Nonetheless, coupling generated mutations for study in the full Gag model, it is possible to investigate the effects of the mutations before they are clinically observed. mutations have been reported on Protease [11,12,13] and Gag [14,15,16,17,18] alone, or concurrently on both Protease and Gag [17,19,20,21,22], revealing an enzyme-substrate synergy to overcome PIs [23] (Physique 1B). Expectedly, Gag cleavage site mutations contribute directly to PI resistance [24], while non-cleavage site mutations contribute to drug resistance by compensating for the loss of viral fitness [22,25,26] that resulted when protease accumulates drug resistant mutations reducing its proteolytic functions. As Gag is usually a larger protein than protease, and mutations (both cleavage and non-cleavage) can contribute to PI resistance, there is thus a need to study the mechanisms to how these mutations work in synergy with protease. Such studies will unravel potential weak points to which Gag can be targeted against, opening more opportunities in drug design. 2. Possible Targets in Gag The Gag polyprotein consists of components matrix (MA), capsid (CA), nucleocapsid (NC), p6, and two spacer peptides p1 and p2. The MA subunit, located at the N-terminus, is essential for targeting Gag to the cell membrane, while the CA forms a shell to protect the viral RNA genome and other core proteins during maturation. The NC is responsible for RNA packing and encapsidation [27] while the two spacer peptides p1 and p2 regulate the rate and the sequential cleavage process of Gag by protease [28]. This process of viral assembly is usually complemented by viral budding moderated by the small Proline-rich p6. Mutations at Etidronate (Didronel) either the N-terminal or C-terminal Rabbit Polyclonal to BAZ2A of these core proteins were reported to block viral assembly and impair Gag binding to plasma membrane, thereby inhibiting viral budding [27]. Since the Gag cleavage sites do not share a consensus sequence (Physique 2), the recognition of the cleavage sites by protease is likely to be based on their asymmetric three-dimensional structures [29] that would fit into the substrate-binding pocket of protease [30]. The cleavage of these scissile bonds (seven-residue peptide sequences unique for each cleavage site) are highly regulated and occur at differing rates [24,28,31]. The first cleavage occurs at the site between the p2 peptide and NC domain name (Physique 2), followed by the MA from CACp2 at a rate that is ~14-fold slower than that of the first cleavage, before proceeding to release p6 from the NC-p1 domain name (at a rate ~9-fold slower than the first cleavage). At the last step, the two spacer peptides p1 and p2 are cleaved from NC-p1 and CACp2 at rates ~350-fold and ~400-fold, respectively, slower than the initial cleavage [24,28,30,31]. Open in a separate window Physique 2 The sequential Gag proteolysis by Protease. The cleavage sites are marked by the 7-residues, along with the estimated cleavage rates [28] marked by arrows. For easy comparison, the initial cleavage site rate is set to Etidronate (Didronel) the value of 1 1, while the other cleavage site values depict the reduced normalized rate. The cleavage site sequences are colored based on their physicochemical properties, e.g., hydrophobic (black), charged (positive: blue, unfavorable: red), polar (other colors), and varied in text sizes based on positional conservation, using WebLogo [32,33]. Structural surface presentations of the cleavage sites are also attached for visualization. To date, there are nine PIs, i.e., Etidronate (Didronel) Saquinavir (SQV), Ritonavir (RTV), Indinavir (IDV), Nelfinavir (NFV), Fos/Amprenavir (FPV/APV), Lopinavir (LPV), Atazanavir (ATV), Tipranavir (TPV), and Darunavir (DRV) in clinical treatment regimes [30]. With increasing PI resistance [34,35,36,37] and cross-resistance [21,24,35,38] conferred by protease mutations that compromise viral fitness, there is a compromise between enzymatic activity and drug inhibition by protease within its 99-residue homodimer subunits. Mapped to the resistance to several current PIs [39,40,41,42], many mutations were found to spontaneously arise as part of the natural variance [43] selected for during the treatment regimes. These mutations directly intervene with PI binding via steric perturbation at the active site, and those distant from the active site allosterically modulated protease activity Etidronate (Didronel) [12,13,44,45,46,47,48,49,50,51,52]. However, such mutations often reduce viral fitness, resulting in future repertoires Etidronate (Didronel) of viruses with compromised fitness [53]. This fitness trade-off is then compensated by additional mutations that restore enzymatic activity to an extent [44,48,49,54]. Reports of Gag PI-resistant mutations [17,19,20,21,22,24], whether impartial or linked to protease mutations, include those that restore the reduced binding affinity to mutated proteases [17,19,20,21,22,23,24,55]. Such mutations were reported throughout the whole Gag structure with the majority on MA and p6 domains, playing a major role towards therapy failure [15,23]. In fact, multiple Gag inhibitors were rendered ineffective due to natural Gag polymorphisms [56]. New clinical protease resistant mutations.