HIV-2 Protease

What are the key structural differences between HIV-1 and HIV-2 proteases?

HIV-1 and HIV-2 proteases share significant structural similarities as they are both symmetrical homodimers composed of two identical 99-amino-acid subunits. Each subunit contributes one aspartic tripeptide motif, and both proteases contain two layers of orthogonally oriented beta sheets forming a hydrophobic core and flexible beta hairpin "flap regions" (residues 42-58) that close upon substrate binding .

Despite these similarities, the two proteases share only 38-49% sequence identity. The most critical differences are found within the ligand-binding pocket, specifically at positions 32, 47, 76, and 82. In HIV-1, these positions contain Val32, Ile47, Leu76, and Val82, while HIV-2 contains Ile32, Val47, Met76, and Ile82 . These amino acid differences, though conservative in nature (all hydrophobic), are sufficient to create distinct binding characteristics that significantly impact inhibitor efficacy.

How do HIV-1 and HIV-2 proteases differ in catalytic efficiency and substrate recognition?

Kinetic studies reveal that HIV-2 protease generally demonstrates higher catalytic efficiency (kcat/Km) compared to HIV-1 protease for several substrates. For instance, with the HIV-2 CA/p2 peptide (KARLM↓AEALK), HIV-2 protease shows a 6.5-fold higher catalytic efficiency than HIV-1 protease . Even more dramatically, for the HIV-2 p2/NC peptide (IPFAA↓AQQRK), HIV-2 protease exhibits a 10.4-fold higher catalytic efficiency .

The two proteases show distinct preferences for amino acids at the P2 and P4 positions of substrates. These preferences correlate with the differences in residues 32, 47, and 82 that form part of the substrate binding cavities. The table below illustrates these differential catalytic efficiencies:

These differences in substrate specificity highlight the evolutionary divergence between the two viral proteases and have important implications for designing specific inhibitors .

Why is HIV-2 protease intrinsically resistant to most FDA-approved HIV-1 protease inhibitors?

HIV-2 shows intrinsic resistance to most FDA-approved HIV-1 protease inhibitors (PIs), retaining clinically useful susceptibility only to lopinavir (LPV), darunavir (DRV), and saquinavir (SQV) . The mechanism of this resistance has been linked to the four amino acid differences in the ligand-binding pocket at positions 32, 47, 76, and 82.

The differential binding of APV in the P2' pocket is particularly notable. In HIV-2, Val47 (compared to Ile47 in HIV-1) creates a less favorable interaction with the aniline ring of APV. Additionally, the position of the main-chain carbonyl of D30 differs between the two proteases, resulting in a longer, weaker hydrogen bond with the aniline NH2 group of APV in HIV-2 (3.7Å vs. 3.2Å in HIV-1) .

These structural differences collectively explain why HIV-2 is naturally resistant to most HIV-1 PIs, providing a molecular basis for the observed clinical differences in drug efficacy.

How do single versus multiple amino acid substitutions in HIV-2 protease affect inhibitor binding and efficacy?

Research has demonstrated that while individual amino acid substitutions at positions 32, 47, 76, and 82 can increase the susceptibility of HIV-2 to certain protease inhibitors, no single change confers class-wide sensitivity. In contrast, the combination of all four substitutions (I32V, V47I, M76L, and I82V - creating the PRΔ4 variant) renders HIV-2 as susceptible as HIV-1 to all nine FDA-approved PIs tested .

Single amino acid replacements showed variable effects:

• I32V increased susceptibility to some PIs

• V47I improved binding of certain inhibitors

• M76L enhanced PI sensitivity for specific compounds

• No single change provided comprehensive sensitivity improvement

• Nelfinavir (NFV): 10-fold increase

• Darunavir (DRV): 26-fold increase

• Amprenavir (APV): >43-fold increase

• Atazanavir (ATV): 60-fold increase

These findings establish that the four amino acid differences in the binding pocket are the primary determinants of differential PI susceptibility between HIV-1 and HIV-2 .

What are the implications of HIV-2 protease studies for understanding drug resistance in HIV-1?

The natural amino acids found in HIV-2 protease at positions 32, 47, 76, and 82 correspond to well-documented drug resistance mutations in HIV-1 protease. Specifically, V32I, I47V, and various substitutions at V82 in HIV-1 are associated with multi-PI resistance .

This remarkable convergence suggests that HIV-2 naturally possesses a partially PI-resistant protease conformation that HIV-1 achieves only through mutations under drug pressure. This provides a unique opportunity to study resistance mechanisms:

• The HIV-2 protease can serve as a natural model system for studying class-wide PI resistance

• Structural and biochemical studies of HIV-2 protease can inform predictions about resistance pathways in HIV-1

• Comparing HIV-1 and HIV-2 binding mechanisms helps elucidate the molecular basis of resistance mutations

Research shows that HIV-1 mutants with substitutions mimicking the HIV-2 binding site (V32I, I47V, V82I) partially replicate the specificity characteristics of HIV-2 protease, both in terms of substrate processing and inhibitor binding . This provides valuable insights into the structural basis of drug resistance mutations at these positions and can guide the development of novel inhibitors effective against resistant strains of both viruses.

What methods are most effective for studying the structural basis of HIV-2 protease inhibitor binding?

Several complementary methodological approaches have proven effective for studying HIV-2 protease and its interactions with inhibitors:

• X-ray Crystallography: High-resolution crystal structures (1.5Å or better) of HIV-2 protease complexed with inhibitors provide detailed atomic-level information about binding modes. This approach has been successfully used to determine structures of HIV-2 protease with APV, DRV, and other inhibitors . Key considerations include:

  • Protein expression and purification must yield highly pure, stable enzyme

  • Crystal growth conditions require optimization for each protease-inhibitor complex

  • Data collection at synchrotron sources provides higher resolution

• Computational Modeling and Energy Minimization: Energy-minimized models based on existing crystal structures can predict the effects of mutations. This approach involves:

  • Introducing mutations using tools like Rotamers in UCSF Chimera

  • Energy minimization with programs such as YASARA

  • Comparing minimized structures to validate consistency

• Enzyme Kinetics and Inhibition Assays: Single-cycle replication assays with replication-competent viruses provide quantitative measures of inhibitor potency (EC50 values). These involve:

  • Construction of molecular clones with specific protease mutations

  • Transfection into appropriate cell lines (e.g., 293T/17 cells)

  • Measurement of viral replication in the presence of inhibitors

• Site-Directed Mutagenesis: Engineering specific amino acid changes in HIV-2 protease enables systematic analysis of their contributions to inhibitor binding and resistance .

The most comprehensive studies combine structural data with functional analyses to correlate structural features with inhibition potency, allowing researchers to identify critical interactions that determine inhibitor efficacy.

How can enzymatic assays be optimized to accurately assess HIV-2 protease activity and inhibition?

Optimizing enzymatic assays for HIV-2 protease requires careful consideration of several factors to ensure reliable and reproducible results:

• Substrate Selection: Choose substrates that represent authentic HIV-2 cleavage sites. Studies show that HIV-2 protease has different specificities compared to HIV-1 protease, particularly at the P2 and P4 positions . Include:

  • HIV-2-specific peptides (e.g., HIV-2 CA/p2: KARLM↓AEALK)

  • HIV-2 p2/NC peptides (e.g., IPFAA↓AQQRK)

  • Comparative HIV-1 peptides with variations at key positions

• Assay Conditions Optimization:

  • Determine optimal pH (typically 5.5-6.0 for retroviral proteases)

  • Establish appropriate buffer conditions

  • Optimize enzyme concentration to achieve linear reaction rates

  • Ensure substrate concentration spans values below and above Km

• Data Analysis Approaches:

  • Calculate both Km and kcat values, not just IC50

  • Compare catalytic efficiency (kcat/Km) across different enzymes and substrates

  • Use relative kcat/Km values to normalize comparisons

• Controls and Validation:

  • Include wild-type HIV-1 protease as a comparison standard

  • Test mutant proteases with specific substitutions (e.g., PRΔ4 mutant)

  • Validate biochemical findings with structural data

For inhibition studies, the data should ideally be reported as both EC50 values (for cellular assays) and Ki values (for enzymatic assays), allowing for more direct comparisons between different studies and inhibitors.

How do differences in the P2 and P2' binding pockets between HIV-1 and HIV-2 proteases influence inhibitor design?

The P2 and P2' binding pockets show subtle but critical differences between HIV-1 and HIV-2 proteases that significantly impact inhibitor binding. Understanding these differences is essential for rational drug design:

• P2' Pocket Differences:

  • In HIV-1, Ile47 forms favorable C—H···π interactions with the aniline ring of inhibitors like APV

  • In HIV-2, Val47 (shorter side chain) results in weaker interactions with the aniline moiety

  • The PRΔ4 mutant (containing V47I) brings the side chain approximately 0.8Å closer to the aniline ring, forming new stabilizing interactions

• Main-Chain Conformational Changes:

  • The main-chain carbonyl of D30 adopts different orientations in HIV-1 versus HIV-2

  • This results in a shorter, stronger hydrogen bond with the aniline NH2 in HIV-1 (3.2Å) compared to HIV-2 (3.7Å)

  • The PRΔ4 mutant shows a ~40° rotation of this carbonyl group, adopting a conformation similar to HIV-1

• P2 Pocket Variations:

  • The combined effects of differences at positions 32 and 82 alter the shape and hydrophobicity of the P2 pocket

  • These changes affect the binding of the THF/bis-THF groups found in APV and DRV

These structural insights suggest specific design strategies for developing inhibitors effective against both HIV-1 and HIV-2:

• Design compounds with flexibility to accommodate differences in the P2' pocket

• Incorporate functional groups that can form additional interactions to compensate for weaker binding in HIV-2

• Optimize bis-THF-like moieties (as in DRV) that maintain efficacy against both proteases

• Target conserved regions of the binding pocket to minimize the impact of sequence variations

Notably, darunavir's bis-THF group forms more extensive contacts than amprenavir's THF group, likely explaining its retained potency against HIV-2 despite the structural differences .

What are the critical hydrogen bonding networks in HIV-2 protease-inhibitor complexes and how do they differ from HIV-1?

Hydrogen bonding networks play crucial roles in stabilizing inhibitor binding to HIV proteases. Comparative structural analyses reveal both conserved and differential hydrogen bonding networks between HIV-1 and HIV-2:

• Conserved Hydrogen Bonds:

  • Interactions involving the catalytic aspartates (D25 and D25')

  • Hydrogen bonds with the flap regions and a coordinating water molecule

  • Interactions at the P1 and P1' sites of the enzymes

• Differential Hydrogen Bonds in the P2' Site:

  • The hydrogen bond between the D30 carbonyl oxygen and the aniline NH2 of APV differs significantly:

    • HIV-1: 3.2Å (strong hydrogen bond)

    • HIV-2: 3.7Å (weaker interaction)

    • PRΔ4 mutant: 3.2Å (restored strong interaction)

  • These differences correlate directly with inhibitor potency

• P2 Site Hydrogen Bonding:

  • The interactions with the THF group of APV or bis-THF group of DRV show subtle variations

  • DRV forms more extensive hydrogen bonds in both proteases, explaining its broader efficacy

• Effect of Mutations on Hydrogen Bonding Networks:

  • The V32I substitution affects the positioning of neighboring residues

  • I47V/V47I alters the geometry of interactions in the P2' pocket

  • M76L influences the orientation of the inhibitor in the binding site

  • I82V/V82I affects hydrophobic packing in the P1 region

Understanding these complex hydrogen bonding networks provides critical insights for rational drug design. Optimizing inhibitors to form strong hydrogen bonds with both HIV-1 and HIV-2 proteases, particularly at positions where differences exist, could lead to broader-spectrum antiretroviral agents.

How can molecular dynamics simulations complement crystallographic studies of HIV-2 protease?

While crystallographic studies provide static snapshots of HIV-2 protease-inhibitor complexes, molecular dynamics (MD) simulations can offer crucial insights into the dynamic behavior of these systems. Combining both approaches provides a more comprehensive understanding:

• Advantages of MD Simulations for HIV-2 Protease Research:

  • Capture dynamic conformational changes in the flap regions

  • Explore water-mediated interactions that may be missed in crystal structures

  • Assess the stability and longevity of hydrogen bonds identified in crystal structures

  • Investigate induced-fit effects upon inhibitor binding

  • Predict the impact of mutations before experimental validation

• Methodological Considerations:

  • Start with high-resolution crystal structures as initial models

  • Use energy minimization (as performed with YASARA in the cited research)

  • Implement explicit solvent models to capture water-mediated interactions

  • Run simulations for sufficient time to observe relevant conformational changes

  • Analyze hydrogen bond networks throughout the simulation trajectory

• Applications to HIV-2 Protease Research:

  • Compare flap dynamics between HIV-1 and HIV-2 proteases

  • Assess the stability of interactions in the P2 and P2' pockets

  • Evaluate the energetic contributions of specific residues to inhibitor binding

  • Predict the effects of designed inhibitors before synthesis

MD simulations can be particularly valuable for studying the differences between HIV-1 and HIV-2 proteases, as they can reveal dynamic aspects of inhibitor binding that may not be apparent from static crystal structures. This approach complements experimental studies and can guide the rational design of inhibitors effective against both proteases.

What are the implications of HIV-2 protease research for clinical treatment of HIV-2 infections?

Research on HIV-2 protease has direct implications for the clinical management of HIV-2 infections, which are prevalent in West Africa and increasingly observed in other regions:

• Limited PI Options for HIV-2 Treatment:

  • Only three FDA-approved PIs (lopinavir, darunavir, and saquinavir) show clinically useful activity against HIV-2

  • Other PIs exhibit significantly reduced efficacy against HIV-2

  • This restricts treatment options for HIV-2-infected patients

• Rational Selection of PIs:

  • Understanding the structural basis of PI resistance helps inform clinical decisions

  • Darunavir should be preferentially considered due to its maintained potency against HIV-2

  • Higher doses of certain PIs might be necessary to achieve therapeutic effects against HIV-2

• Combination Therapy Considerations:

  • The limited PI options necessitate careful selection of companion drugs

  • Research on HIV-2 protease resistance mechanisms should inform regimen design

• Monitoring Considerations:

  • HIV-2 viral load assays must be validated specifically for HIV-2

  • Genotypic resistance testing interpretations differ between HIV-1 and HIV-2

  • The natural presence of resistance-associated amino acids in HIV-2 protease requires different interpretation algorithms

This research underscores the need for HIV-2-specific treatment guidelines and the development of antiretrovirals specifically validated against HIV-2, rather than simply extrapolating from HIV-1 studies.

How can structural insights into HIV-2 protease guide the development of dual-active inhibitors?

Structural studies comparing HIV-1 and HIV-2 proteases provide valuable insights that can guide the rational design of dual-active inhibitors effective against both viruses:

• Target Conserved Binding Features:

  • Focus on interactions with catalytic aspartates and flap regions

  • Maintain critical interactions at P1 and P1' sites where binding modes are conserved between HIV-1 and HIV-2

• Optimize P2/P2' Interactions:

  • Design larger, more flexible moieties for the P2 site to accommodate differences at positions 32 and 82

  • Incorporate functional groups that can form stronger interactions with Val47 in HIV-2

  • Consider bis-THF-like groups (as in darunavir) that maintain efficacy despite structural differences

• Leverage Structural Water Networks:

  • Design inhibitors that can utilize conserved water-mediated hydrogen bonding networks

  • Target interactions that are less dependent on specific amino acid side chains

• Apply Substrate-Based Design Principles:

  • Consider the differential substrate preferences of HIV-1 and HIV-2 proteases

  • Design inhibitors that incorporate elements recognized by both enzymes

  • Focus on scaffolds that accommodate the higher catalytic efficiency of HIV-2 protease

• Utilize the PRΔ4 Model System:

  • Test candidate compounds against both wild-type HIV-2 and the PRΔ4 mutant

  • Compounds showing similar potency against both variants may be more robust against resistance

These strategies, informed by detailed structural comparisons between HIV-1 and HIV-2 proteases and their interactions with inhibitors, provide a roadmap for developing next-generation antiretrovirals with broad efficacy against diverse HIV strains.

What novel methodological approaches could advance our understanding of HIV-2 protease dynamics and inhibition?

Several innovative methodological approaches could significantly enhance our understanding of HIV-2 protease:

• Cryo-Electron Microscopy (Cryo-EM):

  • Apply cryo-EM to study HIV-2 protease in the context of larger assemblies

  • Investigate the dynamics of protease within the Gag-Pol polyprotein

  • Examine conformational ensembles that may not be captured in crystal structures

• Advanced Spectroscopic Techniques:

  • Nuclear Magnetic Resonance (NMR) to study protease dynamics in solution

  • Single-molecule Förster Resonance Energy Transfer (FRET) to monitor flap movements

  • Hydrogen-deuterium exchange mass spectrometry to probe conformational flexibility

• Integrative Structural Biology:

  • Combine multiple experimental techniques (X-ray, NMR, SAXS)

  • Develop computational frameworks to integrate diverse structural data

  • Build more comprehensive models of protease function

• Machine Learning Approaches:

  • Apply deep learning to predict inhibitor binding affinities

  • Develop models that can predict cross-reactivity between HIV-1 and HIV-2 proteases

  • Use generative models to design novel inhibitors targeting both proteases

• Time-Resolved Crystallography:

  • Study the dynamics of inhibitor binding and release

  • Capture intermediate states in the catalytic cycle

  • Understand the conformational changes associated with substrate recognition

These approaches would complement existing methodologies and provide new insights into the dynamic behavior of HIV-2 protease, potentially revealing novel strategies for inhibitor design.

What are the research priorities for understanding HIV-2 protease evolution under drug pressure?

Understanding how HIV-2 protease evolves under selective pressure from protease inhibitors represents a critical research priority:

• Longitudinal Studies of HIV-2 Patients on PI Therapy:

  • Track protease sequence evolution during long-term PI treatment

  • Identify emerging resistance mutations specific to HIV-2

  • Compare resistance pathways with those observed in HIV-1

• In Vitro Selection Experiments:

  • Perform serial passage of HIV-2 in the presence of increasing PI concentrations

  • Characterize emerging resistance mutations

  • Determine the genetic barrier to resistance for different PIs

• Structural Studies of Resistant Variants:

  • Solve crystal structures of drug-resistant HIV-2 protease variants

  • Compare with corresponding HIV-1 resistant structures

  • Identify compensatory mechanisms that maintain catalytic efficiency

• Functional Characterization of Resistant Enzymes:

  • Measure catalytic parameters (Km, kcat) of resistant variants

  • Determine fitness costs associated with resistance mutations

  • Assess substrate specificity changes in resistant enzymes

• Computational Prediction of Resistance Pathways:

  • Develop models to predict likely resistance mutations

  • Simulate evolutionary trajectories under different drug pressures

  • Design inhibitors with higher genetic barriers to resistance

Given that HIV-2 naturally contains amino acids associated with resistance in HIV-1 (at positions 32, 47, 76, and 82), it presents a unique opportunity to study how protease evolves under additional selective pressure, potentially revealing novel resistance mechanisms relevant to both HIV types.