HIV-1 gp41 16kDa

What is the structural organization of the HIV-1 gp41 protein and its 16kDa domain?

HIV-1 gp41 forms a trimeric structure with each monomer consisting of several distinct domains. The full gp41 ectodomain adopts a six-helix bundle (6-HB) conformation in its post-fusion state, where three N-terminal heptad repeat (HR1) helices form a central coiled-coil surrounded by three C-terminal heptad repeat (HR2) helices packed in an antiparallel fashion . The 16kDa domain typically refers to a specific construct used in research that includes key functional regions.

Crystallographic studies of gp41 constructs reveal that:

• The N-terminal helices (residues 30-80) form a parallel coiled-coil structure in the interior

• The C-terminal helices (residues 107-147) are located on the exterior

• A 26-residue loop (residues 81-106) connects these helical regions

• The membrane-proximal external region (MPER) follows the C-terminal helix

The solution structure of the SIV gp41 ectodomain (44 kDa) determined by NMR spectroscopy confirms this arrangement and additionally characterizes the connecting loop as ordered with numerous intermolecular and non-sequential intramolecular contacts .

How does the structure of gp41 change during the fusion process?

The gp41 protein undergoes significant conformational changes during viral fusion:

• In the native state, gp41 is associated with gp120 in a metastable conformation with gp120 shielding gp41 .

• Upon binding of gp120 to CD4 and a co-receptor (CCR5 or CXCR4), gp41 undergoes a large conformational change forming the prehairpin fusion intermediate, where:

  • The N-terminal and C-terminal helices separate

  • The fusion peptide extends toward the host membrane

• The final postfusion conformation involves the formation of a six-helix bundle that brings the viral and cellular membranes into close proximity .

Recent research has focused on designing mutations that destabilize the postfusion conformation (6-helix bundle-destabilizing or 6-HBD mutations) to stabilize the prefusion state of the Env trimer. These mutations prevent sCD4-induced gp120 shedding by stabilizing the native conformation .

What are the optimal methods for producing recombinant HIV-1 gp41 constructs for structural studies?

Structural studies of HIV-1 gp41 require carefully designed constructs and expression systems:

For crystallography studies, constructs like HR1-54Q can be prepared by:

• PCR amplification of the HR1 fragment from templates like Mcon6gp160

• Specific primer design (e.g., 5′-CCATGGATCCGGCATCGTGCAGCAG-3′ and 5′-CCATGGATCCTCCTCCTCCCTGCTTGATGCCCCACAC-3′)

• Restriction enzyme digestion (e.g., BamHI)

• Insertion into expression vectors (e.g., pET-gp41-54Q)

For studies requiring properly folded, glycosylated protein, mammalian expression systems are preferred:

• 293 cells can be used to produce secreted, soluble recombinant gp41

• The resulting protein forms trimers and can be purified to homogeneity

• Glycosylation and proper folding can be verified by biochemical and immunological methods

To enhance gp120-gp41 cleavage efficiency, the natural cleavage site (REKR) can be replaced with six arginine residues (R6), while cleavage-defective variants can be created by mutating the site to SEKS .

What challenges arise in expressing the membrane-proximal external region (MPER) of gp41, and how can they be overcome?

The MPER of gp41 presents several challenges for structural and functional studies:

• Flexibility: Crystallographic studies show poor electron density for C-terminal MPER regions beyond residue 86 (corresponding to gp41 Ala 667), indicating significant flexibility .

• Aggregation: gp41 has a strong propensity to aggregate and is typically expressed at low levels, making studies with authentic gp41 produced in eukaryotic cells rare .

• Conformational heterogeneity: The MPER region may adopt different conformations in various states of gp41.

Strategies to overcome these challenges include:

• Crystal dehydration treatment, which can improve resolution and allow building of additional MPER residues

• Use of mammalian expression systems to produce properly folded, glycosylated protein

• Introduction of stabilizing mutations or disulfide bonds to lock specific conformations

• Combined methodological approaches (X-ray crystallography, NMR, X-ray footprinting) to validate structural findings

How does the fusion domain (FD) of HIV-1 gp41 interact with cellular membranes?

The fusion domain of gp41 mediates critical interactions with cellular membranes during viral entry:

• Membrane binding: The fusion domain interacts with phospholipid membranes, as demonstrated by fluorescence experiments using fluorescein phosphatidylethanolamine (FPE)-labeled membranes. The fluorescence signal increases following exposure to gp41 FD, consistent with binding of the positively charged N-terminus to the membrane surface .

• Cooperative binding: Titration of membranes with gp41 FD produces a binding profile with a Hill coefficient close to 1.5, indicating cooperative binding rather than a simple hyperbolic binding isotherm .

• Heparan sulfate interaction: On T cells, gp41 FD specifically interacts with heparan sulfate on the cell surface. This interaction is blocked by interleukin-8 and abolished by pre-treatment with heparitinase .

• Structural transitions: The secondary structure of the fusion peptide during membrane interaction remains controversial, with some studies describing β-structures and others reporting obliquely oriented α-helices .

Importantly, the behavior of gp41 FD differs between artificial membranes and T cells. While IL-8 treatment of Jurkat T cells abolishes gp41 FD-membrane interaction, similar treatment does not affect interaction with phospholipid membranes, suggesting specific cellular components mediate the interaction in T cells .

What immunosuppressive properties does gp41 exhibit, and what are their implications for HIV pathogenesis?

HIV-1 gp41 exhibits significant immunomodulatory effects that may contribute to viral pathogenesis:

• Lymphocyte inhibition: Both inactivated virus particles and recombinant gp41 inhibit lymphocyte proliferation and alter cytokine release and gene expression .

• Cell type-specific binding: Purified gp41 binds preferentially to monocytes and to a lesser extent to lymphocytes, triggering the production of specific cytokines when added to peripheral blood mononuclear cells .

• T cell suppression: When expressed on target cells, gp41 inhibits the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production .

• Conserved immunosuppressive domain: A peptide corresponding to a highly conserved domain present in all retroviral TM proteins (the immunosuppressive domain) shows similar effects to full-length gp41 .

These findings suggest that gp41 may play a direct role in HIV-1 immunopathogenesis through modulation of immune responses, potentially contributing to the progressive immunodeficiency characteristic of AIDS. Understanding these mechanisms could lead to novel therapeutic approaches targeting gp41-mediated immunosuppression .

What epitopes in gp41 are targeted by broadly neutralizing antibodies, and how are they structurally characterized?

Several broadly neutralizing monoclonal antibodies (bnmAbs) target epitopes within gp41:

• MPER-targeting antibodies: Three bnmAbs (2F5, 4E10, and Z13e1) bind to the membrane-proximal external region (MPER) of gp41, which encompasses approximately 30 residues located between the HR2 region and the transmembrane domain .

• Conformational epitopes: Another bnmAb, m44, interacts with a conformational epitope located in the HR2 and the neighboring loop region upstream .

These epitopes make the MPER a desirable template for developing immunogens that could elicit antibodies with broadly neutralizing activities for HIV vaccine development .

Structural characterization of these epitopes involves:

• X-ray crystallography of antibody-epitope complexes

• Surface plasmon resonance to measure binding kinetics

• ELISA binding studies with conformation-specific monoclonal antibodies

How can mutations be designed to stabilize specific conformations of gp41 for immunogen development?

Strategic mutations can stabilize specific conformations of gp41 for immunogen design:

• Six-helix bundle-destabilizing (6-HBD) mutations:

  • Target residues at the interface of the N-heptad and C-heptad repeat regions

  • Prevent postfusion six-helix bundle formation

  • Retain conformational integrity of the native Env trimer

  • Prevent soluble CD4 (sCD4)-induced gp120 shedding

• Specific mutations with demonstrated effects:

  • V570D appears to destabilize the postfusion conformation

  • E168K confers binding of bnmAbs PG9 and PG16 to JRFL Env

  • SOSIP mutations introduce disulfide bonds that stabilize specific conformations

• Cleavage site modifications:

  • Replacement of the REKR cleavage site with six arginine residues (R6) improves cleavage efficiency

  • Mutation of REKR to SEKS creates cleavage-defective variants for specific studies

These mutation strategies can be assessed using mammalian cell surface display to probe conformational changes of the native Env trimer. The goal is to design immunogens that present epitopes in conformations recognized by broadly neutralizing antibodies, potentially leading to more effective HIV vaccines .

What are the optimal techniques for studying the structure-function relationship of HIV-1 gp41?

Multiple complementary techniques are essential for comprehensive structure-function analysis of HIV-1 gp41:

For structural studies, a combination of X-ray crystallography and NMR spectroscopy has proven particularly valuable. The crystal structure of HR1-54Q provides atomic-level detail , while NMR studies of the 44 kDa ectodomain of SIV gp41 establish the connectivity of helical domains and characterize the conformation of the intervening loop .

For functional studies, fluorescence-based assays and cell-based systems help understand membrane interactions and immunological effects .

How can contradictory findings about gp41 structure and function be reconciled through methodological approaches?

Researchers face several contradictions in the gp41 literature that require careful methodological consideration:

• Secondary structure discrepancies:

  • Some studies describe the fusion peptide as forming β-structures while others report α-helices during membrane interaction

  • Resolution: Combined use of multiple spectroscopic techniques (CD, FTIR, NMR) under identical conditions can help resolve these contradictions

• Membrane interaction differences:

  • Behavior of gp41 FD differs between artificial membranes and T cells

  • Resolution: Parallel studies using both model systems with equivalent methodologies can identify specific cellular components mediating differences

• Conformational state representation:

  • Most structural studies capture the postfusion conformation, while the prefusion and intermediate states remain less characterized

  • Resolution: Design of stabilized constructs that lock specific conformational states, combined with rapid kinetic methods to capture transient intermediates

• Aggregation effects:

  • Peptide aggregates in aqueous preparations complicate structural interpretations

  • Resolution: Careful sample preparation, characterization of aggregation state, and accounting for aggregation in data analysis

These methodological considerations are essential for developing a comprehensive understanding of gp41 structure and function, particularly when working with constructs like the 16kDa domain that may represent specific functional states of the protein .

How are destabilizing mutations in the six-helix bundle advancing our understanding of gp41 function and immunogen design?

Recent research on six-helix bundle-destabilizing (6-HBD) mutations has provided significant insights:

• Counterintuitive stabilization effects:

  • Mutations that destabilize the postfusion conformation paradoxically stabilize the prefusion native trimer

  • This challenges previous assumptions about Env stability and suggests new approaches to immunogen design

• Prevention of conformational changes:

  • 6-HBD mutations prevent sCD4-induced gp120 shedding

  • This suggests that stabilizing the prefusion state can block progression through the fusion pathway

• Combined mutation strategies:

  • Integration of 6-HBD mutations with other approaches (e.g., SOSIP, E168K) may create optimally stabilized immunogens

  • These combinations can enhance both stability and antibody recognition

Future directions include:

• Structural characterization of 6-HBD mutants in different conformational states

• Assessment of these mutants' ability to elicit broadly neutralizing antibodies

• Development of rationally designed immunogens incorporating 6-HBD mutations for HIV vaccine candidates

What are the implications of gp41 immunosuppressive properties for HIV therapy and vaccine development?

The immunosuppressive properties of gp41 have significant implications for HIV research:

• Therapeutic targets:

  • Blocking gp41-mediated immunosuppression could potentially restore immune function in HIV patients

  • Peptide inhibitors or antibodies targeting the immunosuppressive domain might serve as novel therapeutic approaches

• Vaccine considerations:

  • Immunogens based on gp41 might inadvertently suppress immune responses

  • Modifications to eliminate immunosuppressive properties while preserving neutralizing epitopes may be necessary

• Pathogenesis insights:

  • Understanding how gp41 modulates cytokine production and impairs T cell responses provides new perspectives on HIV immunopathogenesis

  • This could explain why some HIV envelope-based vaccines have shown limited efficacy

Future research should focus on:

• Mapping the specific domains and mechanisms responsible for immunosuppression

• Developing modified gp41 constructs that maintain immunogenicity without immunosuppressive effects

• Exploring adjuvants that might counteract the immunosuppressive properties

• Investigating the role of gp41-mediated immunosuppression in HIV persistence and latency

Understanding and addressing these immunosuppressive properties will be crucial for developing effective HIV vaccines and therapeutic strategies targeting gp41.