Direct evidence for the absence of intercellular trafficking of VP22 fused to GFP or to the herpes simplex virus thymidine kinase

Abstract

The treatment of solid tumors by retroviral delivery of the herpes simplex virus thymidine kinase (TK) followed by ganciclovir (GCV) treatment has so far shown only limited success in patients. One major drawback in this approach is the lack of efficient in vivo gene delivery to cancer cells. Although, the transduction of every single tumor cell is not a requirement since the bystander effect (BE) mediated by gap junctions allows the diffusion of the toxic GCV metabolites from TK-expressing cells toward untransduced cells. To render the TK/GCV approach more potent, and independent of the level of gap junctions, we have tested the efficiency of a TK mutant (TK30) fused to VP22, a herpes simplex protein that seems to be capable of intercellular trafficking. We failed to detect an increase in the BE with cells expressing VP22 fused to TK30 versus cells containing TK30 alone, and this result forced us to reinvestigate the trafficking properties of VP22. Using very sensitive Western blot and fluorescence assays, we were not able to detect the spread of VP22 fused either to TK30 or GFP. These results indicate that VP22 cannot be used as a cargo to translocate TK30 or GFP.

Fusogenic membrane glycoproteins induce syncytia formation and death in vitro and in vivo: a potential therapy agent for lung cancer

“…29 Fusion proteins between VP22 and a therapeutic polypeptide were thus successfully used for in vivo tumor therapy although conflicting results have also been reported. 27,28,[30][31][32][33][34][35] Positive gene therapy results using suicide genes like the herpes simplex type-1 thymidine …”

Suicide genes for cancer therapy

“…Initial data revealed that a fusion protein of HSVtk and the HSV-1 tegument protein VP22 mediated more efficient cell-killing than HSVtk alone due to an improved bystander effect (Dilber et al, 1999;Elliott and O'Hare, 1997). However, more recent findings indicate that any differences between the action of VP22-HSVtk and wild-type HSVtk are not actually mediated by intercellular trafficking of the fusion protein (Roy et al, 2005). Fusion proteins consisting of HSVtk and 8-11 amino acids from the human immunodeficiency virus (HIV)-1 TAT protein have been shown to retain full catalytic activity while also being capable of gap-junction independent intercellular trafficking (Tasciotti and Giacca, 2005).…”

The taming of the cell penetrating domain of the HIV Tat: Myths and realities

“…Though documented evidence exists for cell permeability of VP22 [23,[38][39][40][41], many studies could not verify this property and have shown failures in transcellular activity [22,42,43]. It seems possible that fusion of VP22 with different proteins will attain a different conformation every time, which obviously will affect the transduction behavior.…”