HIV-2 gp160

What is HIV-2 gp160 and how does it differ structurally and functionally from HIV-1 gp160?

HIV-2 gp160 is an envelope glycoprotein precursor that is cleaved into gp105 (surface) and gp36 (transmembrane) components, analogous to HIV-1's gp120 and gp41. Despite sharing only approximately 40% sequence identity with HIV-1 gp160, both proteins perform similar functions in viral entry - binding to CD4 receptors and co-receptors (CCR5 or CXCR4) to facilitate membrane fusion .

The key structural differences include:

• HIV-2 gp140 demonstrates greater stability than HIV-1 gp160, making it potentially more suitable for structural studies

• Both envelope proteins form similar 6-helix bundles from N-terminal and C-terminal regions of the ectodomain, a structural feature common to many viral fusion proteins

• HIV-2 gp160 shows evidence of CD4-independence in some contexts, unlike HIV-1

• HIV-2 subtype A sequence contains specific regions covering C4, V5, and C5 from HIV-2 gp120 and extends to HIV-2 gp36

Functionally, HIV-2 gp160 is associated with reduced pathogenicity compared to HIV-1 gp160, resulting in slower progression to immunodeficiency and lower infectious potential during early stages of infection .

How does HIV-2 gp160 contribute to the distinct pathogenicity and transmission dynamics of HIV-2?

HIV-2 gp160 plays a central role in the notably different infection profile of HIV-2 compared to HIV-1. Though transmitted through identical routes (exposure to bodily fluids including blood, semen, tears, and vaginal fluids), HIV-2 demonstrates distinct characteristics in disease progression .

The envelope glycoprotein contributes to these differences in several ways:

• HIV-2 shows reduced pathogenicity relative to HIV-1, with immunodeficiency developing more slowly

• HIV-2 demonstrates enhanced immune control of infection, possibly related to envelope characteristics

• HIV-2 is less infectious in early stages of viral infection, though infectiousness increases as the virus progresses

• HIV-2 exhibits a degree of CD4-independence not typically seen with HIV-1, potentially influencing transmission dynamics and cellular tropism

These characteristics have important epidemiological implications, explaining in part why HIV-2 has remained largely confined to West Africa while HIV-1 has spread globally. The reduced early-stage infectiousness of HIV-2, to which gp160 properties contribute, creates a narrower window for transmission.

What experimental systems are available for studying HIV-2 gp160 binding to receptors?

Several experimental approaches have been developed to study HIV-2 gp160 interactions with cell surface receptors:

• ELISA-based binding assays: Partially purified HIV-2 gp120 can be assessed for human CD4-binding in ELISA-based assays. These systems allow quantification of binding affinities and can demonstrate that expressed proteins maintain native conformations and functional activity .

• Mammalian cell expression systems: HIV-2 env-gene constructs have been generated that allow expression of soluble gp120 (gp105 with truncated gp36) in mammalian cells, including Human Embryonic Kidney 293T cells for transient expression and Chinese Hamster Ovary (CHO K1) cells for stable expression lines .

• Antibody recognition panels: Expressed HIV-2 gp120 can be tested against panels of mapped anti-gp105 monoclonal antibodies to verify correct conformational folding and epitope presentation .

• Viral entry inhibition assays: Similar to approaches used with HIV-1, inhibition of viral entry can be assessed using soluble CD4 preparations or antibodies that target either the gp160 or CD4 molecules .

For successful experimental design, it's essential to consider that HIV-2 gp140 shows greater stability than HIV-1 gp160, which may affect experimental conditions including buffer composition, temperature ranges, and storage considerations .

What are the optimal methods for producing HIV-2 gp160 constructs for structural and functional studies?

Based on documented approaches, optimal methods for producing HIV-2 gp160 constructs involve:

• Construct design modifications: Several strategic modifications improve expression and stability:

  • Truncation at specific positions in the gp36 coding region upstream of the membrane anchor

  • Removal of the gp105/36 processing site to maintain the complex

  • Introduction of a trimer-stabilizing motif from Bacteriophage T4 fibritin (GYIPEAPRDGQAYVRKDGEVWLLSTFL) at the C-terminus of gp15

  • Addition of C-terminal hexa-His tags to facilitate purification

• Expression system selection: While E. coli systems can produce HIV-2 gp160 (42kDa), mammalian expression systems more reliably generate properly folded and glycosylated proteins:

  • Transient expression in Human Embryonic Kidney 293T cells for initial screening

  • Stable cell line development in Chinese Hamster Ovary Cells (CHO K1) for constitutive expression and secretion

• Purification strategy: Multi-step purification yields properly folded, oligomeric proteins:

  • Initial lectin (GNA)-affinity chromatography leveraging glycosylation

  • Followed by monoclonal antibody immunoaffinity chromatography (e.g., ARP 3085, NIBSC)

  • Final polishing via gel filtration (Superose 6)

• Quality control: Several approaches verify structural integrity:

  • Dynamic light scattering (ideal polydispersity ~23.3%)

  • Circular dichroism up to 85°C to assess thermal stability

  • Antibody recognition panels to verify conformational epitopes

  • Functional binding assays to confirm CD4 interaction

This approach has successfully generated HIV-2 gp160 proteins suitable for crystallization trials and functional studies.

How do polymorphisms in HIV-2 gp160 influence susceptibility to attachment inhibitors compared to HIV-1?

While direct data on HIV-2 gp160 polymorphisms affecting attachment inhibitor susceptibility is limited in the provided search results, we can extrapolate from HIV-1 data to understand potential parallel mechanisms.

For HIV-1, specific polymorphisms in gp160 are known to reduce susceptibility to attachment inhibitors like temsavir. These occur at discrete positions in gp160: S375H/I/M/N/T, M426L/P, M434I/K and M475I . Analysis of the Los Alamos National Laboratory (LANL) HIV Sequence Database revealed a generally low prevalence of these polymorphisms across most common HIV-1 subtypes .

For HIV-2 gp160, several considerations apply:

• Given the ~40% sequence identity between HIV-1 and HIV-2 envelope proteins, analogous resistance-conferring positions likely exist but may not map identically .

• HIV-2's different RNA packaging strategy (preferential binding to mRNA that creates the Gag protein) compared to HIV-1 (binds to any appropriate RNA) results in lower mutation rates, potentially leading to slower development of resistance-associated polymorphisms .

• The distinct structural features of HIV-2 gp160, including enhanced stability and partial CD4-independence, suggest that polymorphisms affecting inhibitor binding might occur at different frequencies or positions compared to HIV-1 .

Research gaps remain in systematically mapping HIV-2 gp160 polymorphisms affecting attachment inhibitor susceptibility, representing an important area for future investigation.

What are the implications of HIV-2 gp160's greater stability for vaccine design approaches?

The documented enhanced stability of HIV-2 gp140 compared to HIV-1 gp160 presents several promising implications for vaccine design strategies:

• Improved antigen preservation: Greater stability likely translates to better retention of native conformational epitopes during vaccine production, storage, and delivery, potentially eliciting more relevant neutralizing antibodies .

• Extended shelf-life potential: Enhanced thermal stability (as demonstrated by circular dichroism studies up to 85°C) suggests vaccines based on HIV-2 gp160 might maintain immunogenicity longer under field conditions .

• Structural studies advantage: The superior stability facilitates more successful structural characterization through methods like X-ray crystallography, providing detailed atomic models to guide rational immunogen design .

• Natural trimeric presentation: HIV-2 gp140's tendency to form stable trimers without extensive modification mimics the native viral spike configuration, potentially eliciting antibodies that better recognize authentic viral targets .

• Cross-reactivity possibilities: Despite sequence differences, structural similarities between HIV-1 and HIV-2 envelope proteins suggest that stabilized HIV-2 gp160-based immunogens might elicit antibodies with cross-reactive neutralizing potential .

These characteristics make HIV-2 gp160 not only valuable for HIV-2-specific vaccine approaches but also as a potential alternative platform for developing broader HIV immunogens.

How can recombinant HIV-2 gp160 be effectively used in diagnostic assay development?

Recombinant HIV-2 gp160 offers potential advantages for diagnostic assay development, particularly for detecting HIV-2 infections and for creating more specific HIV testing algorithms. Based on parallel experiences with HIV-1 gp160 in diagnostic contexts and the specific properties of HIV-2 gp160, the following approaches are indicated:

• Enzyme Immunoassay Development: Native or recombinant HIV-2 gp160 can serve as a specific capture antigen for detecting HIV-2 antibodies. In HIV-1, similar approaches using purified native gp160 demonstrated 100% specificity and sensitivity, with superior performance in detecting early seroconversion compared to virus lysate-based assays .

• Improved Specificity for HIV-2 Detection: Given the structural differences between HIV-1 and HIV-2 envelope proteins despite functional similarities, HIV-2 gp160-based assays could reduce cross-reactivity issues that sometimes occur with whole virus lysate assays .

• Applications in Research and Clinical Settings:

  • ELISA tests using purified HIV-2 gp160 as capture antigen

  • Lateral flow immunoassays for rapid point-of-care testing

  • Western blot confirmatory tests with greater specificity

• Production Considerations: Expression in E. coli systems yields HIV-2 gp160 (42kDa) suitable for lateral flow immunoassay and ELISA applications, though mammalian cell expression systems may provide better conformational epitope presentation for certain applications .

The enhanced stability of HIV-2 gp160 compared to HIV-1 gp160 suggests potential benefits for assay shelf-life and performance consistency across varied storage conditions .

What protein engineering strategies optimize HIV-2 gp160 expression and stability for structural studies?

Several protein engineering approaches have been documented to optimize HIV-2 gp160 for structural and functional studies:

These engineering approaches have successfully generated HIV-2 gp160 constructs that maintain proper folding, CD4 binding capability, and recognition by conformational antibodies while providing sufficient stability for structural studies .

What analytical methods are most effective for characterizing HIV-2 gp160 structural integrity and function?

A multi-faceted analytical approach is necessary to comprehensively characterize HIV-2 gp160:

• Biochemical Characterization:

  • SDS-PAGE (10% PAGE) with Coomassie staining for purity assessment (>90% purity achievable)

  • Western blot detection using anti-gp120 antibodies to confirm identity and integrity

  • Size exclusion chromatography (using Superose 6) to assess oligomeric state

• Biophysical Analyses:

  • Dynamic light scattering to evaluate sample homogeneity and oligomeric state (polydispersity ~23.3% indicating well-formed trimers)

  • Circular dichroism up to 85°C to assess thermal stability and secondary structure characteristics

  • Differential scanning calorimetry to determine precise melting temperatures and stability parameters

• Functional Assessments:

  • ELISA-based CD4 binding assays to confirm functional activity

  • Antibody binding panels using well-characterized monoclonal antibodies targeting different epitopes

  • Surface plasmon resonance to determine binding kinetics and affinities

• Structural Studies:

  • Negative-stain electron microscopy for initial structural assessment

  • X-ray crystallography trials to determine atomic-level structure

  • Hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

These complementary approaches provide a comprehensive profile of protein quality, confirming both structural integrity and functional activity before proceeding to more resource-intensive studies like crystallization trials or immunization experiments.

How can HIV-2 gp160 be effectively used in comparative studies with HIV-1 to advance broader HIV vaccine strategies?

HIV-2 gp160 offers unique advantages for comparative studies that could inform broader HIV vaccine development:

• Structural Comparison Approaches:

  • Despite sequence divergence, HIV-1 and HIV-2 envelope proteins share conserved structural elements, particularly in the CD4 binding site and fusion machinery

  • Parallel crystallization and structural analysis of both proteins can identify conserved epitopes potentially vulnerable to cross-neutralizing antibodies

  • Enhanced stability of HIV-2 gp140 versus HIV-1 gp160 may facilitate structural determination that has been challenging with HIV-1 alone

• Immunological Cross-Reactivity Assessment:

  • Testing sera from HIV-1 and HIV-2 infected individuals against both antigens to identify antibodies targeting conserved epitopes

  • Immunization studies comparing responses to homologous regions of both proteins to identify universally immunogenic elements

  • Evaluating the potential for a single immunogen to protect against both viruses

• Fusion Mechanism Studies:

  • Both HIV-1 and HIV-2 envelope proteins form similar 6-helix bundles from N-terminal and C-terminal regions of the ectodomain

  • Comparative analysis of fusion kinetics and inhibition could reveal conserved mechanisms and intervention points

• Chimeric Construct Approaches:

  • Engineering chimeric envelope proteins containing elements from both HIV-1 and HIV-2

  • Assessing whether HIV-2 gp160's enhanced stability can be transferred to HIV-1 constructs

  • Evaluating whether more stable chimeras better present conserved neutralization epitopes

Previous vaccine efforts, such as the ALVAC-HIV strategy tested in combination with gp120 or gp160 protein boosts, provide precedent for combining vectors and envelope proteins in prime-boost strategies, an approach that could be extended to include HIV-2 components .

What are the key knowledge gaps in HIV-2 gp160 research that require further investigation?

Despite advances in HIV-2 gp160 research, several critical knowledge gaps remain that warrant focused investigation:

• Structural Determination: While HIV-2 gp140 demonstrates enhanced stability compared to HIV-1 gp160, complete high-resolution structural data is still lacking. Determining the atomic structure would provide crucial insights for vaccine design and understanding neutralization mechanisms .

• Neutralization Epitope Mapping: Comprehensive mapping of neutralizing epitopes on HIV-2 gp160 and their comparison with HIV-1 counterparts would identify conserved vulnerabilities potentially targetable by broad-spectrum interventions .

• CD4-Independence Mechanisms: The documented "some degree of CD4-independence" of HIV-2 requires deeper mechanistic understanding, as this property impacts cell tropism, transmission dynamics, and potential escape from antibody neutralization .

• Polymorphism Impact Analysis: While polymorphisms affecting HIV-1 susceptibility to attachment inhibitors have been characterized, parallel comprehensive analysis for HIV-2 gp160 is needed to guide therapeutic development specific to HIV-2 infections .

• Cross-Protective Potential: Systematic evaluation of whether HIV-2 gp160-based immunogens can elicit antibodies neutralizing both HIV-2 and HIV-1 would advance universal vaccine approaches .

These research priorities would advance both fundamental understanding of HIV-2 biology and accelerate translational applications in diagnostics, therapeutics, and preventive strategies.

How might advanced computational approaches enhance HIV-2 gp160 research?

Advanced computational methodologies offer promising avenues to accelerate HIV-2 gp160 research across multiple domains:

• Structure Prediction and Molecular Dynamics:

  • Leveraging AlphaFold and RoseTTAFold to predict structural features of HIV-2 gp160 regions not yet crystallized

  • Molecular dynamics simulations to model conformational changes during receptor binding and fusion

  • Virtual screening to identify potential small molecule inhibitors targeting HIV-2 gp160

• Sequence-Structure-Function Relationships:

  • Deep learning approaches to identify correlations between sequence polymorphisms and functional outcomes

  • Computational epitope mapping to predict antibody binding sites

  • Analysis of coevolution patterns between envelope regions to identify functionally linked residues

• Immunogen Design:

  • Computational immunogen design to create HIV-2 gp160 variants with enhanced presentation of conserved epitopes

  • Optimization algorithms to improve stability while maintaining antigenic properties

  • In silico germline-targeting approaches to engage specific B-cell receptors

• Evolutionary Analysis:

  • Phylogenetic comparisons between HIV-1 and HIV-2 envelope sequences to identify convergent solutions

  • Analysis of selection pressures on HIV-2 gp160 in different geographical regions

  • Predictive modeling of potential future evolutionary trajectories and resistance development

These computational approaches, integrated with experimental validation, can significantly accelerate progress in understanding HIV-2 gp160 and developing interventions against HIV-2 infection.

What are the most promising research applications for HIV-2 gp160?

Based on the current state of knowledge, several research applications for HIV-2 gp160 show particular promise:

• Structural biology platform: The enhanced stability of HIV-2 gp140 compared to HIV-1 gp160 makes it an attractive target for high-resolution structural studies that have proven challenging with HIV-1 envelope proteins. Success here could provide templates for understanding both viruses .

• Comparative virology model: HIV-2's reduced pathogenicity despite using similar entry mechanisms offers a valuable comparative model for understanding HIV-1 pathogenesis. The envelope proteins play central roles in these differences .

• Diagnostic development: HIV-2 gp160-based assays could improve specific detection of HIV-2 infections, addressing limitations in current testing algorithms that sometimes miss or misclassify HIV-2 cases .

• Cross-protective vaccine exploration: Given structural similarities despite sequence divergence, HIV-2 gp160 could serve as an alternative immunogen potentially capable of eliciting antibodies with cross-neutralizing activity against conserved epitopes .

• Novel inhibitor discovery: The CD4-binding region and fusion machinery of HIV-2 gp160 represent targets for inhibitor development, potentially leading to therapeutics effective against both HIV types .

These applications leverage HIV-2 gp160's unique properties while addressing important gaps in HIV research and intervention development.

What methodological considerations are most critical for researchers beginning work with HIV-2 gp160?

Researchers initiating work with HIV-2 gp160 should consider several critical methodological factors:

• Expression system selection: While E. coli systems can produce HIV-2 gp160 (42kDa) useful for certain applications, mammalian expression systems (particularly CHO K1 stable cell lines) better preserve conformational epitopes critical for structural and immunological studies .

• Storage and stability: HIV-2 gp160 preparations demonstrate optimal stability when:

  • Stored at 4°C if used within 2-4 weeks

  • Stored frozen at -20°C for longer periods

  • Supplemented with carrier protein (0.1% HSA or BSA) for long-term storage

  • Protected from multiple freeze-thaw cycles

• Purification strategy: Multi-step purification yields best results:

  • Initial lectin (GNA)-affinity chromatography

  • Followed by monoclonal antibody immunoaffinity chromatography

  • Final polishing via gel filtration (Superose 6)

• Functional validation: Confirming biological activity through:

  • CD4-binding in ELISA-based assays

  • Recognition by conformational antibodies

  • Proper oligomerization (likely trimeric) confirmed by biophysical methods

• Collaborative approach: Given the specialized nature of HIV-2 research, new investigators benefit from collaborations with established HIV-2 research groups and accessing standardized reagents from repositories like the NIH AIDS Reagent Program.