HIV-1 reverse transcriptase
Created by George Waldenmaier
HIV-1 is the major causative agent behind the AIDS pandemic which has been ongoing over the past three decades; and within HIV-1, the reverse transcriptase enzyme plays one of the most integral roles in its reproduction. HIV-1 reverse transcriptase (3NBP) catalyzes the creation of double-stranded DNA from the single-stranded viral RNA genome, which can then be incorporated into the host’s genome to code for the production new viral particles1. As it is such a key enzyme in the reproductive cycle of HIV, reverse transcriptase has become the focus of significant research, and most current treatment strategies for HIV infection rely on the inhibition of reverse transcriptase2. Aminopyrimidine inhibitor 2 is one such example; functioning as a non-competitive, allosteric inhibitor which has shown to be highly effective at blocking reproduction of HIV-1 and various mutational strains2.
HIV-1 reverse transcriptase is comprised of two separate subunits, referred to as the p66 and p51 subunits according to their molecular weights (66 and 51 kDa respectively)3. The isoelectric point was determined to be at a pH of 8.53 and 8.60 for the p66 subunit and p51 subunit respectively4. HIV-1 reverse transcriptase is originally translated as a part of single, 160 kD polyprotien and is subjected to post-translational modification to form the two required subunits1. Neither of the monomeric subunits shows any form of significant catalytic activity until binding with its partner to form reverse transcriptase1. The p66 subunit is further divided into two domains: the N-terminal 440 amino acid residues comprise the polymerase domain of the subunit, where the viral RNA binds and complementary double-stranded DNA is produced3. The 120 C-terminal amino acid residues comprise the RNase H domain, which plays a significant role in enzymatic digestion of the viral RNA, along with removal of RNA primers during DNA synthesis5. The p51 subunit is identical in primary structure to the 440 residue polymerase domain of the p66 subunit; however it lacks the C-terminal 120 residue RNase H domain6. The p51 subunit further differs in its tertiary structure, in which several of the key catalytic amino acids which are exposed in the p66 subunit, are buried in the interior of the p51 subunit6.
All catalytic behavior of reverse transcriptase is localized to the active sites on the p66 subunit; the p51 subunit serves primarily only for structural and conformational support4. The polymerase domain of the p66 subunit is divided into 4 subdomains; referred to as the fingers, palm, thumb, and connection3. The fingers, palm, and thumb subdomains are arranged so as to form a cleft to which the primer template can bind, with the actual active site located within the palm subdomain3. The “floor” of the cleft is comprised of the connection subdomains from both the p66 and p51 subunits6. As the p51 subunit contains the same primary structure as the p66 polymerase domain, it too contains each of the four subdomains; however they are arranged in manner which is very distinct from the p66 polymerase domain. The palm region of the p51 subunit lacks a discernable cleft, and the active site located on the palm subdomain is buried within its interior7. Additionally, the connection subdomain is located centrally in the p51 subunit and contacts each of the 4 subdomains in p66 subunit. The p66 connection subdomain contacts only the thumb, and links the polymerase domain to the RNase H domain7.
In terms of secondary structure, both the palm and connection subdomains are very similar7. Both regions are comprised of a three stranded antiparallel β-sheet with an internal haipin, flanked on one side up several successive α-helices6. The thumb and fingers subdomains function as “clamps” in the polymerase domain, and serve hold the substrate in place during catalysis. The thumb region consists of three α-helices, which work in tandem with the fingers region, which is composed of several individual β-strands and a single α-helix, to hold the substrate in place and position it over the active site7.
The active site itself is located in the palm subdomain, and is characterized by a few highly conserved residues. The three most important of the residues are Asp-110, Asp-185 and Asp-186, which are integral to the start of the catalytic process, as without them, enzyme function is greatly reduced3. Asp-185 is noted to form a hydrogen bond with the 3’-OH group at the primer terminus, and to act as a general base to undergo a nucleophilic attack on the α-phosphate group of a nucleoside triphosphate on an incoming primer6. Additionally, the side chain of Tyr-183 also functions to hydrogen bond with the nucleotide bases in the second base pair position6. Each of these residues is contained in a general motif which is highly conserved throughout all HIV reverse transcriptases. This motif is characterized by an YMDD or YXDD amino acid pattern, and has been shown to undergo significant conformational change upon binding of a substrate6. This conformation change is thought to have a significant effect on the mechanism of polymerization and on the mechanism of inhibition by non-nucleoside inhibitors, as the conformation change seems to position the substrate for optimal base pairing6.
The RNase domain structure is characterized by a cleft which contains its active site and is contiguous with the cleft in the polymerase domain7. Its secondary structure consists of a five –stranded β-sheet flanked by four α-helices in an asymmetric distribution8. The active site is characterized by a cluster of four highly conserved acidic amino acid residues, which function to catalyze degradation of the RNA template via hydrolysis8. The binding clefts within both domains of the p66 subunit interface with each other, however the subunit itself has no catalytic function in the absence of the p51 subunit. Thus, it has been suggested that the p51 subunit causes a key conformational change in the p66 subunit, allowing the two domains to properly interface and become functionally active8.
The highly conservative nature of several key amino acid sequences within the binding clefts and active sites in the p66 subunit make it vulnerable to both competitive and non-competitive inhibition. The inhibitors are divided into two categories: nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs)2. NRTIs function as direct competitors for the substrate binding site, whereas NNRTIs, such as aminopyrimidine 2, function as non-competitive allosteric inhibitors2. Aminopyrimidine 2 functions via attachment into the binding cleft of the polymerase domain by hydrogen bonding to some specific conserved residues. The major hydrogen bond complexes that typically form are with the carbonyl backbone of Lys-101 and Lys-103, as well as with Trp-2299. Through this binding, it elicits a different conformational change from the YXDD binding motif, which significantly degrades reverse transcriptase’s ability to bind to its substrate, and thus greatly reduces its enzymatic ability.
The high conservation of sequence and structure was shown through both a BLAST protein sequence analysis and a DALI server structural analysis. BLAST analysis showed extremely high primary structure conservation, with an E value of zero in all comparisons of both subunits to the HIV-2 strain (1MU2) and other retroviruses10. DALI server analysis confirmed the BLAST findings by yielding many high Z values and low root mean square deviations, which indicates high conformity in tertiary structure to HIV-2 and other retrovirus reverse transcriptases11. Further investigation revealed significant homology between the catalytic subunits, most specifically in area of the active sites.
HIV-1 is the most common cause of development and transmission of acquired immune deficiency syndrome (AIDS), and has reached a pandemic scale over the past two decades2. Consequently, a significant amount of research has been poured into discerning the structure and exact function of HIV-1 reverse transcriptase, as it is one of the most critical proteins in the virus’s life cycle and the primary cause behind the virus’s ability to replicate. Having the virus’s reproductive cycle reliant on a single protein, however, provides some advantage as the inhibition of that enzyme can bring the virus’s life cycle to a standstill. Even so, retroviruses like HIV are notoriously difficult to control due to their highly mutation-prone RNA genomes, which can eventually make most inhibitors ineffective. Therefore, it is crucial that continuing research be conducted in the areas of the conserved structures and sequences between the various reverse transcriptases of various strains. Aminopyrimidine inhibitor 2 is one such inhibitor that makes use of homologous structures across strains; however it is still likely that eventually HIV will mutate and decreased the efficacy of aminopyrimide as well. Thus continued research and understanding of HIV and its critical components are necessary to produces increasingly effective AIDS treatments.