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EFFECT OF INCORPORATION OF DEOXYNUCLEOTIDE BORANOTRIPHOSPHATE ON RNASE H CLEAVAGE BY VIRAL REVERSE TRANSCRIPTASES
- Abstract:
Human Immunodeficiency Virus Type 1 (HIV-1) is the major cause for acquired immunodeficiency syndrome (AIDS), a disease that has caused great impact on human health and society. Since the HIV-1 genome is subject to frequent mutations, current therapeutic agents meet great challenges. In this research, we targeted one of the most highly conserved sequences in the HIV-1 genome, 3’ polypurine tract (3’ PPT), which is responsible for the plus-strand pro-viral DNA synthesis and the definition of the 5’ end of the pro-viral double-strand DNAs. In nature, this 3’ PPT is resistant to internal cleavage by HIV-1 RT and must be precisely cleaved from the viral RNA. Otherwise, truncated or elongated 3’ PPT would result in lower viability of the virus.
The objectives of this research were to investigate: (1) if deoxynucleoside boranotriphosphates (B-dNTPs) can be incorporated into the complementary strand of the 3’ PPT-containing RNA region; (2) whether the incorporation of B-dNTPs can alter the cleavage specificity of the 3’ PPT-containing region; and (3) which part of the PPT is most sensitive to B-dNTP substitution and the possible explanation.
We conducted in vitro studies using two model systems with Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (RT) and HIV-1 RT respectively. Our results showed that: (1) Both MMLV RT and HIV-1 RT were able to recognize the HIV-1 PPT-containing region and cleaved it with high specificity. (2) MMLV RT cleaved HIV-1 PPT one nucleotide downstream from the 3’ end of PPT. (3) Incorporation of B-dATP was able to alter the cleavage specificity by one nucleotide (upstream from the 3’ end of PPT) and lead to an internal cleavage catalyzed by HIV-1 RT. (4) A novel cleavage, at the 7th nucleotide position downstream from the 3’ end of PPT, was found in the HIV-1 RT model system, with or without the B-dNTPs substitution.
These results suggested that the incorporation of B-dNTPs could have changed the special structure of the PPT-containing RNA/DNA hybrid, from which the high cleavage specificity results. The U-tract next to the 5’ end of the PPT is sensitive to the substitution by B-dATPs in the complementary strand, which is in support of a previous model by Rausch et al (Rausch et al., J. Biol. Chem., 2004, 279: 37095-37102). The novel cleavage position could facilitate the reorientation of the RT during reverse transcription, which was first proposed in a model by Dash et al (Dash et al., Nucleic Acids Res., 2004, 32: 1539-1547). Further investigation of other nucleotide modifications (e.g., base or ribose modifications) along with the boranophosphate modification could make B-dNTPs potential anti-viral agents.
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