HIV-1 Ribonuclease H: Structure, Catalytic Mechanism and Inhibitors

Abstract

Since the human immunodeficiency virus (HIV) was discovered as the etiological agent of acquired immunodeficiency syndrome (AIDS), it has encouraged much research into antiviral compounds. The reverse transcriptase (RT) of HIV has been a main target for antiviral drugs. However, all drugs developed so far inhibit the polymerase function of the enzyme, while none of the approved antiviral agents inhibit specifically the necessary ribonuclease H (RNase H) function of RT. This review provides a background on structure-function relationships of HIV-1 RNase H, as well as an outline of current attempts to develop novel, potent chemotherapeutics against a difficult drug target.

Keywords: HIV; RNase H; drug resistance; inhibitors; reverse transcriptase.

Figure 1.

Schematic of the process of reverse transcription. A The viral RNA genome is shown as a thick red line. Reverse transcription is initiated by the binding of an endogenous tRNA^lys3 molecule to the primer binding site (PBS) on the genome. B RT elongates the tRNA primer to the 5′ end of the genome, creating a fragment called (−)-strand strong stop DNA ((−)ssDNA). The RNase H activity of RT concomitantly degrades the RNA genome during DNA synthesis. The degradation of the 5′ end of the genome is necessary for (−)-strand transfer, and failure to degrade the RNA at this point results in the arrest of reverse transcription. C First or (−)-strand transfer. The (−)ssDNA fragment dissociates from the PBS sequence and re-associates with the repeat (R) sequence at the 3′ end of the genome. This step is capable of both intrastrand and interstrand transfer. D Continuation of (−)-strand DNA synthesis. RT extends the 3′ end of the (−)ssDNA fragment toward the PBS sequence, while the RNase H activity concomitantly degrades the RNA genome, which the exception of the polypurine tract (PPT). E The PPT is used as the primer for the initiation of (+)-strand DNA synthesis. The PPT primer is extended by the RT polymerase activity. F After approximately 12 nucleotides have been added, the PPT primer is removed by RNase H activity. The nascent (+)-strand DNA is extended to the 5′ end of the (−)-strand DNA, copying the PBS sequence from the tRNA that is still associated with the (−)-strand DNA. Here, the tRNA is removed by the RNase H activity, leaving a single ribonucleotide (rA) at the 3′ end of the U5 sequence (shown in red). G In the second, or (+)-strand transfer, the PBS sequences on both strands associate. This step occurs predominantly in an intrastrand fashion. H Both DNA strands are extended to the ends of their templates, forming the provirus that is ready to be integrated into the host genome by integrase. The long terminal repeats (LTRs) that are formed as a result of reverse transcription are shown.

Figure 2.

A Crystal structure of HIV-1 RT (PDB code: 1RTD. The p51 subunit is shown in orange, while the p66 subunit is divided into the fingers (cyan), connection (blue) and RNase H (grey) subdomains. The residues of the conserved DEDD motif are shown and red and marked with arrows. B Crystal structures of the RNase H domain of HIV-1 RT (PDB code:1RTD) [5], human RNase H1 (PDB code: 2QKB) [12] and E. coli RNase H1 (PBD code: 1WSJ). All three show the same mixed beta-sheet with asymmetric alpha helices, while the human and E. coli RNases H contain the C-helix, or basic loop.

Figure 3.

The chemistry of RNase H cleavage is believed to be a two-metal ion mechanism. A Two divalent metal ions (red spheres, marked A and B) are coordinated by the active site residues D549, D443, D498 and E478 approximately 4Å apart. Metal ion A activates a water molecule. B The activated water molecule carries out a nucleophilic attack (blue arrow) driving the phosphoryl transfer reaction. C In the putative transition state, the metal ions move toward each other to bring the nucleophile within range of the scissile phosphate. D The reaction products consist of a 3′ OH group and a 5′ phosphate group, and the metal ions are again likely to be re-positioned.

Figure 4.

HIV RT can exist in two distinct binding modes when bound to a nucleic acid substrate. The polymerase-dependent mode is characterized by the polymerase active site being in contact with the 3′ primer terminus. All other possible conformations are considered polymerase-independent.

Figure 5.

Polymerase-dependent binding can occur in two distinct positions. Post-translocation, where the 3′ primer terminus occupies the P site, leaving the N site available for nucleotide binding, or pre-translocation, where the N-site is occupied by the 3′ primer terminus and the incoming nucleotide is blocked by the primer terminus. An RT enzyme bound in a polymerase-dependent mode is in thermodynamic equilibrium between both pre- and post-translocational positions. The equilibrium is sequence-dependent.

Figure 6.

A During (−)-strand DNA synthesis, the PPT region of the RNA genome is resistant to RNase H cleavage, while a portion of the RNA genome is degraded concomitantly with (−)-strand DNA synthesis. Here, a specific cleavage is made to create the PPT primer. B The RNase H-resistant PPT sequence forms the primer for (+)-strand DNA synthesis when the rest of the genome is completely degraded by RNase H. C The RNA primer is extended 12 nucleotides, D then RT pauses and changes orientations to a polymerase-independent binding mode in order to cleave at the DNA:RNA junction, and remove the PPT primer. E The 12-mer fragment is extended toward the 5′ end of the (−)-strand DNA (see Figure 1F). After the second strand transfer event, the (+)-strand DNA is fully synthesized resulting in a fully double-stranded provirus. Adapted from [49].

Figure 7.

Examples of the main classes of small molecule inhibitors against HIV-1 RT-associated RNase H activity. A N-hydroxyimides. Developed from influenza inhibitors and among the first to use the 3-oxygen pharmacophore [84]. B Pyrimidinol carboxylic acid derivatives [85]. Potent inhibitors based on the successful scaffold of the metal-chelating active site inhibitors. C Diketo acid derivatives are also active in some cases against the viral integrase. RDS1643 shown here is the only diketo acid to have antiviral activity in vivo [86]. D The vinylogous ureas (NSC727447 pictured here) represent a different kind of inhibitor that binds allosterically near the p51 thumb [87]. E N-acyl hydrazones appear to bind to multiple binding sites depending on the specific inhibitor, including the RNase H domain and a site that overlaps the NNRTI binding pocket (DHBNH picture above) [88]. F Hydroxylated tropolones (β-thujaplicinol pictured here) are the subject of several studies that have provided the basis for a biochemical mechanism of inhibition by active site RNase H inhibitors [39,89,90].

Figure 8.

A schematic of possible models of active site inhibitor binding, based on studies with the tropolone derivative β-thujaplicinol (blue sphere) [39,90]. Evidence suggests that the inhibitor is unable to bind to an enzyme-substrate (E-S) complex (top left), only to free enzyme forming (top right) and enzyme-inhibitor (E-I) complex (bottom right). However, the substrate might be able to bind to this E-I complex, forming an E-S-I complex that is not productive with respect to RNase H cleavage (bottom left). As suggested by Himmel et al., the inhibitor occupies the position normally claimed by the scissile phosphate [90]. As such, it is possible that the substrate undergoes a change in trajectory in relation to the scissile phosphate and the RNase H active site [39]. Then eventually, the inhibitor dissociates and RT is allowed to cleave the uninhibited substrate (E-S complex, top left).