Recombinant Human adenovirus B serotype 3 Early E3 9.0 kDa glycoprotein

Basic Research Questions

Advanced Research Questions

• What are the technical challenges in expressing and purifying functional recombinant E3 9.0 kDa glycoprotein?

  Expressing and purifying transmembrane glycoproteins presents several technical challenges:

  1. Proper folding and glycosylation: Bacterial expression systems (E. coli) lack glycosylation machinery, so mammalian or insect cell systems are preferred

  2. Membrane integration: The hydrophobic transmembrane domain makes solubilization difficult

  3. Protein yield: Small proteins often have lower expression yields

  4. Conformational integrity: Maintaining native structure during purification

  Methodological solution: A comprehensive approach involves:

  • Using mammalian expression systems (like HEK293F cells) for proper glycosylation

  • Employing detergent screening (DDM, CHAPS, Triton X-100) for optimal solubilization

  • Implementing affinity tags (His-tag) for purification while confirming they don't interfere with function

  • Validating protein conformation using circular dichroism or limited proteolysis

  Based on available protocols, recombinant E3 9.0 kDa glycoprotein has been successfully expressed in E. coli with an N-terminal His-tag , but mammalian expression might better preserve glycosylation patterns essential for function.

• How can researchers determine if the E3 9.0 kDa glycoprotein interacts with components of the host immune system?

  Investigating potential immune interactions requires multiple complementary approaches:

  Approach	Methodology	Expected Outcome
  Protein-protein interaction screening	Co-immunoprecipitation, yeast two-hybrid, proximity labeling (BioID)	Identification of binding partners
  Flow cytometry	Binding assays with recombinant protein and immune cells	Cell type-specific binding patterns
  Surface plasmon resonance	Quantitative binding kinetics	Affinity constants (Kd) for interactions
  Functional immune assays	NK cell cytotoxicity, T cell activation, cytokine production	Immunomodulatory effects

  Methodological solution: Based on studies of other E3 proteins like E3/49K, which binds to CD45 on leukocytes , researchers should first conduct binding assays with recombinant E3 9.0 kDa glycoprotein and various immune cell populations. Positive interactions can be further characterized by pull-down assays followed by mass spectrometry to identify binding partners, with subsequent validation using surface plasmon resonance or bio-layer interferometry to determine binding kinetics.

• What structural determinants of the E3 9.0 kDa glycoprotein are essential for its function?

  Understanding structure-function relationships requires systematic mutagenesis and functional assessment. Studies with other E3 proteins like E3/19K have identified specific conserved amino acids critical for function .

  Methodological solution: Implement a comprehensive mutagenesis strategy:

  1. Generate alanine substitution mutants for conserved residues

  2. Create deletion mutants targeting predicted functional domains

  3. Exchange domains with homologous proteins from other adenovirus serotypes

  4. Assess each mutant for:

     • Proper protein expression and folding

     • Subcellular localization

     • Binding to target molecules

     • Immunomodulatory function

  For example, studies with E3/19K identified that substitutions of W52, M87, and W96 abrogated HLA-I complex formation, suggesting these residues make direct contacts with HLA-I molecules . Similar approaches could identify key functional residues in the E3 9.0 kDa glycoprotein.

• How does the evolutionary conservation of the E3 9.0 kDa glycoprotein compare across different adenovirus serotypes?

  The E3 region represents one of the most divergent regions of adenoviruses , with species-specific variations in coding capacity. Understanding evolutionary conservation provides insights into functional importance.

  Methodological solution:

  1. Perform comprehensive bioinformatic analysis using:

     • Multiple sequence alignment of E3 9.0 kDa homologs across serotypes

     • Phylogenetic tree construction

     • Calculation of selection pressure (dN/dS ratios) on different protein domains

     • Identification of conserved motifs

  2. Map conservation patterns onto predicted structural models

  3. Correlate conservation with known or predicted functional domains

  The Ad3 9-kDa protein shares homology primarily with the C-terminal region of the Ad2/5 11.6-kDa adenovirus death protein (ADP) , suggesting functional divergence across serotypes while maintaining core functions.

• What methods are best suited for investigating the immunomodulatory effects of the E3 9.0 kDa glycoprotein?

  Investigating immunomodulatory effects requires systems that can detect subtle changes in immune response parameters:

  Methodological solution: A multi-tiered approach is recommended:

  1. In vitro immune cell assays:

     • NK cell cytotoxicity assays using 51Cr-release or flow cytometry-based methods

     • T cell activation assays measuring CD69/CD25 expression, proliferation, and cytokine production

     • Antigen presentation assays with dendritic cells and T cell responders

  2. Reporter systems:

     • NF-κB, NFAT, or AP-1 luciferase reporter assays to detect changes in immune signaling pathways

     • HLA-I surface expression monitoring using flow cytometry

  3. Ex vivo systems:

     • Human PBMCs exposed to recombinant protein or infected with E3 9.0 kDa-expressing vs. knockout viruses

     • Cytokine profiling using multiplex bead arrays or ELISA

  These approaches would mirror those successfully used to characterize other E3 proteins, such as E3/49K, which was shown to suppress NK cell cytotoxicity and T cell activation .

• How can CRISPR-Cas9 genome editing be applied to study the E3 9.0 kDa glycoprotein in the context of viral infection?

  CRISPR-Cas9 technology offers powerful approaches for precise manipulation of viral genomes.

  Methodological solution:

  1. Direct viral genome editing:

     • Clone the adenovirus genome into a bacterial artificial chromosome (BAC)

     • Design guide RNAs targeting the E3 9.0 kDa gene

     • Introduce precise mutations or deletions

     • Reconstitute infectious virus from the modified BAC

  2. Host factor manipulation:

     • Create CRISPR knockout cell lines lacking potential interaction partners

     • Screen for host factors affecting E3 9.0 kDa function using CRISPR libraries

     • Generate reporter cell lines to monitor E3 9.0 kDa activity

  3. Applications:

     • Create point mutations to test structure-function hypotheses

     • Generate tagged versions for localization studies

     • Produce chimeric proteins to map interaction domains

  This approach has been successfully applied to other adenovirus genes, as demonstrated in studies using BAC technology to generate recombinant E1-deleted Ad3 vectors .

• What is the role of glycosylation in the function of the E3 9.0 kDa glycoprotein?

  As a glycoprotein, post-translational modifications likely play important roles in protein folding, stability, and function.

  Methodological solution:

  1. Glycosylation site identification:

     • Bioinformatic prediction of N-linked and O-linked glycosylation sites

     • Mass spectrometry analysis of purified protein

     • Site-directed mutagenesis of predicted glycosylation sites

  2. Functional impact assessment:

     • Expression in glycosylation-deficient cell lines

     • Treatment with glycosidases or glycosylation inhibitors (tunicamycin, kifunensine)

     • Lectin binding assays to characterize glycan composition

  3. Structural analysis:

     • Comparative modeling of glycosylated vs. non-glycosylated forms

     • Stability and binding assays with differentially glycosylated variants

  While the E3 9.0 kDa protein contains an N-linked glycosylation motif, its utilization may be limited due to the absence of a signal sequence , requiring experimental verification of its glycosylation status.

• How do the mechanisms of immune evasion differ between the E3 9.0 kDa glycoprotein and other well-characterized E3 proteins?

  Comparative analysis of immune evasion mechanisms provides insights into the diversity of viral strategies to counteract host defenses.

  Methodological solution:

  1. Comprehensive functional comparison:

     E3 Protein	Known Mechanism	Experimental Approach for E3 9.0 kDa
     E3/19K	MHC-I retention in ER	Flow cytometry for MHC-I surface expression
     E3/49K	CD45 binding on leukocytes	CD45 binding assays, phosphatase activity tests
     E3 10.4K-14.5K	Apoptosis receptor downregulation	Surface receptor analysis, apoptosis assays
     E3 13.7K (PAdV-3)	Virion structural component	Virion incorporation analysis

  2. Pathway analysis:

     • Transcriptomic profiling (RNA-seq) comparing E3 9.0 kDa with other E3 proteins

     • Phosphoproteomic analysis to identify affected signaling pathways

     • Temporal analysis of immune response modulation

  This comparative approach would establish whether the E3 9.0 kDa glycoprotein employs novel mechanisms or variations of known E3 protein strategies.

• What are the implications of E3 9.0 kDa glycoprotein research for adenovirus-based vector development?

  Understanding E3 function has direct applications for improving adenoviral vectors used in gene therapy and vaccination.

  Methodological solution:

  1. Vector optimization strategies:

     • Retention or deletion of E3 9.0 kDa based on desired immune profile

     • Modification of E3 9.0 kDa to enhance vector persistence or immunogenicity

     • Creation of chimeric E3 proteins with tailored functions

  2. Application-specific considerations:

     • For oncolytic vectors: Assess impact on tumor microenvironment and anti-tumor immunity

     • For vaccine vectors: Evaluate effect on antigen-specific immune responses

     • For gene therapy: Determine influence on vector persistence and expression

  3. Experimental evaluation:

     • Compare vectors with and without E3 9.0 kDa in relevant preclinical models

     • Assess immunogenicity, efficacy, and safety parameters

     • Conduct long-term persistence studies

  Studies have shown that the presence of the E3 region can enhance the oncolytic potency of conditionally replicative adenoviruses by 1.6-20 times in different cell lines , suggesting that understanding E3 9.0 kDa function could lead to improved vector design for specific applications.