Structural characterization of B and non-B subtypes of HIV-protease: insights into the natural susceptibility to drug resistance development

Although a majority of HIV-1 infections in Brazil are caused by the subtype B virus (also prevalent in the United States and Western Europe), viral subtypes F and C are also found very frequently. Genomic differences between the subtypes give rise to sequence variations in the encoded proteins, including the HIV-1 protease. The current anti-HIV drugs have been developed primarily against subtype B and the effects arising from the combination of drug-resistance mutations with the naturally existing polymorphisms in non-B HIV-1 subtypes are only beginning to be elucidated. To gain more insights into the structure and function of different variants of HIV proteases, we have determined a 2.1 A structure of the native subtype F HIV-1 protease (PR) in complex with the protease inhibitor TL-3. We have also solved crystal structures of two multi-drug resistant mutant HIV PRs in complex with TL-3, from subtype B (Bmut) carrying the primary mutations V82A and L90M, and from subtype F (Fmut) carrying the primary mutation V82A plus the secondary mutation M36I, at 1.75 A and 2.8 A resolution, respectively. The proteases Bmut, Fwt and Fmut exhibit sevenfold, threefold, and 54-fold resistance to TL-3, respectively. In addition, the structure of subtype B wild type HIV-PR in complex with TL-3 has been redetermined in space group P6(1), consistent with the other three structures. Our results show that the primary mutation V82A causes the known effect of collapsing the S1/S1' pockets that ultimately lead to the reduced inhibitory effect of TL-3. Our results further indicate that two naturally occurring polymorphic substitutions in subtype F and other non-B HIV proteases, M36I and L89M, may lead to early development of drug resistance in patients infected with non-B HIV subtypes.     

Effectiveness of commercial inhibitors against subtype F HIV-1 protease

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K(i) and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.     

Effectiveness of commercial inhibitors against subtype F HIV-1 protease

Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K_i and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.     

The contribution of naturally occurring polymorphisms in altering the biochemical and structural characteristics of HIV-1 subtype C protease

Fourteen subtype B and C protease variants have been engineered in an effort to study whether the preexistent baseline polymorphisms, by themselves or in combination with drug resistance mutations, differentially alter the biochemical and structural features of the subtype C protease when compared with those of subtype B protease. The kinetic studies performed in this work showed that the preexistent polymorphisms in subtype C protease, by themselves, do not provide for a greater level of resistance. Inhibition analysis with eight clinically used protease inhibitors revealed that the natural polymorphisms found in subtype C protease, in combination with drug resistance mutations, can influence enzymatic catalytic efficiency and inhibitor resistance. Structural analyses of the subtype C protease bound to nelfinavir and indinavir showed that these inhibitors form similar interactions with the residues in the active site of subtype B and C proteases. It also revealed that the naturally occurring polymorphisms could alter the position of the outer loops of the subtype C protease, especially the 60's loop.     

Computational characterization of structural role of the non-active site mutation M36I of human immunodeficiency virus type 1 protease

A prominent characteristic of human immunodeficiency virus type 1 (HIV-1) is its high genetic variability, which generates diversity of the virus and often causes a serious problem of the emergence of drug-resistant mutants. Subtype B HIV-1 is dominant in advanced countries, and the mortality rate due to subtype B HIV-1 has been decreased during the past decade. In contrast, the number of patients with non-subtype B viruses is still increasing in developing countries. One of the reasons for the prevalence of non-subtype B viruses is lack of information about the biological and therapeutic differences between subtype B and non-subtype B viruses. M36I is the most frequently observed polymorphism in non-subtype B HIV-1 proteases. However, since the 36th residue is located at a non-active site of the protease and has no direct interaction with any ligands, the structural role of M36I remains unclear. Here, we performed molecular dynamics (MD) simulations of M36I protease in complex with nelfinavir and revealed the influence of the M36I mutation. The results show that M36I regulates the size of the binding cavity of the protease. The reason for the rare emergence of D30N variants in non-subtype B HIV-1 proteases was also clarified from our computational analysis.     

Analysis of HIV-1 CRF_01 A/E protease inhibitor resistance: structural determinants for maintaining sensitivity and developing resistance to atazanavir

A series of HIV-1 protease mutants has been designed in an effort to analyze the contribution to drug resistance provided by natural polymorphisms as well as therapy-selective (active and non-active site) mutations in the HIV-1 CRF_01 A/E (AE) protease when compared to that of the subtype B (B) protease. Kinetic analysis of these variants using chromogenic substrates showed differences in substrate specificity between pretherapy B and AE proteases. Inhibition analysis with ritonavir, indinavir, nelfinavir, amprenavir, saquinavir, lopinavir, and atazanavir revealed that the natural polymorphisms found in A/E can influence inhibitor resistance. It was also apparent that a high level of resistance in the A/E protease, as with B protease, is due to it aquiring a combination of active site and non-active site mutations. Structural analysis of atazanavir bound to a pretherapy B protease showed that the ability of atazanavir to maintain its binding affinity for variants containing some resistance mutations is due to its unique interactions with flap residues. This structure also explains why the I50L and I84V mutations are important in decreasing the binding affinity of atazanavir.     

A structural and thermodynamic escape mechanism from a drug resistant mutation of the HIV-1 protease

The efficacy of HIV-1 protease inhibition therapies is often compromised by the appearance of mutations in the protease molecule that lower the binding affinity of inhibitors while maintaining viable catalytic activity and substrate affinity. The V82F/I84V double mutation is located within the binding site cavity and affects all protease inhibitors in clinical use. KNI-764, a second-generation inhibitor currently under development, maintains significant potency against this mutation by entropically compensating for enthalpic losses, thus minimizing the loss in binding affinity. KNI-577 differs from KNI-764 by a single functional group critical to the inhibitor response to the protease mutation. This single difference changes the response of the two inhibitors to the mutation by one order of magnitude. Accordingly, a structural understanding of the inhibitor response will provide important guidelines for the design of inhibitors that are less susceptible to mutations conveying drug resistance. The structures of the two compounds bound to the wild type and V82F/I84V HIV-1 protease have been determined by X-ray crystallography at 2.0 A resolution. The presence of two asymmetric functional groups, linked by rotatable bonds to the inhibitor scaffold, allows KNI-764 to adapt to the mutated binding site cavity more readily than KNI-577, with a single asymmetric group. Both inhibitors lose about 2.5 kcal/mol in binding enthalpy when facing the drug-resistant mutant protease; however KNI-764 gains binding entropy while KNI-577 loses binding entropy. The gain in binding entropy by KNI-764 accounts for its low susceptibility to the drug-resistant mutation. The heat capacity change associated with binding becomes more negative when KNI-764 binds to the mutant protease, consistent with increased desolvation. With KNI-577, the opposite effect is observed. Structurally, the crystallographic B factors increase for KNI-764 when it is bound to the drug-resistant mutant. The opposite is observed for KNI-577. Consistent with these observations, it appears that KNI-764 is able to gain binding entropy by a two-fold mechanism: it gains solvation entropy by burying itself deeper within the binding pocket and gains conformational entropy by losing interaction with the protease.     

New Insights into the In Silico Prediction of HIV Protease Resistance to Nelfinavir

The Human Immunodeficiency Virus type 1 protease enzyme (HIV-1 PR) is one of the most important targets of antiretroviral therapy used in the treatment of AIDS patients. The success of protease-inhibitors (PIs), however, is often limited by the emergence of protease mutations that can confer resistance to a specific drug, or even to multiple PIs. In the present study, we used bioinformatics tools to evaluate the impact of the unusual mutations D30V and V32E over the dynamics of the PR-Nelfinavir complex, considering that codons involved in these mutations were previously related to major drug resistance to Nelfinavir. Both studied mutations presented structural features that indicate resistance to Nelfinavir, each one with a different impact over the interaction with the drug. The D30V mutation triggered a subtle change in the PR structure, which was also observed for the well-known Nelfinavir resistance mutation D30N, while the V32E exchange presented a much more dramatic impact over the PR flap dynamics. Moreover, our in silico approach was also able to describe different binding modes of the drug when bound to different proteases, identifying specific features of HIV-1 subtype B and subtype C proteases.     

Structural insights into the South African HIV-1 subtype C protease: impact of hinge region dynamics and flap flexibility in drug resistance

The HIV protease plays a major role in the life cycle of the virus and has long been a target in antiviral therapy. Resistance of HIV protease to protease inhibitors (PIs) is problematic for the effective treatment of HIV infection. The South African HIV-1 subtype C protease (C-SA PR), which contains eight polymorphisms relative to the consensus HIV-1 subtype B protease, was expressed in Escherichia coli, purified, and crystallized. The crystal structure of the C-SA PR was resolved at 2.7?Å, which is the first crystal structure of a HIV-1 subtype C protease that predominates in Africa. Structural analyses of the C-SA PR in comparison to HIV-1 subtype B proteases indicated that polymorphisms at position 36 of the homodimeric HIV-1 protease may impact on the stability of the hinge region of the protease, and hence the dynamics of the flap region. Molecular dynamics simulations showed that the flap region of the C-SA PR displays a wider range of movements over time as compared to the subtype B proteases. Reduced stability in the hinge region resulting from the absent E35-R57 salt bridge in the C-SA PR, most likely contributes to the increased flexibility of the flaps which may be associated with reduced susceptibility to PIs.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:36     

Molecular dynamics simulations applied to the study of subtypes of HIV-1 protease common to Brazil, Africa, and Asia

Africa accounts for the majority of HIV-1 infections worldwide caused mainly by the A and C viral subtypes rather than B subtype, which prevails in the United States and Western Europe. In Brazil, B subtype is the major subtype, but F, C, and A also circulate. These non-B subtypes present polymorphisms, and some of them occur at sites that have been associated with drug resistance, including the HIV-1 protease (PR), one important drug target. Here, we report a Molecular Dynamics study of the B and non-B PR complexed with the inhibitor ritonavir to delineate the behavior of each subtype. We compare root mean squared deviation, binding free energy by linear interaction energy approach, hydrogen bonds, and intermolecular contact surface area between inhibitor and PR. From our results, we can provide a basis to understand the molecular mechanism of drug resistance in non-B subtypes. In this sense, we found a decrease of approx 4 kcal/mol in ΔG of binding between B and non-B subtypes. This corresponds to the loss of one hydrogen bond, which is in agreement with our H-bond analysis. Previous experimental affinity studies reported analogous results with inhibition constant values for non-B PR.     

The binding energetics of first- and second-generation HIV-1 protease inhibitors: implications for drug design

KNI-764 is a powerful HIV-1 protease inhibitor with a reported low susceptibility to the effects of protease mutations commonly associated with drug resistance. In this paper the binding thermodynamics of KNI-764 to the wild-type and drug-resistant mutant V82F/I84V are presented and the results compared to those obtained with existing clinical inhibitors. KNI-764 binds to the wild-type HIV-1 protease with very high affinity (3.1 x 10(10) M(-1) or 32 pM) in a process strongly favored by both enthalpic and entropic contributions to the Gibbs energy of binding (Delta G = -RTlnK(a)). When compared to existing clinical inhibitors, the binding affinity of KNI-764 is about 100 fold higher than that of indinavir, saquinavir, and nelfinavir, but comparable to that of ritonavir. Unlike the existing clinical inhibitors, which bind to the protease with unfavorable or only slightly favorable enthalpy changes, the binding of KNI-764 is strongly exothermic (-7.6 kcal/mol). The resistant mutation V82F/I84V lowers the binding affinity of KNI-764 26-fold, which can be accounted almost entirely by a less favorable binding enthalpy to the mutant. Since KNI-764 binds to the wild type with extremely high affinity, even after a 26-fold decrease, it still binds to the resistant mutant with an affinity comparable to that of other inhibitors against the wild type. These results indicate that the effectiveness of this inhibitor against the resistant mutant is related to two factors: extremely high affinity against the wild type achieved by combining favorable enthalpic and entropic interactions, and a mild effect of the protease mutation due to the presence of flexible structural elements at critical locations in the inhibitor molecule. The conclusions derived from the HIV-1 protease provide important thermodynamic guidelines that can be implemented in general drug design strategies.     

HIV-1 protease molecular dynamics of a wild-type and of the V82F/I84V mutant: possible contributions to drug resistance and a potential new target site for drugs

The protease from type 1 human immunodeficiency virus (HIV-1) is a critical drug target against which many therapeutically useful inhibitors have been developed; however, the set of viral strains in the population has been shifting to become more drug-resistant. Because indirect effects are contributing to drug resistance, an examination of the dynamic structures of a wild-type and a mutant could be insightful. Consequently, this study examined structural properties sampled during 22 nsec, all atom molecular dynamics (MD) simulations (in explicit water) of both a wild-type and the drug-resistant V82F/I84V mutant of HIV-1 protease. The V82F/I84V mutation significantly decreases the binding affinity of all HIV-1 protease inhibitors currently used clinically. Simulations have shown that the curling of the tips of the active site flaps immediately results in flap opening. In the 22-nsec MD simulations presented here, more frequent and more rapid curling of the mutant's active site flap tips was observed. The mutant protease's flaps also opened farther than the wild-type's flaps did and displayed more flexibility. This suggests that the effect of the mutations on the equilibrium between the semiopen and closed conformations could be one aspect of the mechanism of drug resistance for this mutant. In addition, correlated fluctuations in the active site and periphery were noted that point to a possible binding site for allosteric inhibitors.     

Impaired fitness of human immunodeficiency virus type 1 variants with high-level resistance to protease inhibitors.

One hope to maintain the benefits of antiviral therapy against the human immunodeficiency virus type 1 (HIV-1), despite the development of resistance, is the possibility that resistant variants will show decreased viral fitness. To study this possibility, HIV-1 variants showing high-level resistance (up to 1,500-fold) to the substrate analog protease inhibitors BILA 1906 BS and BILA 2185 BS have been characterized. Active-site mutations V32I and I84V/A were consistently observed in the protease of highly resistant viruses, along with up to six other mutations. In vitro studies with recombinant mutant proteases demonstrated that these mutations resulted in up to 10(4)-fold increases in the Ki values toward BILA 1906 BS and BILA 2185 BS and a concomitant 2,200-fold decrease in catalytic efficiency of the enzymes toward a synthetic substrate. When introduced into viral molecular clones, the protease mutations impaired polyprotein processing, consistent with a decrease in enzyme activity in virions. Despite these observations, however, most mutations had little effect on viral replication except when the active-site mutations V32I and I84V/A were coexpressed in the protease. The latter combinations not only conferred a significant growth reduction of viral clones on peripheral blood mononuclear cells but also caused the complete disappearance of mutated clones when cocultured with wild-type virus on T-cell lines. Furthermore, the double nucleotide mutation I84A rapidly reverted to I84V upon drug removal, confirming its impact on viral fitness. Therefore, high-level resistance to protease inhibitors can be associated with impaired viral fitness, suggesting that antiviral therapies with such inhibitors may maintain some clinical benefits.     

Evolution of the human immunodeficiency virus type 1 subtype-specific V3 domain is confined to a sequence space with a fixed distance to the subtype consensus.

Human immunodeficiency virus type 1 (HIV-1) strains can be separated into genetic subtypes based on phylogenetic analysis of the envelope gene. Once it had been shown that population-wide intrasubtype genetic variation of HIV-1 strains increases in the course of the AIDS epidemic, it remained uncertain whether HIV-1 subtypes are phenotypic entities spreading as distinct virus populations. To examine this, we applied Eigen's concepts of sequence geometry and fitness topography to the analysis of intrasubtype evolution of the gp120 V3 domain of HIV-1 subtypes A, B, C, and D in the course of the global AIDS epidemic. We observed that despite the high evolution rate of HIV-1, the nonsynonymous distances to the subtype consensus of sequences obtained early in the epidemic are similar to those obtained more than 10 years later, in contrast to the synonymous distances, which increased steadily over time. For HIV-1 subtype B, we observed that the evolution rate of the individual sequences is independent of their distance from the subtype B consensus, but for the individual sequences most distant from the consensus evolution away from the consensus is constrained. As a result, individual HIV-1 genomes fluctuate within a sequence space with fixed distance to the subtype consensus. Our findings suggest that the evolution of the V3 domain of HIV-1 subtypes A, B, C, and D is confined to an area in sequence space within a fixed distance to the consensus of a respective subtype. This in turn indicates that each HIV-1 subtype is a distinct viral quasispecies that is well adapted to the present environment, able to maintain its identity in the V3 region over time, and unlikely to merge during progression of the AIDS epidemic.     

Structural basis for coevolution of a human immunodeficiency virus type 1 nucleocapsid-p1 cleavage site with a V82A drug-resistant mutation in viral protease

Maturation of human immunodeficiency virus (HIV) depends on the processing of Gag and Pol polyproteins by the viral protease, making this enzyme a prime target for anti-HIV therapy. Among the protease substrates, the nucleocapsid-p1 (NC-p1) sequence is the least homologous, and its cleavage is the rate-determining step in viral maturation. In the other substrates of HIV-1 protease, P1 is usually either a hydrophobic or an aromatic residue, and P2 is usually a branched residue. NC-p1, however, contains Asn at P1 and Ala at P2. In response to the V82A drug-resistant protease mutation, the P2 alanine of NC-p1 mutates to valine (AP2V). To provide a structural rationale for HIV-1 protease binding to the NC-p1 cleavage site, we solved the crystal structures of inactive (D25N) WT and V82A HIV-1 proteases in complex with their respective WT and AP2V mutant NC-p1 substrates. Overall, the WT NC-p1 peptide binds HIV-1 protease less optimally than the AP2V mutant, as indicated by the presence of fewer hydrogen bonds and fewer van der Waals contacts. AlaP2 does not fill the P2 pocket completely; PheP1' makes van der Waals interactions with Val82 that are lost with the V82A protease mutation. This loss is compensated by the AP2V mutation, which reorients the peptide to a conformation more similar to that observed in other substrate-protease complexes. Thus, the mutant substrate not only binds the mutant protease more optimally but also reveals the interdependency between the P1' and P2 substrate sites. This structural interdependency results from coevolution of the substrate with the viral protease.     

A major role for a set of non-active site mutations in the development of HIV-1 protease drug resistance

A major problem in the chemotherapy of HIV-1 infection is the appearance of drug resistance. In the case of HIV-1 protease inhibitors, resistance originates from mutations in the protease molecule that lower the affinity of inhibitors while still maintaining a viable enzymatic profile. Drug resistance mutations can be classified as active site or non-active site mutations depending on their location within the protease molecule. Active site mutations directly affect drug/target interactions, and their action can be readily understood in structural terms. Non-active site mutations influence binding from distal locations, and their mechanism of action is not immediately apparent. In this paper, we have characterized a mutant form of the HIV-1 protease, ANAM-11, identified in clinical isolates from HIV-1 infected patients treated with protease inhibitors. This mutant protease contains 11 mutations, 10 of which are located outside the active site (L10I/M36I/S37D/M46I/R57K/L63P/A71V/G73S/L90M/I93L) and 1 within the active site (I84V). ANAM-11 lowers the binding affinity of indinavir, nelfinavir, saquinavir, and ritonavir by factors of 4000, 3300, 5800, and 80000, respectively. Surprisingly, most of the loss in inhibitor affinity is due to the non-active site mutations as demonstrated by additional experiments performed with a protease containing only the 10 non-active site mutations (NAM-10) and another containing only the active site mutation (A-1). Kinetic analysis with two different substrates yielded comparable catalytic efficiencies for A-1, ANAM-11, NAM-10, and the wild-type protease. These studies demonstrate that non-active site mutations can be the primary source of resistance and that their role is not necessarily limited to compensate deleterious effects of active site mutations. Analysis of the structural stability of the proteases by differential scanning calorimetry reveals that ANAM-11 and NAM-10 are structurally more stable than the wild-type protease while A-1 is less stable. Together, the binding and structural thermodynamic results suggest that the non-active site mutants affect inhibitor binding by altering the geometry of the binding site cavity through the accumulation of mutations within the core of the protease molecule.     

Viability of a drug-resistant human immunodeficiency virus type 1 protease variant: structural insights for better antiviral therapy

Under the selective pressure of protease inhibitor therapy, patients infected with human immunodeficiency virus (HIV) often develop drug-resistant HIV strains. One of the first drug-resistant mutations to arise in the protease, particularly in patients receiving indinavir or ritonavir treatment, is V82A, which compromises the binding of these and other inhibitors but allows the virus to remain viable. To probe this drug resistance, we solved the crystal structures of three natural substrates and two commercial drugs in complex with an inactive drug-resistant mutant (D25N/V82A) HIV-1 protease. Through structural analysis and comparison of the protein-ligand interactions, we found that Val82 interacts more closely with the drugs than with the natural substrate peptides. The V82A mutation compromises these interactions with the drugs while not greatly affecting the substrate interactions, which is consistent with previously published kinetic data. Coupled with our earlier observations, these findings suggest that future inhibitor design may reduce the probability of the appearance of drug-resistant mutations by targeting residues that are essential for substrate recognition.     

Human leukocyte antigen-associated gag and nef polymorphisms in HIV-1 subtype A/E-infected individuals in Vietnam

Numbers of HLA-associated polymorphisms have been reported on HIV-1 subtypes B and C, but few on other subtypes. Here, we analyzed HLA-associated gag and nef polymorphisms in HIV-1 subtype A/E prevalent in Vietnam. We determined HLA-A, B and C genotypes in 179 HIV-1-infected Vietnamese by next generation sequencing and analyzed proviral genome sequences in 144 of them, showing that 142 of the 144 were subtype A/E. Analysis revealed HLA-associated subtype A/E gag and nef polymorphisms at nineteen residues including those newly determined. Accumulation of these data would contribute to our understanding of HIV-1 subtype A/E and host immune interaction.     

Secondary mutations M36I and A71V in the human immunodeficiency virus type 1 protease can provide an advantage for the emergence of the primary mutation D30N

Development of resistance mutations in enzymatic targets of human immunodeficiency virus 1 (HIV-1) hampers the ability to provide adequate therapy. Of special interest is the effect mutations outside the active site of HIV-1 protease have on inhibitor binding and virus viability. We engineered protease mutants containing the active site mutation D30N alone and with the nonactive site polymorphisms M36I and/or A71V. We determined the K(i) values for the inhibitors nelfinavir, ritonavir, indinavir, KNI272, and AG1776 as well as the catalytic efficiency of the mutants. Single and double mutation combinations exhibited a decrease in catalytic efficiency, while the triple mutant displayed catalytic efficiency greater than that of the wild type. Variants containing M36I or A71V alone did not display a significant change in binding affinities to the inhibitors tested. The variant containing mutation D30N displayed a 2-6-fold increase in K(i) for all inhibitors tested, with nelfinavir showing the greatest increase. The double mutants containing a combination of mutations D30N, M36I, and A71V displayed -0.5-fold to +6-fold changes in the K(i) of all inhibitors tested, with ritonavir and nelfinavir most affected. Only the triple mutant showed a significant increase (>10-fold) in K(i) for inhibitor nelfinavir, ritonavir, or AG-1776 displaying 22-, 19-, or 15-fold increases, respectively. Our study shows that the M36I and A71V mutations provide a greater level of inhibitor cross-resistance combined with active site mutation D30N. M36I and A71V, when present as natural polymorphisms, could aid the virus in developing active site mutations to escape inhibitor binding while maintaining catalytic efficiency.