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Papers In Press, published online ahead of print February 22, 2006
Microbiology and Immunology, University of Rochester, Rochester, NY 14642
Corresponding Author: baek_kim{at}urmc.rochester.edu
Retroviruses and DNA viruses utilize cellular dNTPs as substrates for their DNA polymerases during viral replication in infected cells. However, due to S phase-dependent dNTP biosynthesis, the availability of cellular dNTPs significantly varies among cell types (e.g. dividing vs. nondividing cells and normal vs. 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 due to 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). While the wildtype 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 due to inefficient reverse transcription in low cellular dNTP environments. We also observed that the Q151N vector expressing HSV-TK 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.
J. Biol. Chem, 10.1074/jbc.M600291200
Submitted on January 11, 2006
Revised on February 8, 2006
Accepted on February 22, 2006
Modification of human immunodeficiency virus type 1 reverse transcriptase to target cells with elevated cellular dntp concentrations
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