Transduction of Vero cells and bovine monocytes with a herpes simplex virus-1 based amplicon carrying the gene for the bovine herpesvirus-1 Circ protein.
Herpes simplex virus-1 (HSV-1) based amplicon vectors are promising gene delivery vehicles because they have a large transgene capacity and can efficiently transduce many different cell types, including non-dividing cells, of various animal species. The Circ protein of bovine herpesvirus-1 (BHV-1) is a myristylated virion component of unknown function. Preliminary experiments with a circ gene deletion mutant indicated that Circ may influence the host's immune response by downregulating MHC-II expression in bovine monocytes. To get more insight into the function of Circ, amplicon vectors were constructed with various open reading frames (ORFs) under the control of the HSV-1 IE4/5 promoter: (i) the Circ ORF alone, (ii) a fusion ORF encoding an N-terminal Circ fused to the enhanced green fluorescent protein (eGFP), (iii) the eGFP ORF alone, and (iv) the Circ ORF in the inverted orientation. Upon helpervirus-free packaging into HSV-1 amplicon particles and transduction of Vero cells, both Circ alone and the Circ-eGFP fusion protein produced a punctate pattern within the cytoplasm, suggesting membrane association of the myristylated protein. In contrast, eGFP alone was evenly distributed over the cytoplasm of transduced cells. Upon infection of bovine buffy-coat cells, it was observed that cells of the monocyte lineage but not lymphocytes were transduced. Transgene expression reached a peak around 20h after transduction and lasted for at least 90h. Transduced monocytes underwent specific morphological changes, which may be attributed to Circ synthesis.
Schwyzer
M
M
Institute of Virology, Faculty of Veterinary Medicine, Winterthurerstrasse 266A, University of Zürich, CH-8057, Zürich, Switzerland. schwyzer@vetvir.unizh.ch
Fischer-Bracher
C
C
Fraefel
C
C
Bächi
T
T
Nuñez
R
R
Engels
M
M
Ackermann
M
M
D000818:Animals; D002417:Cattle; D002522:Chlorocebus aethiops; D003588:Cytopathogenic Effect, Viral / Q000235:genetics; D005434:Flow Cytometry / Q000662:veterinary; D015316:Genetic Therapy / Q000379:methods / Q000662:veterinary*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007242:Herpesvirus 1, Bovine / Q000737:chemistry / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000737:chemistry / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry; D009000:Monocytes; D010957:Plasmids / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*
D008164:Luminescent Proteins; D014764:Viral Proteins; C090374:circ protein, bovine herpesvirus type 1; D049452:Green Fluorescent Proteins