Retroviral RNase H: Structure, mechanism, and inhibition

Abstract

All retroviruses encode the enzyme, reverse transcriptase (RT), which is involved in the conversion of the single-stranded viral RNA genome into double-stranded DNA. RT is a multifunctional enzyme and exhibits DNA polymerase and ribonuclease H (RNH) activities, both of which are essential to the reverse-transcription process. Despite the successful development of polymerase-targeting antiviral drugs over the last three decades, no bona fide inhibitor against the RNH activity of HIV-1 RT has progressed to clinical evaluation. In this review article, we describe the retroviral RNH function and inhibition, with primary consideration of the structural aspects of inhibition.

Keywords: Dynamics; HIV-1; Retrovirus; Reverse transcriptase; Ribonuclease H; Structure.

Figure 1.

RNH domains in retroviruses and retrotransposon: (a) HIV-1 RT in the Pol region of GagPol, (b) mature RT, comprising p66 and p51 subunits, (c) M-MLV RT that functions as a monomer, (d) Ty3/Gypsy RT in retrotransposon. Domains are indicated with the following colors: PR, pink; polymerase, blue; connection (Conn.), yellow; RNH, orange; IN, dark green; RNH-fold, cyan. (See text for the references)

Figure 2.

Comparison of the RNH folds in HIV-1 Pol: (a) Connection domain (residues 329 to 426 in PDB 3LP2 [78]), (b) RNH domain (residues 427 to 557 in PDB 3LP2 [78]), and (c) IN central domain (residues 56 to 190 in PDB 1EX4 [123]). In each ribbon presentation, α-helices are shown by yellow. The active site residues D443, E478, D498 and D549 in (b) and D64 D116, E152 in (c) are shown with sticks.

Figure 3.

Metal coordination in HIV-1 RNH domain: (a) the entire RNH domain with 2 Mg²⁺ bound (inhibitor bound form), (b-d) close-ups of the RNH domain active site in: (b) 2 Mg²⁺ bound form (inhibitor bound form), (c) one Mg²⁺ bound form (with dsDNA), and (d) one Mn²⁺ bound form, with gapped RNA/DNA and NNRTI. In (a) and (b), structural presentations were generated using PDB 5J1E [84]. In (c) and (d), structural presentations were generated using PDB 3KK2 [124] and 4Q0B [125], respectively. The active site residues D443, E478, D498 and D549, are shown with sticks. In (a), a short substrate binding region, compared to that of E. Coli is marked with a dashed red circle (see text).

Figure 4.

RNH titration with Mg²⁺ monitored by NMR. (a-c) ¹H-¹⁵N HSQC spectra, i.e. signals of backbone amides, showing an example of the chemical shift changes of a single residue, T477, at different Mg²⁺ concentrations and (d-f) cartoons indicating possible roles of cations in each experiment. The NMR experiments were performed in (a) 20 mM bisTris buffer without any additional salt, (b) 20 mM bisTris buffer containing 50 mM NaCl, and (c) 20 mM bisTris buffer at constant Cl⁻ concentration (160 mM) [73]. In (a)-(c), dash lines indicate 0, 10 mM and 80 mM Mg²⁺ concentrations. Cartoons envision the following: (d) at low MgCl₂ concentration (< 20 mM), titrated MgCl₂ binds charged protein surfaces, as a salt, as well as the active site, resulting in the relatively slow migration of NMR peaks that is observed in (a), compared with (b) or (c); (e) in the presence of NaCl, as in (b), Na⁺ interacts with charged protein surfaces, leaving more Mg²⁺ available to bind the active site; (f) in the constant Cl⁻ condition of (c), the number of cations is larger than in (a) or (b), and Na⁺ ions can temporarily occupy the active site, even in the presence of Mg²⁺, which weakly binds the site (K_D > mM). An abundance of cations, not limited to Mg²⁺, may enhance the on/off exchange rate at the active site, resulting in sharp NMR signals, compared to (b).

Figure 5.

Active-site inhibitor interaction with the RNH: (a) β-thujaplicinol and (b) naphthyridinone-based inhibitor. Active site residues D443, E478, D498 and D549 are shown by stick presentation. The active site Mn²⁺ ions are shown by pink spheres. Protein surfaces are shown for residues within 6 Å from the inhibitor. The structural presentation was generated with (a) PDB 3IG1 [77] and (b) PDB 3LP0 [78], using VMD software [126].