Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation

Abstract

Structures of type-1 human immunodeficiency virus (HIV-1) reverse transcriptase (RT) have been determined in several forms, but only one contains an RNA/DNA hybrid. Here we report three structures of HIV-1 RT complexed with a non-nucleotide RT inhibitor (NNRTI) and an RNA/DNA hybrid. In the presence of an NNRTI, the RNA/DNA structure differs from all prior nucleic acid bound to RT including the RNA/DNA hybrid. The enzyme structure also differs from all previous RT–DNA complexes. As a result, the hybrid has ready access to the RNase H active site. These observations indicate that an RT–nucleic acid complex may adopt two structural states, one competent for DNA polymerization and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites often map to the unique RT–hybrid interface that undergoes conformational changes between two catalytic states.

Examining the Role of the HIV-1 Reverse Transcriptase p51 Subunit in Positioning and Hydrolysis of RNA/DNA Hybrids

“…5, panel ii). With respect to Tyr-427, such observations indeed lend support to the notion that a bulky aromatic side chain may be required to preserve the architecture of the p51 C terminus to facilitate RNA/DNA hybrid binding and impose the correct orientation on the RNA strand for hydrolysis in the p66 RNase H active site (23). At the same time, the data of Figs.…”

“…Our studies were prompted by observations that the p51 and p66 are further separated ϳ2.0 Å and that p51 helix ␣-helix MЈ and surrounding residues are in the core of such movement (23). The RT-RNA/DNA cocrystal structure also demonstrated hydrogen bonding between p51 residues Asn-348 and Tyr-427, in contrast to previous structures where Asn-348 was surrounded by hydrophobic side chains (23).…”

“…In this study, we have taken advantage of our recently solved structure of HIV-1 RT containing an NNRTI and a non-polypurine tract RNA/DNA hybrid (PDB ID 4B3O) that differs structurally from all previously reported RT-nucleic acid complexes and is compatible with RNA degradation (23) to evaluate the effect of point mutations, deletions, and alanine scanning mutagenesis on positioning of ␣-helix MЈ (Pro-421-Tyr-427), which constitutes the last structural element of the p51 subunit visible from crystallography. Our studies were prompted by observations that the p51 and p66 are further separated ϳ2.0 Å and that p51 helix ␣-helix MЈ and surrounding residues are in the core of such movement (23).…”

“…Our studies were prompted by observations that the p51 and p66 are further separated ϳ2.0 Å and that p51 helix ␣-helix MЈ and surrounding residues are in the core of such movement (23). The RT-RNA/DNA cocrystal structure also demonstrated hydrogen bonding between p51 residues Asn-348 and Tyr-427, in contrast to previous structures where Asn-348 was surrounded by hydrophobic side chains (23). Lastly, the peptide comprising Phe-416 -Pro-421 preceding ␣-MЈ was shown to interact with the DNA strand and cross the shallow minor groove to form Van der Waals contacts with the RNA strand adjacent to the scissile phosphate ( Fig.…”

The Nature of the N-Terminal Amino Acid Residue of HIV-1 RNase H Is Critical for the Stability of Reverse Transcriptase in Viral Particles

“…The RNA-dependent DNA polymerase and RNase H activities of HIV-1 RT are mainly carried out by the p66 subunit, while p51 was thought to be enzymatically inactive and serve only a structural role (5,(7)(8)(9)(10). However, recent structural and biochemical evidence suggests that the C-terminal end of the p51 subunit is involved in hydrolysis and positioning of the RNA/DNA hybrid formed during the reverse transcription process (11)(12)(13).…”