Evaluation of VP22 Spread in Tissue Culture

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.VP22, the product of the UL49 gene of herpes simplex virus (8), is a major structural component of the virion. The protein is 301 residues in length, basic, and subject to a number of posttranslational modifications including phosphorylation (7) and nucleotidylylation (2). We previously reported that VP22 exhibits the unusual property of transport between cells (5). Transport was observed after introduction of the VP22 gene by several routes, including transfection or microinjection of the isolated gene in plasmid constructs or by infection with a nonreplicating herpesvirus encoding the native VP22 gene. One of the features of transport was that in cells actively synthesizing the protein, VP22 was located predominantly in the cytoplasm, where it could be observed in filamentous arrays colocalizing with bundled microtubules (4), while in the surrounding cells, VP22 was observed mainly in the nucleus, where it could also be observed colocalizing with chromatin in mitotic cells. A short C-terminal deletion mutant of VP22 lacking 34 residues was expressed normally and exhibited unaltered cytoplasmic localization in the primary cells expressing VP22 but failed to spread to the surrounding cells. Spread of VP22 was also sensitive to treatment of cells with cytochalasin D (5). In addition, we found that this transport activity was retained in a fusion protein consisting of VP22 linked to green fluorescent protein (GFP) which behaved essentially like the native protein with respect to expression, localization, and spread (5).We subsequently reported that trafficking of the GFP-VP22 fusion protein could not be readily observed in living cells (6), in agreement with the results of Fang and colleagues (9), but was detected in methanol-fixed cells either by examining intrinsic GFP fluorescence or by immunofluorescence (IF) analysis with anti-GFP antibodies (6). More recently, other laboratories have observed spread of a VP22-GFP fusion protein in fixed but ...

Enhanced suicide gene effect by adenoviral transduction of a VP22-cytosine deaminase (CD) fusion gene

“…39 Others concluded that methanol fixation is merely mimicking VP22-mediated protein transport. 40 This is contradicted by a report only investigating the fixation procedures of VP22 chimaeric proteins 31 together with our present analysis on quantitating a significant enhancement of the suicide gene effect. Additionally, many studies are dealing with the GFP-VP22 fusion being the only transgene tested.…”

Mapping of herpes simplex virus-1 VP22 functional domains for inter- and subcellular protein targeting

“…In previous studies it has been shown that a GFP-VP22 fusion protein retains the properties of the native VP22 in a recombinant HSV-1 virus, 5 and allowed the characterisation in detail of the subcellular localisation of VP22 1 and its intercellular transfer. [6][7][8][9] GFP-VP22 fusion protein has been found to retain the intercellular trafficking properties of VP22 in all the cell lines studied.…”

“…Plasmids pEGFP-VP22 was created by cloning into the BamHI site of pEGFP-C1 (Clontech, Palo Alto, CA, USA) a BglIIBamHI fragment from pUL49epB 7 and adding the BamHI-XbaI fragment from pINSHNES (both kind gifts from Dr P O'Hare, Marie Curie Research Institute, Oxted, UK) containing the hemaglutinin (HA) epitope. Deletion constructs fused to EGFP were created by the following strategy: pEGFP-VP22 was cleaved with ApaI and XbaI.…”

Intercellular trafficking and enhanced in vivo antitumour activity of a non-virally delivered P27-VP22 fusion protein

“…4,25,26 This phenomenon could lead to an artefactual VP22 nuclear homing 18 not observed with paraformaldehyde fixation. 25 Conversely, other reports showed that efficient transport can be observed with both fixation methods 5 and in living cells. 7 It is noteworthy that we were able to detect intercellular trafficking of VP22 and P27VP22 using paraformaldehyde (Figures 1 and 3; cytometry) and methanol fixation (Figures 1 and 3; immunofluorescence).…”

“…3 Furthermore, VP22 retains its trafficking ability when genetically fused to an effector protein like the green fluorescent protein (GFP). [3][4][5][6][7] Thus, linkage to VP22 has permitted to enhance the biological activity of several proteins such as the tumour suppressor p53 8,9 or the products of the suicide genes thymidine kinase 10,11 and cytosine deaminase. 12 It was also used to deliver functional Flp recombinase 13 and to carry SV40 large T antigen to induce cell cycle reentry in terminally differentiated muscle cells.…”