HIV-2 gag

What is the basic structure of HIV-2 Gag and how does it differ from HIV-1 Gag?

HIV-2 Gag is a polyprotein that serves as the main structural component of HIV-2 virions. Similar to HIV-1 Gag, it consists of several domains including matrix (MA), capsid (CA), nucleocapsid (NC), and smaller peptides (SP1, SP2, p6). While HIV-1 and HIV-2 CA proteins share approximately 70% sequence identity, subtle amino acid variations create significant differences in their structural organization and lattice formation .

The HIV-2 Gag hexamer exhibits a "wineglass-shaped" structure where CA proteins form the cup and the CA-SP1 six-helix bundle represents the stem. The N-terminal domain (NTD) and C-terminal domain (CTD) of HIV-2 CA twist in a left-handed manner, while the six-helix bundle twists in a right-handed direction . These similarities to HIV-1 Gag indicate conserved structural elements important for immature particle formation across lentiviruses.

What experimental techniques are most effective for studying HIV-2 Gag structure?

Multiple complementary structural biology techniques have proven effective for studying HIV-2 Gag:

How does HIV-2 Gag organize in immature viral particles?

HIV-2 Gag assembles into a remarkably complete hexagonal lattice in immature particles. Cryo-ET and subtomogram averaging have demonstrated that:

• HIV-2 Gag forms an almost complete hexagonal lattice pattern with only small, interspersed gaps or crevice regions .

• The Gag coverage area underneath the viral membrane can reach as high as ~90%, with an average membrane coverage ratio of 76 ± 8% .

• The number of Gag molecules in immature HIV-2 particles ranges from approximately 1,700 to 5,700, with an average of ~3,900 ± 1,000 .

This high Gag occupancy contrasts significantly with HIV-1, where the Gag lattice typically covers only about 60% of the viral membrane . This structural difference may contribute to the distinct pathogenic properties observed between HIV-1 and HIV-2.

What are the most effective methods for purifying HIV-2 particles for structural studies?

For high-quality structural studies of HIV-2 Gag, researchers should implement a multi-step purification process:

• Cell culture and transfection - HEK 293T/17 cells have been successfully used for HIV-2 particle production through transfection with proviral constructs containing protease inactivation mutations to generate immature particles .

• Harvesting and filtration - Culture supernatants should be harvested 48-72 hours post-transfection and filtered through 0.45 μm filters to remove cellular debris.

• Ultracentrifugation - Particles should be concentrated through a 20% sucrose cushion by ultracentrifugation (typically 100,000-120,000 × g for 2 hours).

• Resuspension and preparation - Virus pellets should be gently resuspended in an appropriate buffer (such as PBS or TNE buffer) and used immediately for cryo-EM/ET specimen preparation or stored at -80°C.

The quality of purified particles should be verified by Western blot analysis for Gag protein and negative staining electron microscopy before proceeding to high-resolution structural studies.

How can researchers effectively analyze HIV-2 Gag lattice coverage and organization in viral particles?

Quantitative analysis of HIV-2 Gag lattice organization requires specialized computational approaches:

• Data acquisition - Collect cryo-ET tilt series (typically ±60° with 2° increments) of purified HIV-2 immature particles .

• Initial reconstruction - Reconstruct tomograms using software such as IMOD .

• Subtomogram averaging workflow:

  • Implement iterative convolution procedures that correlate sub-volumes from reconstruction maps with averaged Gag hexamer models using Dynamo .

  • Refine structures using RELION-4.0 or similar software .

  • Calculate positional cross-correlations to identify Gag hexamer positions and lattice gaps .

• Coverage calculation - Determine the Gag lattice coverage by calculating the ratio of the area occupied by the lattice to the total inner membrane surface area .

This analytical pipeline has revealed that HIV-2 immature particles have significantly higher Gag coverage (up to ~90%) compared to HIV-1 (~60%), providing important insights into viral assembly differences .

What mutagenesis approaches are most informative for studying HIV-2 Gag function?

Strategic mutagenesis studies of HIV-2 Gag have proven valuable for understanding structure-function relationships:

• Interface mutations - Target residues at two-fold and three-fold symmetric axes of the Gag lattice, which maintain inter-hexamer interfaces .

• Domain-swapping experiments - Exchange homologous regions between HIV-1 and HIV-2 Gag to identify critical determinants of virus-specific properties .

• Specific residue targets:

  • Mutations at the Gag lattice three-fold interface (e.g., HIV-2 CA G38M) can reduce virus particle production by 50% and eliminate infectivity .

  • Non-conserved residues in helix 2 of CA proteins are particularly informative .

  • The HIV-2 CA N127E mutant, which introduces the HIV-1-encoding residue into HIV-2 CA, severely reduces particle production .

• Helix 10-12 interface - Residues at this interface are critical for maintaining HIV-2 particle release and infectivity .

These mutagenesis approaches have helped identify key structural elements that distinguish HIV-1 and HIV-2 Gag function, providing insights into their different pathogenic properties.

What are the key structural differences between HIV-1 and HIV-2 Gag lattices in immature particles?

HIV-1 and HIV-2 Gag lattices exhibit several important structural differences:

While both viruses form hexagonal Gag lattices with similar molecular structures, the significantly higher coverage and more complete lattice in HIV-2 represents a key distinguishing feature . This difference may contribute to the distinct pathogenic properties and transmission dynamics of the two viruses.

How do sequence variations between HIV-1 and HIV-2 CA impact Gag lattice stability and viral infectivity?

Despite ~70% sequence identity between HIV-1 and HIV-2 CA proteins, specific amino acid variations significantly impact lattice stability and viral function:

• Three-fold interface residues - Exchanging HIV-1 and HIV-2 residues at these positions significantly reduces virus particle production and eliminates infectivity .

• Helix 2 variations - Non-conserved residues in CA helix 2 (e.g., HIV-2 CA G38M and HIV-1 CA M39G) are critical for virus production and infectivity .

• CTD structure - HIV-2 CA CTD contains a novel, extended 3₁₀ helix (H12) that interacts with H10, with residues at this interface being critical for maintaining HIV-2 particle infectivity .

• Inhibitor sensitivity - HIV-2 is insensitive to the HIV-1 maturation inhibitor bevirimat (BVM), highlighting functional consequences of structural differences in the CA-SP1 junction region .

These sequence-specific differences demonstrate how subtle variations in CA structure can have profound effects on viral assembly, stability, and infectivity.

What methodological considerations are important when comparing HIV-1 and HIV-2 Gag structures?

Comparative studies of HIV-1 and HIV-2 Gag require careful methodological considerations:

• Consistent sample preparation - Use identical cell types (e.g., HEK 293T/17), transfection methods, and purification protocols to minimize experimental variables.

• Matched imaging conditions - Employ consistent cryo-EM/ET data collection parameters, including defocus ranges, total electron dose, and tilt series acquisition strategies.

• Resolution matching - When directly comparing structures, ensure they are filtered to comparable resolutions to avoid biases from resolution differences.

• Software consistency - Use the same computational tools and parameters for reconstruction and analysis to enable valid comparisons.

• Statistical rigor - Analyze sufficient numbers of particles from multiple independent preparations to account for biological variability (e.g., the study of HIV-2 analyzed 25 particles to calculate average Gag coverage) .

• Validation controls - Include appropriate control experiments, particularly when conducting mutagenesis studies that compare HIV-1 and HIV-2 Gag variants.

These methodological considerations ensure that observed differences reflect genuine biological distinctions rather than technical artifacts.

What is the relationship between HIV-2 Gag structure and viral pathogenicity?

The structural differences between HIV-1 and HIV-2 Gag may contribute to their distinct pathogenic properties:

• Complete lattice formation - The nearly complete Gag lattice in HIV-2 (up to ~90% coverage) compared to HIV-1 (~60%) suggests differences in particle stability and potentially viral dynamics .

• Particle size consistency - HIV-2 immature particles exhibit a narrower range of particle sizes with consistent electron density distribution beneath the viral membrane, suggesting more regulated assembly .

• Critical residues - Mutations in HIV-2 CA that affect the Gag lattice structure, particularly at three-fold interfaces and the H10-H12 interaction region, severely impact viral infectivity, demonstrating structure-function relationships .

These structural differences may help explain the generally lower transmissibility and slower disease progression associated with HIV-2 compared to HIV-1, though direct mechanistic links require further investigation.

How can structural insights into HIV-2 Gag inform antiviral development strategies?

Structural studies of HIV-2 Gag provide valuable insights for antiviral development:

• Differential inhibitor sensitivity - HIV-2's insensitivity to the HIV-1 maturation inhibitor bevirimat (BVM) highlights the importance of virus-specific structural knowledge for targeted drug development .

• Conserved interfaces - The identification of conserved structural elements at Gag lattice interfaces across HIV-1 and HIV-2 points to potential broad-spectrum antiviral targets .

• Critical interactions - The H10-H12 interface in HIV-2 CA CTD represents a potential target for disrupting virus assembly, as residues in this region are critical for particle infectivity .

• IP6 binding site - Both HIV-1 and HIV-2 Gag incorporate inositol hexakisphosphate (IP6) in their six-helix bundles, suggesting this interaction could be targeted for broad inhibition of lentiviral assembly .

These structural insights can guide rational design of antivirals that either target both HIV types or exploit their differences for virus-specific inhibition.

What advanced imaging techniques are emerging for studying HIV-2 Gag dynamics during assembly and maturation?

Several cutting-edge methodologies are advancing our understanding of HIV-2 Gag dynamics:

• Time-resolved cryo-EM - By capturing viral particles at different stages of assembly and maturation through rapid freezing, researchers can create temporal snapshots of structural transitions.

• Correlative light and electron microscopy (CLEM) - Combining fluorescence microscopy with cryo-EM/ET allows tracking of specific labeled Gag components during assembly and maturation in cellular contexts.

• Single-molecule localization microscopy - Super-resolution techniques like PALM and STORM can visualize the distribution and dynamics of individual Gag molecules with nanometer precision.

• In situ cryo-electron tomography - Direct visualization of Gag assembly within intact cellular environments using cryo-focused ion beam (cryo-FIB) milling and tomography reveals native assembly contexts.

• 4D cryo-electron microscopy - Emerging techniques that combine multiple time points with 3D structural analysis to understand the dynamics of Gag assembly and reorganization during maturation.

These advanced imaging approaches, when applied to HIV-2 Gag, will provide unprecedented insights into the temporal and spatial dynamics of viral assembly and maturation processes.

What are the main technical challenges in obtaining high-resolution structures of HIV-2 Gag in immature particles?

Researchers face several technical challenges when studying HIV-2 Gag structure:

• Sample heterogeneity - Despite having more ordered lattices than HIV-1, HIV-2 particles still exhibit biological variability that limits resolution in structural studies.

• Incomplete lattices - Even in HIV-2, the Gag lattice contains gaps and edge effects that complicate image processing and reconstruction.

• Specimen thickness - Whole virus particles (typically 100-120 nm diameter) approach the practical limits for cryo-EM, resulting in decreased signal-to-noise ratios in thicker regions.

• Beam-induced motion - Electron beam exposure causes specimen movement during data collection, requiring sophisticated motion correction algorithms.

• Conformational flexibility - Some regions of Gag, particularly linking domains and terminal regions, exhibit conformational heterogeneity that limits achievable resolution.

Overcoming these challenges requires optimized specimen preparation, state-of-the-art imaging hardware, and advanced computational approaches for data processing.

How can researchers troubleshoot problems with HIV-2 virus-like particle production for structural studies?

When encountering difficulties producing HIV-2 virus-like particles (VLPs) for structural studies, researchers should consider:

• Plasmid design issues:

  • Verify the integrity of the HIV-2 Gag expression construct by sequencing

  • Ensure appropriate protease mutations are introduced for immature particle production

  • Check promoter elements and regulatory sequences

• Cell culture optimization:

  • Test different cell lines (HEK293T, COS-7)

  • Optimize cell density and culture conditions

  • Adjust transfection reagents and protocols

• Harvesting timing:

  • Collect culture supernatants at different time points (24-72h) post-transfection

  • Consider multiple harvests from the same culture

• Purification troubleshooting:

  • Adjust ultracentrifugation parameters (speed, duration, cushion composition)

  • Test different resuspension buffers and techniques

  • Implement additional purification steps if necessary

• Quality assessment:

  • Use negative stain EM to verify particle morphology before cryo-EM

  • Perform Western blot analysis to confirm Gag expression and processing

These systematic troubleshooting approaches can help resolve common issues in HIV-2 VLP production for structural studies.

What are the emerging research questions about HIV-2 Gag that remain to be addressed?

Despite recent advances, several important questions about HIV-2 Gag remain unanswered:

• Maturation dynamics - How does the nearly complete HIV-2 Gag lattice reorganize during viral maturation, and how does this differ from HIV-1?

• Host factor interactions - Which host cellular factors specifically interact with HIV-2 Gag during assembly, and how do these differ from HIV-1 interactions?

• RNA binding specificity - What are the specific RNA binding properties of HIV-2 Gag, and how do they influence genome packaging and assembly?

• Membrane interactions - How do the membrane-binding properties of HIV-2 MA compare to HIV-1, and what role do these play in assembly site selection?

• Evolutionary significance - What evolutionary pressures have shaped the structural differences between HIV-1 and HIV-2 Gag, and what advantages might these provide?

Addressing these questions will require integrating structural biology with cell biology, virology, and evolutionary approaches to develop a comprehensive understanding of HIV-2 Gag biology.

How might integrative structural biology approaches advance our understanding of HIV-2 Gag function?

Future advances in HIV-2 Gag research will likely come from integrating multiple structural biology techniques:

• Hybrid methods - Combining cryo-EM/ET with X-ray crystallography, NMR spectroscopy, and mass spectrometry can provide multi-scale structural insights from atomic to whole-particle levels.

• Molecular dynamics simulations - Using experimentally determined structures as starting points for simulations can reveal dynamic aspects of Gag assembly and maturation not captured by static structures.

• In-cell structural biology - Techniques like cryo-focused ion beam milling combined with tomography enable visualization of Gag assembly in native cellular environments.

• Cross-linking mass spectrometry - Identifying interaction interfaces between Gag domains and with host factors through chemical cross-linking followed by mass spectrometry analysis.

• Single-molecule approaches - Techniques like FRET and optical tweezers can reveal dynamic properties of individual Gag molecules during assembly.

Integrating these diverse approaches will provide a more complete understanding of HIV-2 Gag structure, dynamics, and function in viral replication.