HSV-1 amplicon peptide display vector.
There are significant uses for expressing foreign peptide epitopes in viral surface attachment proteins in terms of investigating viral targeting, biology, and immunology. HSV-1 attachment, followed by fusion and entry, is mediated in large part by the binding of viral surface glycoproteins to cell surface receptors, primarily through heparan sulfate (HS) glycosaminoglycan residues. We constructed a HSV-1 amplicon plasmid (pCONGA) carrying the gC primary attachment protein gene with unique restriction sites flanking the HS binding domain (HSBD) (residues 33-176) to allow rapid, high efficiency substitution with foreign peptide domains. To test this system, a His tag with an additional unique restriction site (for selection and assay digests) was recombined into the pCONGA HSBD site to create pCONGAH. Infection of pCONGAH transfected Vero cells with HSV-1 helper virus (gCdelta2-3 or hrR3) produced His-modified gC as demonstrated by western blot analysis with co-localization of anti-gC and anti-His tag antibodies to a protein of appropriate molecular weight (50 kd). As CONGA and CONGAH amplicons carry a GFP transgene and the gCdelta2-3 and hrR3 viruses carry a lacZ transgene, vector stocks produced from 1 x 10(5) Vero cells could be titered for competent vector on cell monolayers and were demonstrated to contain 2 x 10(5) amplicon vector transducing units (t.u.)/ml and 1 x 10(7) virus t.u./ml. As the amplicon plasmids also contain the neomycin resistance gene (neo(r)), long term vector producer cell lines were created using G418 selection. This amplicon system provides means to rapidly and efficiently generate HSV-1 amplicon and viral vector expressing surface attachment proteins modified with different peptide epitopes for investigational and therapeutic uses, with the advantages of an amplicon plasmid that can be used with interchangeable helper virus vectors, is designed specifically for easy manipulation, and carries GFP and neo(r) transgenes for marker and selection functions.
Spear
Matthew A
MA
Gene 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
Schuback
Deborah
D
Miyata
Kenichi
K
Grandi
Paola
P
Sun
Fang
F
Yoo
Linda
L
Nguyen
Anh
A
Brandt
Curtis R
CR
Breakefield
Xandra O
XO
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000235:genetics*; D019151:Peptide Library; D010957:Plasmids; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics
D019151:Peptide Library; D014759:Viral Envelope Proteins; C038535:glycoprotein gC, herpes simplex virus type 1