Abstract
Purpose: :
To compare the ability of adenovirus and lentivirus to enhance the actin stress fiber disrupting activity of the C. botulinum exoenzyme C3 transferase in differentiated human trabecular meshwork cells.
Methods: :
Human adenovirus type 5 was produced by the AdEasy Adenoviral Vector System (Agilent Technologies, La Jolla, CA) and purified by the Adenopure adenovirus purification kit (Puresyn, Inc, Malvern, PA). Feline Immunodeficiency virus pseudotyped with VSV-G was produced by the Lentivector Expression System (System Biosciences, Mountain View, CA). Recombinant Clostridium botulinum exoenzyme C3 transferase bearing a GST tag was overexpressed from cDNA in E. coli and purified by binding to glutathione sepharose and elution by thrombin cleavage. Differentiated monolayers of human trabecular meshwork (HTM) primary cells on coverslips were transfected with 5 µg of C3 transferase and/or virus (adenovirus or FIV) at an MOI of 25. Cells were incubated at 37°C in 5% CO2 and fixed and stained for actin 4 and 24 hrs post-transfection.
Results: :
HTM cells transfected with C3 transferase alone did not display a disruption of actin stress fibers 4 hrs post transfection, but showed significant stress fiber disruption and cell rounding 24 hrs post-transfection. Cells infected with adenovirus or lentivirus alone also showed no effect on actin organization 4 hrs or 24 hrs post-infection. Cells transfected with C3 transferase and either virus showed significant disruption of the actin cytoskeleton within 4 hrs post-transfection, which persisted at 24 hrs.
Conclusions: :
The C. botulinum exoenzyme C3 transferase disrupts actin stress fiber formation, and results in significant cell rounding in HTM cells. This activity is enhanced when cells are simultaneously transduced with adenovirus or lentivirus during transfection, as the stress fiber disruption and cell rounding are observed much earlier. The potential for a viral vector to increase the bioavailability of proteins may be a useful therapeutic tool for cells that are difficult to transfect.
Keywords: gene transfer/gene therapy • adenovirus • cytoskeleton