Abstract:
: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.
journal_name
J Virol Methodsjournal_title
Journal of virological methodsauthors
Spear MA,Schuback D,Miyata K,Grandi P,Sun F,Yoo L,Nguyen A,Brandt CR,Breakefield XOdoi
10.1016/s0166-0934(02)00193-3subject
Has Abstractpub_date
2003-01-01 00:00:00pages
71-9issue
1eissn
0166-0934issn
1879-0984pii
S0166093402001933journal_volume
107pub_type
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