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[摘要]：Human immunodeficiency virus type 1 (HIV-1) requires reverse transcriptase (RT) and HIV-1 nucleocapsid protein (NCp7) for proper viral. replication. HIV-1 NCp7 has been shown to enhance various steps in reverse transcription including tRNA initiation and strand transfer, which may be mediated through interactions with RT as well as RNA and DNA oligonucleotides. With the use of DNA oligonucleotides, we have examined the interaction of NCp7 with RT and the kinetics of reverse transcription during (+)-strand synthesis with an NCp7-facilitated annealed primer template. Through the use of a pre-steady-state kinetics approach, the NCp7-annealed primer template has a substantial increase (3- to 7-fold) in the rate of incorporation (k(pol)) by RT as compared to heat-annealed primer template with single-nucleotide incorporation. There was also a 2-fold increase in the binding affinity constant (K-d) of the nucleotide. These differences in k(pol) and K-d were not through direct interactions between HIV-1 RT and NCp7. When extension by RT was examined, the data suggest that the NCp7-annealed primer template facilitates the formation of a longer product more quickly compared to the heat-annealed primer template. This enhancement in rate is mediated through interactions with NCp7's zinc fingers and N-terminal domain and nucleic acids. The NCp7-annealed primer template also enhances the fidelity of RT (3-fold) by slowing the rate of incorporation of an incorrect nucleotide. Taken together, this study elucidates a new role of NCp7 by facilitating DNA-directed DNA synthesis during reverse transcription by HIV-1 RT that may translate into enhanced viral fitness and offers an avenue to exploit for targeted therapeutic intervention against HIV. (C) 2012 Elsevier Ltd. All rights reserved. |
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