[Show abstract][Hide abstract] ABSTRACT: Previously we showed that addition of purified VP22, a structural protein of herpes simplex virus, to short oligonucleotides (ODN) induced the spontaneous assembly of novel particles incorporating both protein and ODN. These particles were not toxic, entered cells, and resided stably in the cytoplasm. Surprisingly the particles could be activated by light in a regulated synchronous manner to release ODN and protein to the cell cytosol and nuclei. Here we construct a fusion protein containing a short peptide from the proapoptotic BH3 domain family member Bak. The BH3-VP22 protein was recruited into particles that entered cells and remained stable in the cytoplasm without toxicity. Light activation rapidly disrupted the particles, a process captured in living cells by time-lapse microscopy, and this synchronized regulated release resulted in subsequent cell death by apoptosis. In control experiments, particles containing a mutant BH3 peptide, although indistinguishable in cell uptake and regulated release, showed no apoptotic effect. Regulated release of VP22-based particles may find application in mechanistic analysis of apoptotic pathways, in cell-based screening assays both of peptides and of oligonucleotides, or as therapeutic agents incorporating specific additional components.
[Show abstract][Hide abstract] ABSTRACT: The low transduction efficiency of viral and nonviral vectors is a major limitation in tumour gene therapy. The HSV-1 tegument protein VP22 has been shown to exhibit a novel intercellular transport property. VP22 wild-type as well as VP22 fusion proteins efficiently spread from the original expressing cell to numerous neighbouring cells, so that protein transport by VP22 chimaeric polypeptides into the surrounding cells offers a possible compensation for the inadequate gene transfer efficiencies. To improve the therapeutic efficacy of the E. coli cytosine deaminase (CD) suicide gene we made use of the VP22 transport property in CD transducing adenoviral (Ad) vectors. C- and N-terminal fusions of CD linked in-frame with VP22 were generated and cloned into recombinant adenoviral vectors. Following in vitro transduction immunofluorescence analysis of Ad-transduced producer cells coplated with naive cells confirmed that the characteristic foci pattern of central producer and adjoining neighbour cells displaying nuclear staining was retained. After transduction of rat hepatoma cells with adenoviral vectors and subsequent incubation with the prodrug 5-FC, we observed enhanced cell cytotoxicity when comparing the CD-VP22 fusion (Ad-CD-VP22) with Ad-vectors expressing the CD gene only (Ad-CD). Thereby employment of Ad-vectors encoding VP22 fusion proteins opens up new possibilities to potentiate the efficiency of suicide gene therapy for the treatment of solid tumours.
[Show abstract][Hide abstract] ABSTRACT: In vitro experiments have demonstrated intercellular trafficking of the VP22 tegument protein of herpes simplex virus type 1 from infected cells to neighboring cells, which internalize VP22 and transport it to the nucleus. VP22 also can mediate intercellular transport of fusion proteins, providing a strategy for increasing the distribution of therapeutic proteins in gene therapy. Intercellular trafficking of the p53 tumor suppressor protein was demonstrated in vitro using a plasmid expressing full-length p53 fused in-frame to full-length VP22. The p53-VP22 chimeric protein induced apoptosis both in transfected tumor cells and in neighboring cells, resulting in a widespread cytotoxic effect. To evaluate the anti-tumor activity of p53-VP22 in vivo, we constructed recombinant adenoviruses expressing either wild-type p53 (FTCB) or a p53-VP22 fusion protein (FVCB) and compared their effects in p53-resistant tumor cells. In vitro, treatment of tumor cells with FVCB resulted in enhanced p53-specific apoptosis compared to treatment with equivalent doses of FTCB. However, in normal cells there was no difference in the dose-related cytotoxicity of FVCB compared to that of FTCB. In vivo, treatment of established tumors with FVCB was more effective than equivalent doses of FTCB. The dose-response curve to FVCB was flatter than that to FTCB; maximal antitumor responses could be achieved using FVCB at doses 1 log lower than those obtained with FTCB. Increased antitumor efficacy was correlated with increased distribution of p53 protein in FVCB-treated tumors. This study is the first demonstration that VP22 can enhance the in vivo distribution of therapeutic proteins and improve efficacy in gene therapy.
Journal of Virology 10/2001; 75(18):8733-41. DOI:10.1128/JVI.75.18.8733-8741.2001 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We compare methods of detection of intercellular transport of the herpes simplex virus protein VP22 and of a green fluorescent protein (GFP)-VP22 fusion protein. Spread of both proteins was observed by immunofluorescence (IF) using organic fixatives. Spread of both proteins was also detected by IF after paraformaldehyde (PFA) fixation and detergent permeabilization, albeit at reduced levels. However, while spread of GFP-VP22 was observed by examining intrinsic GFP fluorescence after methanol fixation, little spread was observed after PFA fixation, suggesting that the levels of the fusion protein in recipient cells were below the detection limits of intrinsic-fluorescence or that PFA fixation quenches the fluorescence of GFP-VP22. We further considered whether elution of VP22 from methanol-fixed cells and postfixation binding to surrounding cells contributed to the increased detection of spread observed after methanol fixation. The results show that while this could occur, it appeared to be a minor effect not accounting for the observed VP22 cell-to-cell spread in culture.
Journal of Virology 02/2000; 74(2):1051-6. DOI:10.1128/JVI.74.2.1051-1056.2000 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We demonstrate that fusion proteins consisting of the herpes simplex virus (HSV) transport protein VP22 linked in frame to HSV thymidine kinase (tk) retain the ability to be transported between cells. In vivo radiolabelling experiments and in vitro assays show that the fusion proteins also retain tk activity. When transfected COS cells, acting as a source of the VP22-tk chimera, were co-plated on to gap junction-negative neuroblastoma cells, ganciclovir treatment induced efficient cell death in the recipient neuroblastoma cell monolayer. No such effect was observed with COS cells transfected with tk alone. Tumours established in mice with neuroblastoma cell lines expressing VP22-tk regressed upon administration of ganciclovir. Furthermore tumours established from 50:50 mixtures of VP22-tk transduced and nontransduced cells also regressed while no significant effect was observed in similar experiments with cells transduced with tk alone. VP22 mediated transport may thus have application in a clinical setting to amplify delivery of the target protein in enzyme-prodrug protocols.
[Show abstract][Hide abstract] ABSTRACT: The herpes simplex virus type 1 (HSV-1) virion protein VP22 exhibits the remarkable property of intercellular trafficking whereby the protein spreads from the cell in which it is synthesized to many surrounding cells. In addition to having implications for protein trafficking mechanisms, this function of VP22 might be exploited to overcome a major hurdle in gene therapy, i.e., efficient delivery of genes and gene products. We show that chimeric polypeptides, consisting of VP22 linked to the entire p53 protein, retain their ability to spread between cells and accumulate in recipient cell nuclei. Furthermore the p53-VP22 chimeric protein efficiently induces apoptosis in p53 negative human osteosarcoma cells resulting in a widespread cytotoxic effect. The intercellular delivery of functional p53-VP22 fusion protein is likely to prove beneficial in therapeutic strategies based on restoration of p53 function. These results, demonstrating intracellular transport of large functional proteins, indicate that VP22 delivery may have applications in gene therapy.