Baculovirus vector requires electrostatic interactions including heparan sulfate for efficient gene transfer in mammalian cells.
BACKGROUND
Recently, several reports have described the ability of recombinant baculoviruses to transduce a variety of mammalian cells. Yet, mechanisms involved in baculovirus entry in those cells remain largely unexplored, particularly at the primary binding step of the virions to the cell membrane.


METHODS
This report focused on the primary virus-cell interactions that lead to in vitro transduction of human 293 cells using a polyhedrin-deleted baculovirus harboring a CMV-driven beta-galactosidase gene (BacLacZ).


RESULTS
Infection rate monitored for 8 h and transduction rate with a multiplicity of infection of up to 800 were, both, non-saturable. Temperatures from 37 degrees C to 4 degrees C dramatically impaired BacLacZ but not adenovirus cell attachment. Competitive infections performed with an excess of a non LacZ-expressing baculovirus hardly competed at a 1/1 ratio. Consistent with an adsorptive binding process onto the cell surface, interactions through electrostatic charges between both viral and cell membranes appeared to be critical for BacLacZ transduction. The addition of polybrene to the cells prior to or during the infection prevented both virus binding and LacZ gene transfer, suggesting the involvement of negatively charged epitopes exposed at the cell surface. The simultaneous presence of the highly charged heparin abrogated BacLacZ binding to the cell surface and subsequent gene transfer. Lastly, direct in vitro binding of BacLacZ to heparin but not BSA columns could be demonstrated after elution of infectious BacLacZ virus in high salt molarity.


CONCLUSIONS
Electrostatic charges play a critical role during the first step in mammalian cell transduction mediated by a recombinant baculovirus.
Duisit
G
G
Laboratoire de Thérapie Génique, CHU Hotel-Dieu, Nantes, France.
Saleun
S
S
Douthe
S
S
Barsoum
J
J
Chadeuf
G
G
Moullier
P
P
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007763:Lac Operon; D018411:Spodoptera; D055672:Static Electricity; D014161:Transduction, Genetic
D006493:Heparin; D006497:Heparan Sulfate