Detecting drug resistance mutations in Gag and protease
2.4 Discussion
Using a large HIV-1 subtype C dataset comprised of publicly available sequences and those of patients recruited in KZN, this study identified various PI-resistance mutations previously linked to both PR and Gag. Additionally, several presumptive Gag mutations associated with PI- resistance/exposure were also identified.
These data revealed that out of the 1,972 PR sequences, only 964 (49%) harboured major PRMs.
As such, more than half (51%) of the PR sequence dataset did not present with drug resistance mutations, suggesting that non-adherence is a prominent factor in the development of resistance mutations, as reported by other studies (Levison et al., 2011; Wallis et al., 2011; Court et al., 2016; Kyaw et al., 2017).
In this study positions 82, 54 and 46, having the highest percentage of variation, are in the active site (82) and flap regions (46, 54) of the viral PR and are therefore integral for substrate cleavage (Saleh et al., 2017). As such, mutations at these positions can aid the virus in outcompeting the inhibitor for more efficient cleavage and subsequently escape drug selection pressure (Liu et al.,
44
2008; Weber and Agniswamy, 2009; Yu et al., 2015). Additionally, the selection of specific mutational variants (M46I, I47A, G50V, I54V, V82A) over others (M46L, I47V, G50L, I54L, V82CMST) at these positions suggests that AA selection also has an important role in maintaining protein functionality when resistance is induced. Dwyer (2001) showed that the electronic characteristics13 of an AA could be comparable to the effect of hydrophobicity on the conformational preferences during protein folding. In this study, the frequently occurring triplet of M46I+I54V+V82A are described as common PI-resistance mutations by the Stanford Drug Resistance Database in ATV/r14, FPV/r, IDV/r, LPV/r2 and NFV/r (Appendix D; Figures 1–2).
Moreover, the selection of L76V in conjunction with the above mutations, as observed in this study, has been shown to not only enhance LPV/r resistance (Young et al., 2010) but also allows for low-level DRV/r resistance to occur (HIVdb algorithm; Appendix D; Figure 3).
Whilst several accessory PI mutations were observed in this study, the most prevalent included L10F/I, A71V and T74S. The L10F/I mutations have been linked to reduced susceptibility and/or increased replication capacity to numerous PIs when found associated with other resistance mutations (Prado et al., 2002; Rhee et al., 2010; De Luca et al., 2016; Aoki et al., 2018). The polymorphic A71V mutation has been found in association with other PI-resistance mutations in numerous studies (Shafer et al., 2007; Lopes et al., 2015; Huang et al., 2017; Pessôa and Sanabani, 2017). Although the role of A71V is largely unknown (Gonzalez et al., 2008), a study conducted by Chang and Torbett (2011) revealed that in combination with I84V, A71V restored the structural stability of the PR to within 1°C of the WT15. The high prevalence of T74S observed in this study was consistent with other studies investigating the selection of drug resistance mutations in patients receiving a PI-inclusive treatment regimen (Taylor et al., 2011; Etta et al., 2017; Lu et al., 2017). Although T74S is polymorphic in most non-B subtypes (Deforche et al., 2006), its presence in treatment-associated outcomes suggests that it provides a selective advantage and can either improve or maintain viral replication capacity in the presence or absence of PR resistance mutations, respectively.
13 Electronic characteristics refers to the physico-chemical nature of the amino acid side chains such as the hydrophobicity, pKa, steric effects (bulk of the side chain), etc. Thus, these characteristics were used to provide a scale: O (strong electron donor), U (weak donor), Z (ambivalent), B (weak electron acceptor) and X (strong acceptor) (Dwyer, 2001).
14 High-level drug resistance.
15 In other words, there was only a 1°C difference in the melting temperature between the WT and mutant when measured using Differential Scanning Calorimetry. Therefore, the mutant is comparatively stable.
45
Of the 11 resistance-associated Gag mutations, only the A431V (NC|p1), L449F (p1|p6) and R452K (p1|p6) mutations were found significantly higher in the treatment exposed versus naïve sequence dataset (Figure 2.4). The A431V mutation has been linked to several PIs, including LPV/r (Mammano et al., 1998; Cote et al., 2001; Mo et al., 2007; Nijhuis et al., 2009). In this study, the A431V mutation was found in 26% (n=107) of the viral Gag sequences. It has been shown to confer resistance in the absence of major PRMs (Dam et al., 2009; Nijhuis et al., 2009) and act in a compensatory manner when PRMs are present by improving viral fitness (Mammano et al., 2000; Cote et al., 2001). This mutation often occurs with major PRMs, V82A (Bally et al., 2000; Malet et al., 2007) and M46I/L (Myint et al., 2004). A study conducted by Nijhuis et al.
(2009) on subtype B infected patients receiving a LPV/r-inclusive treatment regimen revealed that A431V was also selected in viruses comprising an M46I+L76V PR double mutant, increasing LPV/r resistance by 10.6-fold and improving viral replication capacity by 10%. Contrastingly, Myint et al. (2004) revealed that A431V had deleterious effects on the replication capacity of D30N, N88D and L90M PR mutants suggesting that A431V is synergistically co-selected with specific PRMs.
Unlike A431V, the L449F mutation increased viral replication capacity in the PR mutants observed in the Myint et al. (2004) study. This mutation has been shown to reduce the susceptibility of various PIs when combined with major PRMs D30N/N88D and with V82A or L90M in subtype B (Fun et al., 2012). However, unlike A431V, L449F cannot confer resistance alone (Fun et al., 2012), and instead acts in a solely compensatory role (Yates et al., 2006; Girnary et al., 2007; Kolli et al., 2009). Recently, in a structural study investigating the effects of p1|p6 CS mutations on an I50V+A71V subtype B PR mutant, Özen et al. (2014) demonstrated that L449F increased the van der Waals forces in the enzyme-substrate complex, thereby restoring PR’s active site dynamics. Similar to L449F, the R452K Gag mutation, frequently occurring with D30N/N88D, I84V and I50V, has a compensatory role during PI-selection pressure (Kolli et al., 2009).
Additionally, several Gag mutations associated with PI exposure were identified in this study, five (G62R, S373P, I401V, R409K and S451T) of which occurred at significantly higher frequencies in the treated dataset (Figure 2.5). Although the G62R mutation occurred in 22% of the Gag sequence dataset in this study, its role in resistance is not yet understood (Koh et al., 2009). In a study investigating a novel PI (GRL-02031) against a multi-drug resistant PR, Koh et al. (2009) observed that G62R along with three other Gag mutations developed upon a 37th passage after exposure to GRL-02031 on MT-4 cell lines. Interestingly, one of the other mutations
46
found in the Koh study included NC mutation, R409K. Despite evidence linking R409K to APV/r (Gatanaga et al., 2002; Aoki et al., 2009), these studies did not report changes to APV susceptibility in vitro. As such, its role in PI-resistance is unclear. However, Stray et al. (2013) reported that R409K together with five other Gag mutations was able to reduce the efficacy of a novel PI (GS-8374) by 14-fold after >300 days of passage on MT-2 cell lines. This study suggests that since only one polymorphism was selected in PR, the Gag mutations were selected to confer resistance, playing a role as primary resistance mutations. In a similar manner to R409K, NC mutation I401V has been linked to treatment failure (Myint et al., 2004), however the mechanism by which it acts is not understood.
This study identified two Gag CS PI-exposure associated mutations (S373P and S451T). Codon 373 of the p2|NC CS is highly variable and comprises three mutational variants (S373P/Q/T) that are associated with PI-exposure (Fun et al., 2012). The S373P mutation has been linked to LPV studies in subtype B (Mckinnon et al., 2011; Sutherland et al., 2014) and in CRF02_AG (Teto et al., 2017). The S451T mutation in the p1|p6 region has been associated with LPV in subtype B (Masse et al., 2007) and DRV in the POWER16 trials (Dierynck et al., 2007).
Finally, 12 presumptive resistance associated mutations spanning the MA, CA and p6 regions of Gag were also observed (Figure 2.6). Previous data from our lab identified only two of the 12 presumptive Gag mutations, namely, Q69K and I256V (Singh, 2015). However, the dataset was considerably smaller (954 subtype C naïve and 54 acutely infected individuals vs. 80 PI-treated individuals) than what was used in the current study. While mutations outside the Gag CSs have been linked to drug resistance (Gatanaga et al., 2002; Callebaut et al., 2011; Parry et al., 2011), the numerous functional roles of MA, CA and p6 as discussed in section 1.10 of the literature review, suggests that mutations in these domains arise to maintain proper function at various stages in the virus’s lifecycle rather than affecting resistance in a more direct manner, like the CS mutations (van Maarseveeen et al., 2012).