HIV-1 gp41 Long, Biotin

What is the role of the long cytoplasmic tail of HIV-1 gp41 in viral replication?

The long cytoplasmic tail (CT) of HIV-1 gp41 plays a crucial cell type-dependent role in viral replication. Unlike most retroviruses, lentiviruses including HIV-1 have transmembrane envelope glycoproteins with unusually long cytoplasmic tails. Research has demonstrated that this long CT is essential for efficient Env incorporation into virions in the majority of T cell lines, peripheral blood mononuclear cells, and monocyte-derived macrophages .

These findings establish that the gp41 cytoplasmic tail is essential for efficient Env incorporation into virions in natural target cells for HIV-1 infection in vivo, making it a crucial determinant of viral fitness .

How does glycosylation affect the antigenicity of HIV-1 gp41 epitopes?

Glycosylation significantly impacts the antigenicity of HIV-1 gp41 epitopes, particularly affecting the binding of broadly neutralizing antibodies (BnAbs). Studies have shown that glycan shielding creates a major obstacle to antibody recognition and binding to critical gp41 epitopes .

When HIV-1 envelope proteins are partially deglycosylated under native, non-denaturing conditions, the binding of monoclonal antibodies (mAbs) such as 4E10 and 2F5 to gp41 is substantially enhanced . Most notably, while the reverted unmutated ancestor antibodies of 2F5 and 4E10 are either non-reactive or poorly reactive with fully glycosylated envelope proteins, they react well with partially deglycosylated gp140 HIV-1 envelope .

These findings suggest that rather than there being a fundamental lack of naïve B cell receptors capable of recognizing gp41 broadly neutralizing epitopes, glycan interference with unmutated antibody binding to gp41 envelope may be a key impediment to the induction of broadly neutralizing antibodies . This insight has significant implications for HIV-1 vaccine design strategies aimed at inducing protective antibody responses.

What is the fusion-intermediate state of HIV-1 gp41 and why is it important?

The fusion-intermediate state of HIV-1 gp41 represents a critical conformational arrangement that occurs during the process of viral entry into target cells. This state emerges following a cascade of events initiated by binding of gp120 to receptor (CD4) and coreceptor (CCR5 or CXCR4) .

When gp120 binds to these receptors, it undergoes conformational changes that lead to dissociation from gp41 and trigger refolding events in gp41. During these rearrangements, the N-terminal fusion peptide of gp41 translocates and inserts into the target-cell membrane. This extended conformation of the gp41 ectodomain, with its fusion peptide inserted into the target cell membrane while the transmembrane anchor remains in the viral membrane, is termed the "prehairpin intermediate" .

This intermediate state is of particular importance because:

• It represents a transient but critical vulnerable phase in the viral entry process

• It is the target of various fusion inhibitors, including T-20/enfuvirtide, the first approved fusion-inhibiting antiviral drug

• Rare broadly neutralizing antibodies can recognize this intermediate state

• The characteristics of this intermediate have informed the development of peptide-based entry inhibitors and vaccine immunogens

Understanding the structural and temporal properties of this fusion-intermediate state provides crucial insights for developing new therapeutic strategies to prevent HIV-1 infection.

What methodologies are effective for studying HIV-1 gp41 protein interactions?

Several sophisticated methodologies have proven effective for studying HIV-1 gp41 protein interactions, with biotin-based approaches being particularly valuable. One powerful technique is biotin ligase tagging, which enables the analysis of protein-protein interactions in virus-producing cells .

This approach involves creating fusion proteins with a biotin ligase (BirA*), which can biotinylate proximal proteins. In research examining HIV-1 Gag protein interactions, MA-BirA* fusion proteins were used to detect proteins in close proximity during trafficking and virus assembly . The detection of biotinylated wild-type precursor Gag proteins in trans demonstrated that MA-BirA* and wild-type HIV-1 Gag proteins were in close proximity during trafficking and/or release .

A similar strategy can be applied to study gp41 interactions by:

• Creating gp41-BirA* fusion constructs

• Expressing these constructs in relevant cell types

• Identifying biotinylated proteins using streptavidin-based detection methods

• Confirming interactions through complementary techniques such as co-immunoprecipitation

For structural studies, techniques such as X-ray crystallography and cryo-electron microscopy have been instrumental in revealing the conformational states of gp41, particularly in complex with broadly neutralizing antibodies . Additionally, surface plasmon resonance and ELISA-based binding assays using purified, biotin-conjugated gp41 proteins provide quantitative measurements of protein-protein interactions .

How can the gp41 C-peptide region be utilized for targeted HIV-1 therapeutics?

The gp41 C-peptide region presents a promising target for HIV-1 therapeutics due to its accessibility on the surface of infected cells. Research has demonstrated that this region is exposed in sufficient quantities on HIV-1-infected cells to allow effective targeting of therapeutic agents .

A particularly promising approach involves the use of a chimeric 5-Helix/Pseudomonas exotoxin protein that recognizes cells expressing Env from a broad spectrum of HIV-1 strains, including primary isolates from clades B, D, E, G, and H . This recombinant toxin demonstrates remarkable specificity, selectively killing HIV-1-infected cells and blocking spreading infection while maintaining potent inhibitory activity against membrane fusion .

The efficacy of this approach is supported by several key findings:

• The C-terminal region of the gp41 ectodomain is accessible on HIV-1-infected cells in a receptor-independent fashion

• The targeting is effective against a genetically diverse range of HIV-1 strains

• The specificity of the targeting minimizes potential off-target effects on uninfected cells

This research establishes the C-terminal region of the gp41 ectodomain as an accessible target on HIV-1-infected cells for the development of both antiviral therapeutics and neutralizing antibodies . Such targeted approaches represent a potentially valuable addition to current therapeutic strategies that primarily target viral enzymes.

What factors influence the binding of broadly neutralizing antibodies to HIV-1 gp41?

Multiple factors influence the binding of broadly neutralizing antibodies (BnAbs) to HIV-1 gp41, creating a complex landscape for antibody recognition. These factors include:

• Glycosylation Status: Envelope glycosylation significantly impacts antibody binding. Partial deglycosylation of HIV-1 envelope proteins enhances the antigenicity of gp41 epitopes for both BnAbs and their unmutated ancestor antibodies . When JRFL gp140 and group M consensus gp140 (CON-S) Env proteins are partially deglycosylated under native conditions, binding of mAbs 4E10 and 2F5 is substantially improved .

• Conformational State: The conformational state of gp41 is critical for antibody recognition. Certain epitopes are only exposed during specific stages of the fusion process, such as in the prehairpin intermediate state . The accessibility of cluster I and II epitopes differs between oligomeric and monomeric forms of gp140, and can be affected by whether the protein is cleaved or uncleaved .

• Antibody Maturation: The extensive somatic hypermutation required for effective BnAb binding presents another barrier. Unmutated ancestor antibodies of 2F5 and 4E10 show poor reactivity with glycosylated Env but react well with deglycosylated forms, suggesting that glycan interference may impede the initial activation of B cells with appropriate receptors .

• Envelope Heterogeneity: The genetic diversity of HIV-1, particularly in the envelope region, creates variations in gp41 epitopes across different viral strains and clades .

Understanding these factors is crucial for designing immunogens that can effectively elicit broadly neutralizing antibodies against gp41 as part of HIV-1 vaccine development strategies.

How does biotin interference affect HIV-1 diagnostic assays, particularly those targeting gp41?

Biotin interference represents a significant challenge for HIV-1 diagnostic assays, especially those that utilize biotin-streptavidin interactions in their detection systems. This interference is particularly problematic for the detection of anti-HIV-1 antibodies, including those targeting gp41 .

Studies have demonstrated that biotin interference in the detection of anti-HIV-1 antibody in seroconversion panel members is due to the low concentration of gp41 antibodies in early infection . Specifically, research has shown that HIV-1 p24 antigen was not detected at 30 pg/mL when biotin was present at 200 ng/mL concentration .

This interference is clinically relevant because biotin supplementation is relatively common. An outpatient survey revealed that 7.7% of individuals attending a clinic used biotin supplementation . Common biotin supplement doses (5-10 mg) can result in serum biotin concentrations well above the levels known to interfere with assays.

The mechanism of interference depends on the assay format:

Researchers and clinicians should consider biotin supplements as potential sources of falsely increased or decreased test results, especially in cases where supplementation cannot be ruled out . Diagnostic laboratories should implement strategies to mitigate biotin interference, such as using alternative detection systems or incorporating steps to remove or neutralize biotin in samples.

What are the optimal conditions for working with recombinant HIV-1 gp41 proteins conjugated with biotin?

Working with recombinant HIV-1 gp41 proteins conjugated with biotin requires specific handling, storage, and experimental conditions to maintain protein integrity and functionality. Based on available data, the following parameters represent optimal conditions:

For experimental applications, biotin-conjugated gp41 proteins are suitable for various biochemical assays, including SDS-PAGE and immunoblotting . These proteins typically show specific reactivity with human HIV-positive serum and can be used in assays to detect anti-HIV antibodies or to study gp41 interactions with potential binding partners .

When designing experiments, researchers should consider that biotin conjugation may affect protein folding or epitope exposure. Additionally, the presence of tags (such as N-terminal β-galactosidase) may influence protein behavior and should be accounted for in experimental design and interpretation .

How can researchers effectively deglycosylate HIV-1 gp41 while maintaining native conformational epitopes?

Effective deglycosylation of HIV-1 gp41 while preserving native conformational epitopes requires a carefully controlled approach that removes glycans without denaturing the protein structure. Based on successful methodologies, the following protocol elements are critical:

• Enzymatic Deglycosylation: Use of specific glycosidases that cleave N-linked glycans (such as PNGase F) under non-denaturing conditions. This approach has been successfully applied to JRFL gp140 and group M consensus gp140 (CON-S) Env proteins .

• Partial vs. Complete Deglycosylation: Partial deglycosylation often preserves conformational epitopes better than complete deglycosylation. The degree of deglycosylation can be controlled by adjusting enzyme concentration and reaction time .

• Native Conditions: Maintaining non-denaturing buffer conditions (neutral pH, physiological salt concentrations, absence of strong detergents) throughout the deglycosylation process is essential for preserving conformational epitopes .

• Confirmation of Structural Integrity: Following deglycosylation, the preservation of conformational epitopes should be confirmed using conformation-dependent antibodies that recognize native structures.

• Functional Validation: Assess the functionality of deglycosylated proteins through binding assays with receptor molecules or conformation-specific antibodies.

The efficacy of this approach is demonstrated by studies showing that partially deglycosylated gp140 proteins exhibit enhanced binding to broadly neutralizing antibodies like 4E10 and 2F5, as well as their unmutated ancestor antibodies, compared to their fully glycosylated counterparts . This indicates that the deglycosylation process successfully removes glycan shields while maintaining the essential conformational epitopes recognized by these antibodies.

What techniques can be used to study the conformational changes in gp41 during the fusion process?

Studying the dynamic conformational changes in gp41 during the fusion process requires specialized techniques that can capture transient intermediates and structural rearrangements. Several complementary approaches have proven effective:

• Fusion Inhibitor-Based Trapping: Peptide inhibitors like T-20/enfuvirtide can trap gp41 in specific conformational states, particularly the prehairpin intermediate. These trapped states can then be studied using various structural and biochemical methods .

• Conformation-Specific Antibodies: Antibodies that specifically recognize distinct conformational states of gp41 can be used to track the progression of structural changes during fusion. These include antibodies targeting the six-helix bundle conformation or the prehairpin intermediate .

• Site-Directed Spin Labeling and EPR Spectroscopy: This approach involves introducing spin labels at specific positions in gp41 and using electron paramagnetic resonance spectroscopy to monitor changes in distances between labeled sites during conformational transitions.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This technique can reveal regions of gp41 that undergo changes in solvent accessibility during conformational transitions, providing insights into structural rearrangements.

• Single-Molecule FRET (smFRET): By labeling different domains of gp41 with fluorescent dyes, Förster resonance energy transfer can be used to measure distances between domains and monitor conformational changes in real-time at the single-molecule level.

• Cryo-Electron Microscopy: This technique can capture different conformational states of gp41 in the context of the entire Env trimer, providing high-resolution structural information .

• Biotin Ligase Proximity Labeling: As demonstrated with HIV-1 Gag proteins, biotin ligase tagging can identify proteins that come into close proximity during conformational transitions .

These techniques have collectively contributed to our understanding of the fusion-intermediate state of gp41, characterized as a "prehairpin intermediate" where the N-terminal fusion peptide is inserted into the target cell membrane while the transmembrane anchor remains in the viral membrane .

How can biotin-labeled HIV-1 gp41 be utilized in proximity-based protein interaction studies?

Biotin-labeled HIV-1 gp41 offers powerful applications in proximity-based protein interaction studies, providing insights into gp41's interactions with viral and cellular proteins. Several key methodological approaches leverage this technology:

• BioID and TurboID Proximity Labeling: By fusing a promiscuous biotin ligase (BirA* variant) to gp41, researchers can identify proteins that come within ~10 nm of gp41 in living cells. When the fusion protein is expressed, the biotin ligase biotinylates nearby proteins, which can then be purified using streptavidin and identified by mass spectrometry .

• APEX2-based Proximity Labeling: Similar to BioID but using the engineered ascorbate peroxidase APEX2, this approach offers faster labeling kinetics, allowing for temporal resolution of dynamic interactions during processes like membrane fusion.

• Split-BioID Systems: This approach involves splitting the biotin ligase between gp41 and a potential interaction partner. Proximity-dependent biotinylation occurs only when the two proteins interact, allowing for validation of specific protein-protein interactions.

• Trans-biotinylation Assays: As demonstrated with MA-BirA* fusion proteins, when a biotin ligase fusion protein (e.g., gp41-BirA*) is co-expressed with wild-type HIV-1 proteins, biotinylation of the wild-type proteins in trans indicates close proximity during trafficking or assembly .

• Pull-down Assays with Biotinylated gp41: Purified biotin-labeled gp41 can be immobilized on streptavidin beads and used to pull down interacting proteins from cell lysates, followed by identification via mass spectrometry or western blotting.

These techniques have revealed that HIV-1 proteins can be in close proximity during trafficking and/or release, as evidenced by the biotinylation of wild-type HIV-1 Gag proteins by MA-BirA* fusion proteins . Similar approaches with gp41 could identify key interactions during envelope processing, trafficking, and viral assembly, potentially revealing new therapeutic targets.

What are the challenges in developing diagnostic assays that detect antibodies against HIV-1 gp41 in the presence of biotin?

Developing diagnostic assays that reliably detect antibodies against HIV-1 gp41 in the presence of biotin presents several significant challenges:

• Direct Interference with Detection Systems: Many HIV diagnostic assays use biotin-streptavidin interactions for signal amplification. Elevated levels of biotin in samples can compete with biotinylated reagents for binding to streptavidin, resulting in false-negative results .

• Variable Biotin Levels in Patient Samples: Biotin supplementation is relatively common (7.7% in one outpatient survey), but the dosage and resulting serum concentration can vary widely between individuals .

• Particularly Problematic for Early Infection Detection: Biotin interference in detection of anti-HIV-1 antibody in seroconversion panel members is particularly challenging due to the low concentration of gp41 antibodies in early infection .

• Threshold Effects: Research has shown that HIV-1 p24 was not detected at 30 pg/mL when biotin was present at 200 ng/mL concentration, suggesting a threshold effect where detection fails completely above certain biotin concentrations .

To address these challenges, several strategies can be implemented:

Researchers must carefully validate any gp41-based diagnostic assay using samples with varying concentrations of biotin to ensure reliable performance across the range of biotin levels likely to be encountered in clinical settings .

What experimental design considerations are important when studying the cell type-dependent requirements of the gp41 cytoplasmic tail?

Studying the cell type-dependent requirements of the gp41 cytoplasmic tail demands careful experimental design to accurately characterize its functions across different cellular contexts. Based on research findings, several critical considerations should guide experimental approaches:

• Cell Type Selection: Include a diverse panel of relevant cell types:

  • T cell lines with varying requirements for the gp41 CT (e.g., MT-4 cells show minimal dependence)

  • Primary cells that are natural targets for HIV-1 infection (PBMCs, monocyte-derived macrophages)

  • Non-lymphoid cells (e.g., HeLa) for comparative analyses

• Viral Constructs:

  • Create matched pairs of wild-type and cytoplasmic tail-truncated viral constructs

  • Ensure that truncations are precise and don't disrupt other functional domains

  • Consider creating a panel of progressive truncations to map functional regions

• Quantification Methods:

  • Employ sensitive biochemical methods to accurately measure Env incorporation into virions

  • Use multiple detection methods (e.g., Western blotting, ELISA) to quantify gp120/gp41 levels

  • Include appropriate loading controls to normalize virion protein content

• Controls for Alternative Explanations:

  • Monitor cell-surface Env expression to ensure differences aren't due to altered expression

  • Assess gp120 shedding to determine if reduced virion incorporation is due to increased shedding

  • Examine intracellular Env trafficking using microscopy or biochemical fractionation

• Expression Systems:

  • Consider using pseudotyping systems (e.g., with VSV-G) for biochemical studies

  • Compare results between transient and stable expression systems

  • Ensure expression levels are physiologically relevant

Research has shown that the gp41 cytoplasmic tail is essential for efficient Env incorporation into virions in the majority of T cell lines and primary cells, but this requirement varies across cell types. In some cell lines (HeLa, MT-4), truncation reduces Env incorporation only 3-fold, while in others and in primary cells, the reduction exceeds 10-fold . These cell type-dependent differences highlight the importance of comprehensive experimental design when studying this aspect of HIV-1 biology.