NECTIN1 Human

What is NECTIN1 and what are its primary functions in human cells?

NECTIN1 (also known as CD111, HVEC, PRR1, or PVRL1) is a type I transmembrane glycoprotein with a molecular weight of approximately 110 kDa that belongs to the nectin family within the immunoglobulin (Ig) superfamily . The name derives from the Latin word "necto" meaning "to connect," reflecting its primary role in calcium-independent cell-cell adhesion .

The primary functions of NECTIN1 include:

• Promotion of cell-cell contacts through formation of homophilic or heterophilic trans-dimers

• Development of the nervous system, particularly in axon guidance

• Involvement in synaptogenesis

• Promotion of neurite outgrowth

• Formation of adherens junctions

• Mediation of heterotypic adhesion between hair cells and supporting cells in the auditory epithelium

• Role in enamel mineralization

NECTIN1 forms homodimers in cis, then interacts in trans with NECTIN1, NECTIN3, or NECTIN4 . These interactions are essential for establishing proper cellular connections during development and in mature tissues.

What is the molecular structure of human NECTIN1?

Human NECTIN1 isoform 1 is a 517 amino acid protein with a well-defined domain architecture consisting of:

• A 30 amino acid signal sequence

• A 325 amino acid extracellular domain (ECD)

• A 21 amino acid transmembrane segment

• A 141 amino acid cytoplasmic region

The extracellular domain contains three immunoglobulin-like domains:

• An N-terminal V-type domain that mediates ligand binding

• Two C2-type domains

Alternative splicing produces multiple isoforms, including:

• Isoform 2 (also called HigR): 458 amino acids with alternate transmembrane and cytoplasmic sequences

• Isoform 3: A shorter variant with different functional properties

The V-domain (residues 59-133) constitutes the primary interaction interface with herpes simplex virus glycoprotein D (HSV gD) and is critically important for viral entry .

How does NECTIN1 function as a viral entry receptor?

NECTIN1 serves as a primary entry receptor for several alphaherpesviruses, including herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), and pseudorabies virus (PRV) . Research indicates that NECTIN1 constitutes the major receptor for HSV-1 entry into host cells .

The mechanism of NECTIN1-mediated viral entry involves:

• Direct interaction between the V-domain of NECTIN1 (residues 59-133) and the viral glycoprotein D (gD)

• This interaction facilitates viral attachment to the cell surface

• Subsequent conformational changes in viral envelope proteins enable membrane fusion and viral entry

Experimental evidence supporting NECTIN1's role as an entry mediator includes:

• Ectopic expression of human NECTIN1 in otherwise resistant CHO-K1 cells confers susceptibility to VZV infection, with dose-dependent increases in viral glycoprotein expression

• Soluble recombinant forms of NECTIN1 can block viral entry when added during infection but have no effect when added post-infection

• Knockdown of NECTIN1 reduces viral infectivity in human neurons

These findings highlight NECTIN1's critical role in the initial stages of viral infection, making it an important target for antiviral research.

How can NECTIN1-virus interactions be targeted for antiviral development?

Targeting NECTIN1-virus interactions represents a promising strategy for developing novel antivirals, particularly against herpes simplex viruses. Several methodological approaches have demonstrated efficacy:

• Competitive inhibition using soluble NECTIN1 proteins:

  • Soluble NECTIN1 (sNectin-1) produced in mouse myeloma cells can effectively reduce HSV-1 and VZV infection in a dose-dependent manner

  • Treatment with 25 μg of sNectin-1 reduced VZV infection by 97% in human neurons

  • Full-length NECTIN1 protein produced in HEK293 cells reduced VZV infection by 86%

• Peptide-based inhibitors:

  • Peptides derived from the NECTIN1 V-domain that constitutes the interaction interface with HSV gD (residues 59-133) show promising antiviral activity

  • In particular, peptide N1 (residues 76-90) demonstrated significant protection against both HSV-1 (66.57%) and HSV-2 (71.12%) infections in CPE inhibition assays

• Peptidomimetic approaches:

  • Modification of identified peptides through peptidomimetic approaches may further enhance activity and stability

  • In silico methods including PEP-FOLD3, ClusPro 2.0, HawkDock, and Desmond can model peptides and confirm binding specificity with viral proteins

The success of these approaches suggests that disrupting NECTIN1-mediated viral entry represents a viable therapeutic strategy for addressing herpesvirus infections, especially in light of emerging resistance to existing antiviral treatments.

What techniques are available for detecting and quantifying NECTIN1 expression?

Several validated methods exist for detecting and quantifying NECTIN1 expression in experimental settings:

• ELISA-based detection:

  • Human NECTIN1 ELISA Kits (e.g., SimpleStep ELISA®) provide quantitative measurement of NECTIN1 in various sample types including:

    • Heparin plasma

    • Citrate plasma

    • EDTA plasma

    • Cell culture supernatant

    • Serum

  • These assays typically employ a 90-minute single-wash protocol for efficient processing

• Flow cytometry:

  • Monoclonal antibodies against human NECTIN1 (e.g., clone 610835) can detect NECTIN1 expression on cell surfaces

  • Demonstrated applications include detection in U937 human histiocytic lymphoma cell lines

  • Protocol involves staining cells with anti-NECTIN1 antibody followed by fluorophore-conjugated secondary antibody (e.g., Allophycocyanin-conjugated Anti-Mouse IgG F(ab')2)

• Reverse transcription-quantitative PCR (RT-qPCR):

  • Enables quantification of NECTIN1 mRNA expression

  • Used to detect changes in expression during viral infection or after siRNA knockdown

  • Can demonstrate upregulation of NECTIN1 during early stages of infection

• Western blotting:

  • Detects NECTIN1 protein expression in cell lysates

  • Can confirm successful transfection of NECTIN1 expression constructs

  • Useful for monitoring protein levels after knockdown attempts

These methods can be employed individually or in combination to comprehensively characterize NECTIN1 expression under various experimental conditions.

How can NECTIN1 knockdown experiments be optimized in neuronal models?

Optimizing NECTIN1 knockdown in neuronal models requires careful consideration of several methodological factors:

• siRNA transfection strategy:

  • Single transfections may achieve significant mRNA reduction (71% reduction observed 3 days post-transfection) but may not efficiently reduce protein levels

  • Serial transfection of siRNAs every 4 days is recommended to achieve substantial protein knockdown

  • Include appropriate controls (e.g., GAPDH siRNA) to validate transfection efficiency

• Verification of knockdown efficacy:

  • RT-qPCR should be performed to assess mRNA levels (ideally 3 days post-transfection)

  • Western blotting and/or flow cytometry should be used to confirm protein reduction

  • Consider the half-life of the NECTIN1 protein when designing experimental timelines

• Cell model selection:

  • Human pluripotent stem cell-derived neurons offer advantages for studying VZV neurotropism

  • These models have yielded insights regarding viral gene expression, neurovirulence, intracellular viral transport, and latency/reactivation

  • Consider using established human ESC-derived neuronal models to ensure reproducibility

• Functional validation:

  • Test the impact of knockdown on viral infection using appropriate viral strains

  • Quantify infection through viral protein expression (e.g., gE for VZV)

  • Include rescue experiments by expressing siRNA-resistant NECTIN1 constructs to confirm specificity

By implementing these methodological considerations, researchers can effectively reduce NECTIN1 expression in neuronal models and reliably investigate its functional significance.

How do homophilic versus heterophilic NECTIN1 interactions differ functionally?

NECTIN1 participates in both homophilic (NECTIN1-NECTIN1) and heterophilic (NECTIN1 with other nectins) interactions, each with distinct functional implications:

Homophilic interactions:

Heterophilic interactions:

• NECTIN1 forms heterophilic trans-dimers with NECTIN3 and NECTIN4

• NECTIN1-NECTIN3 interactions:

  • Particularly important in the auditory epithelium, where NECTIN1 on hair cells interacts with NECTIN3 on supporting cells

  • Mediate the formation of a checkerboard-like cellular pattern of hair cells and supporting cells

  • Essential for proper auditory system development

• Heterophilic interactions generally display stronger binding affinities than homophilic interactions

• These interactions are crucial for establishing asymmetrical junctions between different cell types

Understanding the structural basis of NECTIN heterophilic interactions has been challenging. While structural characterizations of homophilic NECTIN interactions are available, the structural basis of heterophilic interactions has remained unclear for decades. Recent research has made progress in this area, such as determining the structure of a related Necl (Nectin-like) heterodimer comprising the ectodomains of mouse Necl4 and Necl1 .

The functional differences between these interaction types highlight NECTIN1's versatile role in tissue organization and cell-type specific adhesion patterns.

What are the species-specific differences in NECTIN1 that impact viral entry studies?

Species-specific differences in NECTIN1 have significant implications for viral entry studies, particularly for viruses with strict human tropism like varicella-zoster virus (VZV):

• Structural and sequence differences:

  • Human NECTIN1 differs from mouse NECTIN1 in key residues that affect viral binding

  • Antibodies against human NECTIN1 show no cross-reactivity with recombinant mouse NECTIN1 in direct ELISAs, highlighting structural differences

  • These differences affect the utility of animal models for studying human-specific viral infections

• Post-translational modifications:

  • Post-translational modifications of NECTIN1 appear critical for its function as a viral entry receptor

  • Soluble NECTIN1 produced in different expression systems shows variable effects on viral entry:

    • NECTIN1 produced in a baculovirus expression system unexpectedly enhanced VZV expression in human ESC-derived neurons

    • In contrast, soluble NECTIN1 produced in mouse myeloma cells (where post-translational modifications are more similar to human endogenous NECTIN1) effectively blocked viral entry

• Methodological considerations for research:

  • Human pluripotent stem cell-derived neurons have become increasingly utilized to study VZV neurotropism due to species barriers

  • When designing anti-viral peptides or inhibitors based on NECTIN1, human-specific sequences must be prioritized

  • Ectopic expression of human NECTIN1 in resistant cell lines (like CHO-K1) can create useful experimental systems for studying viral entry mechanisms

  • Human NECTIN1 expression confers susceptibility to VZV infection in otherwise resistant cell lines

These species-specific differences underscore the importance of using appropriate human cell models or humanized systems when studying NECTIN1-mediated viral entry, particularly for strictly human-tropic viruses.

How can NECTIN1-derived peptides be optimized for antiviral efficacy?

Optimizing NECTIN1-derived peptides for antiviral efficacy involves a systematic approach combining in silico prediction, design optimization, and experimental validation:

• Identification of binding interfaces:

  • The V-domain of NECTIN1 (residues 59-133) constitutes the critical interaction interface with HSV gD

  • Within this region, residues 76-90 (peptide N1) have demonstrated significant antiviral activity against both HSV-1 (66.57% protection) and HSV-2 (71.12% protection)

• In silico modeling and optimization:

  • Use computational tools to model peptide structure and binding:

    • PEP-FOLD3 for peptide modeling

    • ClusPro 2.0 for protein-peptide docking

    • HawkDock for binding energy calculation

    • Desmond for molecular dynamics simulations to confirm binding stability

  • These tools help predict peptide-viral protein interactions and enable iterative design improvements

• Peptidomimetic approaches:

  • Modification strategies to enhance stability and efficacy include:

    • N- and C-terminal capping to prevent proteolytic degradation

    • Incorporation of D-amino acids or non-natural amino acids to improve stability

    • Cyclization techniques to constrain peptides in bioactive conformations

    • Addition of cell-penetrating sequences for improved delivery

• Experimental validation workflow:

  • Synthesize candidate peptides using solid-phase peptide synthesis

  • Evaluate binding affinity to viral proteins using biophysical techniques (SPR, ITC)

  • Assess antiviral activity using CPE inhibition assays against relevant viruses

  • Determine mechanism of action (competition assays, time-of-addition studies)

  • Evaluate pharmacokinetic properties and stability in biological fluids

This multifaceted approach to peptide optimization offers the potential to develop highly effective antivirals targeting the NECTIN1-virus interaction with improved stability and efficacy compared to first-generation peptide inhibitors.

What are the methodological challenges in targeting NECTIN1 for therapeutic purposes?

Targeting NECTIN1 for therapeutic purposes presents several methodological challenges that researchers must address:

• Balancing specificity with physiological functions:

  • NECTIN1 plays critical roles in normal cell adhesion, neural development, and synaptogenesis

  • Therapeutic interventions must selectively block viral interactions without disrupting essential physiological functions

  • Research approaches should include:

    • Detailed mapping of interaction domains specific to viral binding versus cellular adhesion

    • Development of targeted compounds that specifically block viral binding epitopes

    • Careful evaluation of potential developmental effects in safety studies

• Delivery challenges:

  • Peptide-based inhibitors face delivery obstacles including:

    • Limited oral bioavailability

    • Potential immunogenicity

    • Poor membrane permeability

    • Rapid clearance and proteolytic degradation

  • Methodological solutions include:

    • Development of non-peptidic small molecule mimetics

    • Exploration of alternative delivery systems (nanoparticles, liposomes)

    • Local delivery approaches for accessible infections (e.g., topical formulations)

• Timing of intervention:

  • NECTIN1-targeting strategies are most effective when administered during viral entry

  • Studies show soluble NECTIN1 effectively blocks infection when added during viral infection but has no effect when added post-infection

  • This necessitates:

    • Early therapeutic intervention

    • Potential prophylactic applications

    • Combination approaches with antivirals targeting different stages of viral replication

• Cross-reactivity and resistance concerns:

  • Different viruses (HSV-1, HSV-2, VZV) may interact with NECTIN1 through slightly different mechanisms

  • Viral mutations could potentially lead to resistance

  • Research strategies should include:

    • Comprehensive testing against multiple viral strains

    • Resistance monitoring in experimental settings

    • Targeting highly conserved interaction domains

Addressing these methodological challenges requires interdisciplinary approaches combining structural biology, medicinal chemistry, drug delivery technology, and virology to develop effective NECTIN1-targeting therapeutics with minimal off-target effects.