Recombinant Human adenovirus C serotype 5 Early E3A 10.5 kDa glycoprotein

What is the biological role of the E3A 10.5 kDa glycoprotein in Adenovirus C serotype 5?

The E3A 10.5 kDa glycoprotein is part of the E3 transcription unit, which plays a critical role in viral pathogenesis. While not essential for viral replication in cultured cells, E3 proteins, including the E3A 10.5 kDa glycoprotein, function primarily to inhibit host immune responses to infection. These proteins specifically target both adaptive immunity (cytotoxic T lymphocytes) and innate immune responses (tumor necrosis factor-α), enabling viral evasion of host defenses .

How is the E3A 10.5 kDa glycoprotein structurally characterized?

The E3A 10.5 kDa glycoprotein is a relatively small protein comprising 93 amino acids in its full-length form. As a glycoprotein, it undergoes post-translational modifications involving carbohydrate attachments that contribute to its function. Recombinant versions can be produced with tags (such as His-tags) to facilitate purification and experimental manipulation in research settings .

How does the E3 region contribute to adenovirus pathogenesis?

The E3 region contains multiple genes whose products collectively modulate host immune responses. While this region is not required for viral replication in cell culture, it significantly enhances viral persistence in vivo. The E3 transcription unit inhibits both specific immunity (cytotoxic T lymphocytes) and innate immune responses (TNF-α), allowing the virus to evade host defense mechanisms during infection .

What methodologies are most effective for studying functional interactions between E3A 10.5 kDa glycoprotein and host immune components?

To effectively study the functional interactions between E3A 10.5 kDa glycoprotein and host immune components, researchers should employ multi-faceted approaches:

• Immunoprecipitation and Co-IP assays: For identifying direct protein-protein interactions

• CRISPR-Cas9 gene editing: To create knockout models for functional validation

• Transcriptomics/proteomics analysis: To evaluate global changes in host response

• Flow cytometry: For quantifying changes in immune cell populations and surface markers

• In vitro cytokine assays: To measure modulation of immune signaling pathways

These approaches should be integrated with bioinformatics analysis to identify potential interacting partners based on structural similarities to other E3 proteins with known immune evasion functions .

How does homologous recombination in E3 genes impact research using recombinant adenoviral vectors?

Homologous recombination in E3 genes presents both challenges and opportunities for research using recombinant adenoviral vectors:

Challenges:

• Investigators using HAdV vectors for transgene delivery should be aware that placement of transgenes within the E3 cassette may leave them susceptible to recombination with wild-type HAdV-D during coinfection

• This could potentially result in infectious, replication-competent viruses expressing the transgene

• This risk is particularly relevant for gene therapy applications, where unintended vector recombination could have safety implications

Research opportunities:

• The high recombination rate of E3 genes can be leveraged to enhance vector diversity

• Understanding recombination mechanisms can inform the design of more stable vectors

• The natural plasticity of this region makes it an attractive site for transgene insertion with minimal impact on viral fitness

What are the current methodological approaches to prevent replication-competent adenovirus (RCA) generation when working with E3-modified vectors?

Preventing RCA generation when working with E3-modified vectors requires strategic approaches:

• Modification of homologous sequences: Shortening the length of homologous sequences between the adenoviral vector and HEK293 genomic DNA significantly reduces RCA production during serial passages

• Alternative cell lines: Using PER.C6 cells instead of HEK293 can minimize recombination events

• Quantitative monitoring: Implementing qPCR-based detection of RCA at various passages to ensure vector quality

• Vector redesign: Creating vectors with minimal sequence overlap with packaging cell lines

• E1/E3 double-deletion: Using vectors with deletions in both regions to reduce packaging capacity for recombined sequences

Experimental data has demonstrated that modified adenoviral vectors with shortened homologous sequences show significantly reduced RCA production through 12 serial passages in HEK293 cells, confirming the effectiveness of this approach .

How does the metabolic impact of adenovirus infection affect experiments using recombinant E3A 10.5 kDa glycoprotein?

Adenovirus infection significantly dysregulates host cell metabolism, which can confound experiments using recombinant E3A proteins:

• Altered cysteine metabolism: Infection changes cysteine utilization pathways that may affect protein folding and function

• Disrupted purine metabolism: Changes in nucleotide availability can impact transcriptional and translational processes

• Unsaturated fatty acid dysregulation: Membrane composition alterations may affect protein localization and trafficking

Researchers should account for these metabolic changes when interpreting results from experiments using recombinant E3A proteins, particularly in the context of intact viral infection models versus isolated protein studies. Control experiments should include metabolic profiling to establish baseline changes induced by viral infection independent of specific E3A protein effects .

What purification strategies yield the highest quality recombinant E3A 10.5 kDa glycoprotein for functional studies?

For obtaining high-quality recombinant E3A 10.5 kDa glycoprotein suitable for functional studies, a multi-step purification process is recommended:

The final purified protein should be assessed for proper folding using circular dichroism and functional activity through appropriate immunological assays that test its ability to modulate immune responses .

How can researchers effectively evaluate E3A 10.5 kDa glycoprotein interactions with host immune components?

To effectively evaluate E3A 10.5 kDa glycoprotein interactions with host immune components, researchers should implement a comprehensive experimental workflow:

• Preliminary in silico analysis:

  • Structural prediction using homology modeling

  • Molecular docking simulations with potential binding partners

  • Identification of putative interaction domains

• In vitro validation:

  • Surface plasmon resonance (SPR) for binding kinetics determination

  • Microscale thermophoresis for quantifying interactions in solution

  • Pull-down assays coupled with mass spectrometry

• Cellular validation:

  • FRET/BRET assays for protein-protein interactions in living cells

  • Immunofluorescence colocalization studies

  • Proximity ligation assays for detecting endogenous interactions

• Functional assessment:

  • Cytokine secretion profile analysis

  • Cell surface receptor modulation assays

  • Immune cell activation/suppression assays

What experimental controls are essential when studying the functions of recombinant E3A 10.5 kDa glycoprotein?

When studying recombinant E3A 10.5 kDa glycoprotein functions, several critical experimental controls must be included:

• Protein-specific controls:

  • Heat-inactivated E3A 10.5 kDa glycoprotein (functional negative control)

  • Tag-only protein preparation (tag interference control)

  • Structurally similar but functionally distinct E3 protein (specificity control)

• Expression system controls:

  • Host cell lysate without recombinant protein expression

  • Expression system-matched control protein

• Functional assay controls:

  • Positive controls using known immune modulators

  • Dose-response relationships to establish specificity

  • Time-course experiments to determine kinetics

• Recombination controls:

  • When using viral vectors, include testing for recombination events

  • PCR verification of genetic stability after multiple passages

How can insights from E3A 10.5 kDa glycoprotein research contribute to improved adenoviral vectors for gene therapy?

The study of E3A 10.5 kDa glycoprotein can significantly enhance adenoviral vector development for gene therapy through several approaches:

• Enhanced immune evasion: By understanding the mechanisms by which E3A 10.5 kDa glycoprotein modulates immune responses, researchers can engineer vectors with improved persistence and reduced immunogenicity

• Vector safety: Knowledge of homologous recombination patterns in E3 regions enables the design of vectors with reduced potential for generating replication-competent adenoviruses during production

• Targeted gene delivery: Insights into E3A protein interactions with host cellular components may permit the development of vectors with enhanced tissue-specific targeting capabilities

• Reduced inflammatory responses: Utilizing modified E3A proteins in vector design could potentially minimize inflammatory responses at the site of vector administration

What are the implications of homologous recombination patterns in E3 genes for designing next-generation adenoviral vectors?

The extensive homologous recombination observed in E3 genes has profound implications for next-generation adenoviral vector design:

• Recombination risk assessment: Researchers must evaluate the propensity for recombination when selecting sites for transgene insertion, particularly when using the E3 region

• Strategic modifications: Vectors should be engineered with strategic nucleotide changes to reduce sequence homology with wild-type adenoviruses while maintaining functional properties

• Monitoring protocols: Implementation of sensitive detection methods for recombination events during vector production and validation

• Alternative insertion sites: For applications where stability is paramount, exploration of less recombination-prone regions may be preferable

These considerations are particularly important for clinical applications where vector stability and safety are critical requirements .