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Related Experiment Videos

HSV-1 amplicon peptide display vector.

Matthew A Spear1, Deborah Schuback, Kenichi Miyata

  • 1Gene Therapy Program, Radiation Oncology, UCSD Cancer Center, UCSD Medical Center, University of California San Diego, MC 8757, 200 West Arbor Drive, La Jolla, CA, USA. mspear@ucsd.edu

Journal of Virological Methods
|November 26, 2002
PubMed
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Researchers developed a novel HSV-1 amplicon system for modifying viral attachment proteins with foreign peptide epitopes. This system enables efficient investigation of viral targeting, biology, and immunology for potential therapeutic applications.

Area of Science:

  • Virology
  • Molecular Biology
  • Immunology

Background:

  • Herpes Simplex Virus type 1 (HSV-1) attachment, fusion, and entry depend on viral glycoproteins binding to cell receptors, primarily heparan sulfate (HS).
  • Modifying viral surface proteins with foreign epitopes is valuable for studying viral targeting, biology, and immunology.

Purpose of the Study:

  • To create a versatile HSV-1 amplicon system for rapid and efficient engineering of viral attachment proteins with foreign peptide epitopes.
  • To facilitate research into viral interactions and develop potential therapeutic strategies.

Main Methods:

  • Constructed a HSV-1 amplicon plasmid (pCONGA) with unique restriction sites in the gC gene's HS binding domain (HSBD) for easy peptide insertion.
  • Recombined a His tag into the HSBD of pCONGA to create pCONGAH for testing the system.

Related Experiment Videos

  • Infected Vero cells with pCONGAH and HSV-1 helper virus, then analyzed for His-modified gC expression using western blot and antibody co-localization.
  • Quantified amplicon and viral vector titers and established long-term vector producer cell lines using neomycin resistance.

    • Main Results:

    • Successfully generated His-modified gC protein (50 kDa) in infected cells, confirmed by western blot and antibody co-localization.
    • Demonstrated high titers for both amplicon (2 x 10^5 t.u./ml) and helper virus (1 x 10^7 t.u./ml) vectors.
    • Established stable, long-term vector producer cell lines using G418 selection.

    Conclusions:

    • The developed HSV-1 amplicon system allows for rapid and efficient generation of viral vectors with modified surface attachment proteins.
    • This system is highly adaptable, utilizing interchangeable helper viruses and incorporating marker/selection genes (GFP, neo(r)).
    • Offers a powerful tool for investigating viral mechanisms and developing novel therapeutic interventions.