Modification of human immunodeficiency virus type 1 reverse transcriptase to target cells with elevated cellular dNTP concentrations.
Retroviruses and DNA viruses utilize cellular dNTPs as substrates for their DNA polymerases during viral replication in infected cells. However, because of S phase-dependent dNTP biosynthesis, the availability of cellular dNTPs significantly varies among cell types (e.g. dividing versus nondividing cells and normal versus tumor cells). Here we tested whether alterations in the dNTP utilization efficiency and dNTP binding affinity of viral DNA polymerases can switch viral infection specificity to cell types with different dNTP concentrations. We employed an HIV-1 reverse transcriptase (RT) mutant (Q151N), which is catalytically active only at high dNTP concentrations because of its reduced dNTP binding affinity. Indeed, the modified HIV-1 vector harboring the Q151N mutant RT preferentially transduced tumor cells containing higher cellular dNTP concentrations than primary cells (e.g. human lung fibroblasts (HLFs) and human keratinocytes). Although the wild type HIV-1 vector transduced both HLFs and tumor cells, the Q151N vector failed to transduce HLFs and keratinocytes but efficiently transduced tumor cells. Pretreatment of HLFs with deoxynucleosides, which increase cellular dNTP pools, enabled the mutant vector to transduce HLFs, suggesting that the transduction failure of the RT mutant vector to primary cells is because of inefficient reverse transcription in low cellular dNTP environments. We also observed that the Q151N vector expressing herpes simplex virus-thymidine kinase renders tumor cells sensitive to gancyclovir. This study validates a novel strategy in which modifications of viral DNA polymerases in various vector systems allow the delivery of target genes exclusively to tumor cells exploiting elevated cellular dNTP concentration as a tumor cell-specific host factor.
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642.
Varuni K
VK
Jamburuthugoda
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642.
Pauline
P
Chugh
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642. Electronic address: baek_kim@urmc.rochester.edu.
Baek
B
Kim
D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000821:virology; D015967:Gene Expression Regulation, Viral; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism / Q000821:virology; D008264:Macrophages / Q000378:metabolism / Q000821:virology; D009154:Mutation; D009705:Nucleosides / Q000378:metabolism*; D009928:Organ Specificity
D009705:Nucleosides; D054303:HIV Reverse Transcriptase