Nucleotide Sugar Pucker Preference Mitigates Excision by HIV-1 RT

Abstract

A series of DNA primers containing nucleotides with various sugar pucker conformations at the 3'-terminus were chemically synthesized by solid-phase synthesis. The ability of wild-type (WT) HIV-1 reverse transcriptase (RT) and AZT-resistant (AZTr) RT to excise the 3'-terminal nucleotide was assessed. Nucleosides with a preference for the North conformation were more refractory to excision by both WT-RT and AZTr-RT. We found that DNA primers that contain North puckered-nucleotides at the 3'-terminus can also affect the translocation status of the RT/template/primer complex, which provides an underlying mechanism to avoid being excised. Together, these results point to a correlation between the sugar conformation of the 3'-terminal nucleotide, the precise position of HIV-1 RT on its nucleic acid substrate, and, in turn, its catalytic function. Nucleotide sugar conformation is therefore an important parameter in defining the susceptibility to RT-catalyzed phosphorolytic excision.

Figure 1.

Schematic illustration of (A) polymerization cycles of HIV-1 RT, (B) sugar conformation of nucleosides, and (C) the excision of a chain terminator (NRTI). Once the NRTI is incorporated into the N-site, further chain extension is stopped due to the lack of a 3’-hydroxyl group in NRTI. HIV-1 RT locates this 3’-terminal NRTI at the N-site. Cellular ATPs bind to the complex and nucleophilically attack the 5’-phosphodiester linkage of the NRTI, excising the NRTI by forming a 5’,5’-linked dinucleoside tetraphosphate (ATP-pNRTI).

Figure 2.

Library of DNA primers that contain unmodified or chemically modified nucleotides at the 3’-terminus.

Figure 3.

Nucleotide excision by WT-RT of primers containing 3′-end North nucleosides (A) and South nucleosides (B). Aliquots were taken at each time point (1, 5, 10, 20, 30, 45, 60, and 90 min, respectively). For the control experiment, aliquots were taken before mixing “ATP-mix” solutions, which were used as aliquots at 0 min.

Figure 4.

Rescue of DNA synthesis of primers G5, G6, G7, and G10 by WT or AZTr-RT. Reactions were monitored in a time course. For the control experiment, aliquots were taken before initiation of the reaction (indicated as 0 min).

Figure 5.

KOONO-mediated site-specific footprinting assays. Highlighted markers in green, red, and blue indicate 50:50 North/South conformation, North conformation preferred, and South conformation preferred respectively. The highlighted marker in black indicates nucleosides containing flexible or size-expanded sugars. (A) Translocation of the complex of DNA primers/template/RT (WT). (B) Translocation of the complex of DNA primers/template/RT (AZTr). For South conformers and AZT: % Pre = Pre/[Pre + Post] × 100 for North conformers, seco-Ur and oxepane-T: % Pre = Pre/[ex-Pre + Pre + Post] × 100.

Figure 6.

(A, left) Modeling of complexes of AZT-terminated DNA primer/DNA template/RT (WT) in the crystal structure of the pretranslocation complex (PDB: 1N6Q) indicating structural fitting to Tyr115, (A, right) Van-der-Waals radii of 2′-(α)-hydrogen in (A) was shown. (B, left) Modeling of complexes of 2′-(α)-F-3′-azido-Ur-terminated DNA primer/DNA template/RT (WT) in the structure of the pretranslocation complex indicates steric clashes to Tyr115, (B, right) Van-der-Waals radii of 2′-(α)-fluorine was shown. (C, left) Modeling of complexes of AZT-terminated DNA primer/DNA template/RT (WT) in the crystal structure of the post-translocation complex indicates structural fitting to Asp185. (C, right) Van-der-Waals radii of 2′-(α)-hydrogen was shown. (D, left) Modeling of complexes of 2′-(α)-F-3′-azido-Ur-terminated DNA primer/DNA template/RT (WT) in the structure of the post-translocation complex indicates steric interaction with Aps185, (D, right) Van-der-Waals radii of 2′-(α)-fluorine was shown. 2′ C–F bond length in C and D was adjusted to 1.399 Å.

Figure 7.

(A) Illustration of the sliding of the RT from ex-Pre to Pre, and to Post translocation. (B, C) Fe-mediated footprintings of the complex containing 2′-F-3′-azido-Ur performed in a different range of concentration of next-incoming dGTP.

Scheme 1.. Synthetic Strategy for the Synthesis of Chemically Modified DNA Primers

(A) Phosphoramidite approach for the chemical incorporation of nucleosides without azido-substituents. (B) H-phosphonate approach for the chemical incorporation of nucleosides containing azido-substituent.