. These results provide an experimental basis for the development of new strategies to therapeutically increase the intracellular concentrations of active nucleoside analogue metabolites as a means to overcome emerging drug resistance.
Lavie
Arnon
A
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60612, USA.
Su
Ying
Y
Section of Infectious Diseases, Immunology and International Medicine, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
Ghassemi
Mahmood
M
Section of Infectious Diseases, Immunology and International Medicine, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
Novak
Richard M
RM
Section of Infectious Diseases, Immunology and International Medicine, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
Caffrey
Michael
M
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60612, USA.
Sekulic
Nikolina
N
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60612, USA.
Monnerjahn
Christian
C
Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Konrad
Manfred
M
Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Cook
James L
JL
Section of Infectious Diseases, Immunology and International Medicine, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
D019380:Anti-HIV Agents / Q000494:pharmacology*; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D002460:Cell Line; D024882:Drug Resistance, Viral; D006678:HIV / Q000187:drug effects* / Q000235:genetics*; D016655:HIV Core Protein p24 / Q000096:biosynthesis; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D014779:Virus Replication / Q000187:drug effects; D015215:Zidovudine / Q000378:metabolism / Q000494:pharmacology*
D019380:Anti-HIV Agents; D016655:HIV Core Protein p24; D015215:Zidovudine; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase