HIV-2 gp-36 397aa

What is HIV-2 gp-36 and what is its role in the viral life cycle?

HIV-2 gp-36 is a transmembrane protein located in the envelope of the HIV-2 virus that plays a crucial role in the viral life cycle . It is the HIV-2 counterpart to the HIV-1 gp41 protein, though they differ significantly in amino acid sequences .

Structurally, HIV-2 gp-36 forms trimers with the gp105 glycoproteins on the virion's membrane . Its primary function is mediating fusion between the viral envelope and the host cell membrane, which is essential for viral entry into target cells . The protein specifically binds to cellular receptor proteins, with studies identifying putative proteins P45 and P62 as important binding partners .

From a methodological perspective, researchers studying gp-36 function typically employ recombinant protein expression systems, such as E. coli-based systems, to produce the protein for structural and functional analyses . The full-length protein contains approximately 397 amino acids, with a complex structure that includes several functional domains critical for the fusion process .

How does HIV-2 gp-36 differ structurally and functionally from HIV-1 gp41?

From a methodological standpoint, researchers investigating these differences typically employ sequence alignment tools such as MEGAv6.0 followed by specialized bioinformatics programs (like EpiMolBio) to identify conserved amino acids and variant-specific markers . These analyses provide critical insights into structural elements that may influence immune recognition, viral fusion kinetics, and potential drug targets specific to each viral type.

What are the key conserved domains in HIV-2 gp-36?

Conservation analysis of HIV-2 gp-36 has identified several highly conserved domains that are critical for viral function . A comprehensive study of 275 gp-36 sequences from three HIV-2 variants found that 59.7% of amino acids are highly conserved (present in ≥90% of sequences), with 12.6% showing complete conservation across all sequences analyzed .

The most conserved regions in gp-36 (similar to those in HIV-1 gp41) include:

• The fusion peptide region

• The fusion peptide proximal region

• The N-heptad repeat domain

• The membrane-proximal external region (MPER)

These domains show ≥84% conservation of amino acids in the consensus sequence . This high level of conservation reflects their critical functional roles in the viral fusion process. The fusion peptide initiates membrane fusion, while the heptad repeat regions form a six-helix bundle structure that brings the viral and cellular membranes into close proximity .

From a research methodology perspective, scientists identify these conserved domains using bioinformatics approaches that analyze large sequence datasets. The most common approach involves downloading sequences from databases like LANL, aligning them using software like MEGAv6.0, and then analyzing conservation patterns using specialized programs that can identify variant-specific markers and conservation levels across structural domains .

What methods are available for detecting HIV-2 gp-36 in laboratory settings?

Several methods are available for detecting and analyzing HIV-2 gp-36 in laboratory settings, each with specific applications in research:

• Western Blotting (WB): Antibodies such as BDI411 (mouse monoclonal IgG2a) can detect HIV-2 gp-36 through western blotting, allowing for protein size verification and relative quantification . This method is particularly useful for confirming protein expression and analyzing protein modifications.

• Immunoprecipitation (IP): Available antibodies like BDI411 can isolate HIV-2 gp-36 from complex protein mixtures through immunoprecipitation, enabling subsequent analysis of protein interactions and modifications .

• ELISA-based methods: Recombinant HIV-2 gp-36 proteins have been demonstrated to be reactive with human HIV-positive serum, indicating their utility in developing ELISA-based detection systems .

• Recombinant protein systems: Expressing HIV-2 gp-36 397aa in systems like E. coli provides research-grade material for various detection and characterization studies . These recombinant proteins are typically produced with greater than 95% purity as determined by HPLC analysis and SDS-PAGE .

For optimal results when working with HIV-2 gp-36, researchers should consider that the protein, while stable at 4°C for up to one week, should generally be stored below -18°C with freeze-thaw cycles minimized . Additionally, when using recombinant proteins in cell culture applications, testing for endotoxin levels is recommended prior to use .

What are the structural characteristics of recombinant HIV-2 gp-36 397aa protein?

Recombinant HIV-2 gp-36 397aa protein is a laboratory-produced version of the viral transmembrane glycoprotein with specific structural characteristics important for research applications . The recombinant protein is typically constructed by creating a cDNA sequence encoding the 397 amino acid sequence of HIV-2 gp-36, which is then expressed in an appropriate host system such as E. coli .

The amino acid sequence of the recombinant protein (as detailed in source ) is:
EQTMVQDDPSTCRGEFLYCNMTWFLNWIENKTHRNYAPCHIKQIINTWHKVGRNVYLPPREGELSCNSTVTSIIANIDWQNNNQTNITFSAEVAELYRLELGDYKLVEITPIGFAPTKEKRYSSAHGRHTRGVFVLGFLGFLATAGSAMGAASLTVSAQSRTLLAGIVQQQQQLLDVVKRQQELLRLTVWGTKNLQARVTAIEKYLQDQARLNSWGCAFRQVCHTTVPWVNDSLAPDWDNMTWQEWEKQVRYLEANISKSLEQAQIQQEKNMYELQKLNSWDIFGNWFDLTSWVKYIQYGVLIIVAVIALRIVIYVVQMLSRLRKGYRPVFSSPPGYIQQIHIHKDRGQPANEETEEDGGSNGGDRYWPWPIAYIHFLIRQLIRLLTRLYSICSQAC

In laboratory settings, recombinant HIV-2 gp-36 appears as a sterile filtered clear solution, typically stored in buffer containing 0.01M Na2CO3, 0.01M Na3EDTA, 0.014 M β-mercaptoethanol, and 0.05% Tween-20 . For structural and functional studies, this recombinant protein needs to maintain proper folding to accurately represent the native viral protein. The purity of commercially available recombinant HIV-2 gp-36 397aa is generally greater than 95% as determined by HPLC analysis and SDS-PAGE, making it suitable for detailed structural investigations .

How do sequence variations in HIV-2 gp-36 impact neutralizing antibody efficacy?

Sequence variations in HIV-2 gp-36 have significant implications for neutralizing antibody efficacy, representing a critical consideration for vaccine design and immunotherapy development . Research has demonstrated that HIV-2 gp-36, like HIV-1 gp41, exhibits considerable variability while maintaining certain highly conserved domains that are essential for viral function .

A comprehensive analysis of 275 gp-36 sequences from three HIV-2 variants revealed that 59.7% of amino acids are highly conserved (present in ≥90% of sequences) . This conservation pattern is non-uniform across the protein structure, with specific domains showing higher conservation than others. The fusion peptide, its proximal region, the N-heptad repeat, and the membrane-proximal external region (MPER) are the most conserved domains, with ≥84% of amino acids maintained in the consensus sequence .

From a methodological perspective, researchers investigating antibody neutralization against HIV-2 gp-36 variants typically employ a combination of:

• Sequence analysis to identify conserved epitopes

• Recombinant protein expression of variant forms

• Antibody binding assays to measure affinity against different variants

• Neutralization assays to assess functional impact of sequence variations

Understanding these sequence-function relationships is crucial for designing immunogens that can elicit broadly neutralizing responses against diverse HIV-2 strains .

What are the challenges in expressing and purifying functional recombinant HIV-2 gp-36 for structural studies?

Expressing and purifying functional recombinant HIV-2 gp-36 presents several technical challenges that researchers must address to obtain high-quality material for structural and functional studies:

• Expression System Selection: While E. coli is commonly used for expressing HIV-2 gp-36 397aa , this prokaryotic system lacks the ability to perform post-translational modifications, particularly glycosylation, which may affect protein folding and function. Researchers must carefully evaluate whether native glycosylation patterns are critical for their specific studies and choose expression systems accordingly.

• Protein Solubility: As a transmembrane protein, HIV-2 gp-36 contains hydrophobic regions that can cause aggregation and inclusion body formation during recombinant expression . Researchers often need to optimize buffer conditions (like the 0.01M Na2CO3, 0.01M Na3EDTA, 0.014 M β-mercaptoethanol, and 0.05% Tween-20 used in some preparations) to maintain solubility .

• Protein Stability: HIV-2 gp-36 has limited stability at higher temperatures, requiring storage below -18°C and minimization of freeze-thaw cycles . This instability complicates purification procedures and subsequent structural studies.

• Functional Verification: Ensuring that recombinant gp-36 retains native-like functionality is essential. Reactivity with human HIV-positive serum is one verification method used , but additional functional assays may be needed to confirm proper folding and activity.

• Endotoxin Contamination: When expressing in bacterial systems like E. coli, endotoxin contamination must be addressed, particularly for cell-based assays . This requires additional purification steps that may affect protein yield and activity.

Methodologically, researchers typically use affinity chromatography for initial purification, followed by size exclusion and/or ion exchange chromatography to achieve high purity (>95% as verified by HPLC and SDS-PAGE) . The balance between maintaining native protein conformation and achieving high purity remains a significant challenge.

How can researchers effectively study gp-36 interactions with cellular receptors?

Studying HIV-2 gp-36 interactions with cellular receptors requires sophisticated methodological approaches to capture these complex molecular events. HIV-2 gp-36 has been shown to bind to putative cellular receptor proteins P45 and P62, which facilitate viral entry into host cells . Additionally, like HIV-1 gp41, gp-36 can bind to human lymphocytes and monocytes . Effectively studying these interactions involves several complementary approaches:

• Protein-Protein Interaction Assays:

  • Co-immunoprecipitation (Co-IP) using specific antibodies like BDI411 can pull down gp-36 along with its binding partners from cell lysates

  • Surface plasmon resonance (SPR) assays with purified recombinant gp-36 and receptor candidates to determine binding kinetics and affinity

  • ELISA-based binding assays to screen for interactions and assess binding strength

• Cellular Binding Studies:

  • Flow cytometry with fluorescently labeled gp-36 to quantify binding to different cell types

  • Confocal microscopy to visualize the localization of gp-36-receptor interactions

  • Cell-cell fusion assays to assess the functional consequences of gp-36-receptor interactions

• Molecular Mapping of Interaction Domains:

  • Mutagenesis studies targeting specific amino acids in gp-36 to identify critical residues for receptor binding

  • Peptide competition assays using synthetic fragments of gp-36 to map binding domains

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions of gp-36 that change conformation upon receptor binding

• Structural Analysis of Complexes:

  • Crystallography or cryo-electron microscopy of gp-36-receptor complexes to determine atomic-level interaction details

  • Computational modeling based on the amino acid sequence of gp-36 (as provided in source ) combined with molecular dynamics simulations

When conducting these studies, researchers should be mindful that recombinant HIV-2 gp-36 may require specific buffer conditions for optimal stability, such as those containing 0.01M Na2CO3, 0.01M Na3EDTA, 0.014 M β-mercaptoethanol, and 0.05% Tween-20 . Additionally, protein storage conditions (below -18°C with minimal freeze-thaw cycles) significantly impact experimental reproducibility .

What conservation patterns in HIV-2 gp-36 might inform targeted drug development?

Conservation analysis of HIV-2 gp-36 reveals specific patterns that provide valuable insights for targeted drug development strategies . Comprehensive examination of 275 gp-36 sequences from three HIV-2 variants has identified that 59.7% of amino acids are highly conserved (present in ≥90% of sequences), with 12.6% showing complete conservation across all analyzed sequences . These conservation patterns have significant implications for drug development:

• Highly Conserved Functional Domains:

  • The fusion peptide, its proximal region, the N-heptad repeat (NHR), and the membrane-proximal external region (MPER) exhibit ≥84% conservation in the consensus sequence

  • These regions represent prime targets for antiviral agents, as mutations in these domains are likely to compromise viral fitness

  • Drugs targeting these conserved regions, similar to the HIV-1 fusion inhibitor enfuvirtide (T-20), are less likely to encounter resistance issues

• Resistance Considerations:

  • Notably, no natural major resistance mutations to T-20 (the HIV-1 fusion inhibitor) were observed in the analyzed HIV-2 gp-36 sequences

  • This suggests that fusion inhibitors designed based on HIV-1 gp41 might have cross-reactivity against HIV-2, though with potential differences in potency

• Variant-Specific Markers (V-markers):

  • The identification of V-markers (amino acid changes specific to certain variants) can guide the development of variant-specific inhibitors when broad coverage is not achieved

  • These variant-specific approaches may be particularly relevant for addressing regional variations in HIV-2 prevalence

From a methodological perspective, researchers investigating gp-36 for drug development typically combine bioinformatics approaches (sequence conservation analysis) with structural biology techniques and functional assays. The conservation data can be mapped onto structural models to identify druggable pockets in highly conserved regions. Subsequent validation typically involves in vitro binding and antiviral assays using recombinant gp-36 proteins .

The complete amino acid sequence of HIV-2 gp-36 397aa, as provided in source , serves as a reference point for these analyses, with particular attention to the functional domains identified through conservation studies.

How do the structural domains of HIV-2 gp-36 influence immune recognition?

The structural domains of HIV-2 gp-36 play crucial roles in determining immune recognition patterns, with significant implications for vaccine design and immunotherapeutic approaches . Research has revealed that gp-36 contains distinct domains with varying levels of conservation and accessibility, each contributing differently to immune system interactions:

From a methodological perspective, researchers investigating immune recognition of HIV-2 gp-36 employ techniques such as:

• Epitope mapping using overlapping peptide arrays derived from the complete gp-36 sequence

• Structural analysis of antibody-peptide complexes via X-ray crystallography or cryo-electron microscopy

• Neutralization assays with monoclonal antibodies targeting specific gp-36 domains

• T-cell proliferation and cytokine production assays using synthetic gp-36 peptides

Understanding these structure-immunogenicity relationships is essential for designing immunogens that present critical gp-36 epitopes in conformations that elicit broadly neutralizing responses against HIV-2.

What are the optimal conditions for working with recombinant HIV-2 gp-36 397aa in laboratory settings?

Working with recombinant HIV-2 gp-36 397aa requires careful attention to storage, handling, and experimental conditions to maintain protein stability and functionality . Based on the available data, the following methodological guidelines should be considered:

• Storage Conditions:

  • Store recombinant HIV-2 gp-36 below -18°C for long-term stability

  • While the protein is stable at 4°C for up to one week, prolonged storage at this temperature is not recommended

  • Avoid repeated freeze-thaw cycles as they can significantly degrade protein quality

• Buffer Composition:

  • The recombinant protein is typically maintained in a buffer containing 0.01M Na2CO3, 0.01M Na3EDTA, 0.014 M β-mercaptoethanol, and 0.05% Tween-20

  • This specific buffer formulation helps maintain protein solubility and stability

  • Any deviation from this buffer composition for experimental purposes should be carefully validated

• Quality Control Measurements:

  • Verify protein purity using methods such as HPLC analysis and SDS-PAGE, with quality recombinant preparations typically showing >95% purity

  • Confirm functionality by testing reactivity with human HIV-positive serum before use in critical experiments

  • For cell culture applications, endotoxin testing is essential, particularly for proteins expressed in bacterial systems like E. coli

• Experimental Considerations:

  • When designing binding studies or immunoassays, consider the protein's natural oligomeric state (trimeric in the viral context)

  • Temperature sensitivity should be factored into experimental design, with reactions preferably conducted at controlled temperatures below 37°C to minimize degradation

  • When coating surfaces for immunoassays, optimize protein concentration and binding conditions empirically for each application

These methodological considerations are crucial for ensuring reproducible results when working with recombinant HIV-2 gp-36 397aa. The protein's relatively complex structure and stability requirements necessitate careful handling to maintain native-like properties for meaningful experimental outcomes.

What analytical techniques are most effective for studying HIV-2 gp-36 structure-function relationships?

Investigating HIV-2 gp-36 structure-function relationships requires a multi-faceted analytical approach that combines structural, biochemical, and functional methodologies. Based on the available literature, the following analytical techniques have proven most effective:

• Structural Analysis Techniques:

  • X-ray Crystallography: While challenging due to the membrane protein nature of gp-36, this technique provides atomic-level resolution of protein structure when successful

  • Cryo-Electron Microscopy (Cryo-EM): Particularly useful for visualizing gp-36 in complex with antibodies or as part of the viral envelope trimer

  • Nuclear Magnetic Resonance (NMR) Spectroscopy: Effective for analyzing the structure of specific domains or peptides derived from gp-36

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Valuable for identifying flexible regions and conformational changes in gp-36 upon binding to partners

• Functional and Binding Assays:

  • Surface Plasmon Resonance (SPR): Provides real-time binding kinetics between gp-36 and potential interaction partners like cellular receptors or antibodies

  • Cell-Cell Fusion Assays: Measures the functional capacity of gp-36 to mediate membrane fusion, its primary biological role

  • Biolayer Interferometry: Offers an alternative to SPR for measuring binding interactions

  • Flow Cytometry: Useful for assessing gp-36 binding to cellular receptors in a native membrane context

• Sequence-Structure-Function Correlation Methods:

  • Alanine Scanning Mutagenesis: Systematically replacing amino acids with alanine to identify functionally critical residues

  • Conservation Analysis: Using bioinformatics tools like those described in source (e.g., MEGAv6.0, EpiMolBio) to correlate sequence conservation with functional domains

  • Molecular Dynamics Simulations: Computational approach to model gp-36 dynamics and predict the effects of mutations or drug binding

• Antibody Epitope Mapping:

  • Peptide Arrays: Systematic analysis of antibody binding to overlapping peptides covering the entire gp-36 sequence

  • Competitive ELISA: Determining if antibodies compete for binding, suggesting overlapping epitopes

  • Escape Mutant Analysis: Identifying mutations that emerge under antibody selection pressure

When implementing these techniques, researchers should consider the complete amino acid sequence of HIV-2 gp-36 397aa as provided in source and the specific buffer requirements for maintaining protein stability (0.01M Na2CO3, 0.01M Na3EDTA, 0.014 M β-mercaptoethanol, and 0.05% Tween-20) .

What are the critical considerations when designing experiments to compare HIV-1 gp41 and HIV-2 gp-36?

Designing experiments to compare HIV-1 gp41 and HIV-2 gp-36 requires careful consideration of several critical factors to ensure valid and informative comparisons. Based on the available literature, researchers should address the following methodological considerations:

• Sequence and Structural Homology Assessment:

  • Begin with comprehensive sequence alignment of gp41 and gp-36 using bioinformatics tools like MEGAv6.0

  • Identify corresponding domains between the two proteins despite their sequence differences

  • Map conserved regions and variant-specific markers (V-markers) as described in the comprehensive analysis cited in source

  • Create domain-specific constructs rather than full-length proteins for more focused comparisons

• Protein Expression and Purification Standardization:

  • Use identical expression systems for both proteins to eliminate system-specific variables

  • While E. coli is commonly used for expressing HIV-2 gp-36 , consider whether the lack of post-translational modifications affects functional comparisons

  • Employ identical purification strategies and quality control measures (>95% purity by HPLC and SDS-PAGE)

  • Verify proper folding through circular dichroism or other structural assessment techniques before functional comparisons

• Functional Assay Considerations:

  • Design cell fusion assays that can accommodate potential differences in fusion kinetics between gp41 and gp-36

  • For receptor binding studies, consider that while both proteins facilitate entry through CD4, they may interact differently with co-receptors or additional cellular factors

  • When testing antibody cross-reactivity, use a panel of antibodies targeting different epitopes, as cross-reactivity may be domain-specific

  • Include appropriate positive and negative controls specific to each protein

• Data Normalization and Analysis:

  • Develop normalization strategies that account for potential differences in baseline activity

  • Consider protein-specific factors that might influence experimental readouts

  • When comparing conservation patterns, use statistical methods that account for different dataset sizes (16,803 gp41 sequences vs. 275 gp-36 sequences in the cited study)

• Reagent Availability Considerations:

  • Note that commercially available reagents are more limited for HIV-2 gp-36 than for HIV-1 gp41

  • For antibody-based studies, options like the mouse monoclonal IgG2a antibody BDI411 are available for HIV-2 gp-36 detection

  • Recombinant HIV-2 gp-36 397aa is available commercially with specific storage and handling requirements

By addressing these methodological considerations, researchers can design robust comparative experiments that account for the intrinsic differences between HIV-1 gp41 and HIV-2 gp-36 while enabling meaningful functional and structural comparisons.

What are the key unanswered questions regarding HIV-2 gp-36 structure and function?

Despite significant advances in our understanding of HIV-2 gp-36, several critical questions remain unanswered, representing important areas for future research:

• Structural Dynamics During Fusion:

  • While the general function of gp-36 in membrane fusion is established, the precise conformational changes and molecular mechanisms during the fusion process remain incompletely characterized

  • How do these dynamics compare to the better-studied HIV-1 gp41? Are there HIV-2-specific features that might explain differences in pathogenicity?

  • What are the intermediate conformational states during the fusion process, and can these be targeted for therapeutic intervention?

• Receptor Interaction Specificity:

  • The specific interactions between gp-36 and the putative cellular receptor proteins P45 and P62 require further characterization

  • What are the binding kinetics and structural bases for these interactions?

  • How do these receptor interactions differ from those of HIV-1 gp41, and what implications does this have for viral tropism and pathogenesis?

• Immune Evasion Mechanisms:

  • How does HIV-2 gp-36 contribute to the observed lower pathogenicity and longer incubation period of HIV-2 compared to HIV-1?

  • What specific features of gp-36 influence immune recognition and potential escape from neutralizing antibodies?

  • How do the conservation patterns identified in previous research relate to immune selection pressure?

• Cross-Reactivity of Neutralizing Antibodies:

  • To what extent do broadly neutralizing antibodies against HIV-1 gp41 cross-react with HIV-2 gp-36?

  • Can the conserved regions in gp-36 (particularly the MPER, fusion peptide, and N-heptad repeat regions) serve as targets for broad-spectrum neutralizing antibodies?

  • What are the structural bases for potential cross-reactivity or lack thereof?

• Therapeutic Target Potential:

  • While no natural major resistance mutations to the HIV-1 fusion inhibitor enfuvirtide (T-20) were observed in HIV-2 gp-36 sequences , what is the actual susceptibility of HIV-2 to this and similar fusion inhibitors?

  • Can HIV-2 gp-36-specific inhibitors be developed that exploit unique structural features of this protein?

  • What is the potential for dual-target inhibitors that could address both HIV-1 and HIV-2 through conserved features of gp41 and gp-36?

Addressing these questions will require integrated approaches combining structural biology, molecular virology, immunology, and computational modeling. The complete amino acid sequence of HIV-2 gp-36 397aa provided in source serves as a valuable reference point for designing such studies.

How might advances in structural biology techniques enhance our understanding of HIV-2 gp-36?

Recent and emerging advances in structural biology techniques offer promising avenues to address longstanding challenges in HIV-2 gp-36 research, potentially transforming our understanding of this critical viral protein:

To maximize the impact of these advanced techniques, researchers should consider the specific characteristics of recombinant HIV-2 gp-36 397aa, including its buffer requirements (0.01M Na2CO3, 0.01M Na3EDTA, 0.014 M β-mercaptoethanol, and 0.05% Tween-20) and storage conditions (below -18°C with minimal freeze-thaw cycles) . Additionally, comparative studies with HIV-1 gp41 could leverage the conservation analysis methodologies described in source to focus structural investigations on functionally critical regions.