Cytoplasm-to-nucleus translocation of a herpesvirus tegument protein during cell division

We have previously shown that the herpes simplex virus tegument protein VP22 localizes predominantly to the cytoplasm of expressing cells. We have also shown that VP22 has the unusual property of intercellular spread, which involves the movement of VP22 from the cytoplasm of these expressing cells into the nuclei of nonexpressing cells. Thus, VP22 can localize in two distinct subcellular patterns. By utilizing time-lapse confocal microscopy of live cells expressing a green fluorescent protein-tagged protein, we now report in detail the intracellular trafficking properties of VP22 in expressing cells, as opposed to the intercellular trafficking of VP22 between expressing and nonexpressing cells. Our results show that during interphase VP22 appears to be targeted exclusively to the cytoplasm of the expressing cell. However, at the early stages of mitosis VP22 translocates from the cytoplasm to the nucleus, where it immediately binds to the condensing cellular chromatin and remains bound there through all stages of mitosis and chromatin decondensation into the G(1) stage of the next cycle. Hence, in VP22-expressing cells the subcellular localization of the protein is regulated by the cell cycle such that initially cytoplasmic protein becomes nuclear during cell division, resulting in a gradual increase over time in the number of nuclear VP22-expressing cells. Importantly, we demonstrate that this process is a feature not only of VP22 expressed in isolation but also of VP22 expressed during virus infection. Thus, VP22 utilizes an unusual pathway for nuclear targeting in cells expressing the protein which differs from the nuclear targeting pathway used during intercellular trafficking.     

Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax

BACKGROUND: Previously, antigens expressed from DNA vaccines have been fused to the VP22 protein from Herpes Simplex Virus type I in order to improve efficacy. However, the immune enhancing mechanism of VP22 is poorly understood and initial suggestions that VP22 can mediate intercellular spread have been questioned. Despite this, fusion of VP22 to antigens expressed from DNA vaccines has improved immune responses, particularly to non-secreted antigens. METHODS: In this study, we fused the gene for the VP22 protein to the gene for Protective Antigen (PA) from Bacillus anthracis, the causative agent of anthrax. Protective immunity against infection with B. anthracis is almost entirely based on a response to PA and we have generated two constructs, where VP22 is fused to either the N- or the C-terminus of the 63 kDa protease-cleaved fragment of PA (PA63). RESULTS: Following gene gun immunisation of A/J mice with these constructs, we observed no improvement in the anti-PA antibody response generated. Following an intraperitoneal challenge with 70 50% lethal doses of B. anthracis strain STI spores, no difference in protection was evident in groups immunised with the DNA vaccine expressing PA63 and the DNA vaccines expressing fusion proteins of PA63 with VP22. CONCLUSION: VP22 fusion does not improve the protection of A/J mice against live spore challenge following immunisation of DNA vaccines expressing PA63.     

Conformational lability of herpesvirus protein VP22

The herpesvirus protein VP22 traffics between cells, being exported from expressing cells in a non-Golgi-dependent manner and localizing in the nuclei of surrounding cells. This transport is retained in certain VP22 fusion proteins, making VP22 a candidate for use in macromolecular drug delivery. In an effort to understand the physical basis for this activity, we have initiated structural studies of VP22.C1, the C-terminal half of VP22, which possesses the full transport activity of the native protein. CD and Fourier transform infrared analyses indicate a secondary structure consisting of approximately 30% alpha-helix, 17% beta-sheet, and 51% disordered and turn structure. Unfolding studies conducted by CD, differential scanning calorimetry, and fluorescence reveal a series of discrete structural transitions in the range of 20-60 degrees C. CD and fluorescence studies of interactions between VP22.C1 and divalent cations and model polyanions indicate that Mg(2+), Zn(2+), oligonucleotides, and heparin interact with the protein, causing changes in secondary structure and thermal stability. Additionally, the interaction of VP22.C1 with model lipids was examined. Fluorescence titrations of the protein with trans-parinaric acid at various temperatures suggest the binding of one to two molecules of parinaric acid to VP22.C1 at temperatures >40 degrees C, suggesting the possibility of conformation dependent membrane interaction under physiological conditions.     

Cellular internalization of green fluorescent protein fused with herpes simplex virus protein VP22 via a lipid raft-mediated endocytic pathway independent of caveolae and Rho family GTPases but dependent on dynamin and Arf6

VP22 is a structural protein of the herpes simplex virus and has been reported to possess unusual trafficking properties. Here we examined the mechanism of cellular uptake of VP22 using a fusion protein between the C-terminal half of VP22 and green fluorescent protein (GFP). Adsorption of VP22-GFP onto a cell surface required heparan sulfate proteoglycans and basic amino acids, in particular, Arg-164 of VP22. Inhibitor treatment, RNA interference, expression of dominant-negative mutant genes, and confocal microscopy all indicated that VP22-GFP enters cells through an endocytic pathway independent of clathrin and caveolae but dependent on dynamin and Arf6 activity. As with CD59 (a lipid raft marker), cell-surface VP22-GFP signals were resistant to Triton X-100 treatment but only partially overlapped cell-surface CD59 signals. Furthermore, unlike other lipid raft-mediated endocytic pathways, no Rho family GTPase was required for VP22-GFP internalization. Internalized VP22 initially entered early endosomes and then moved to lysosomes and possibly recycling endosomes.     

Membrane association of VP22, a herpes simplex virus type 1 tegument protein

Tegument proteins of herpes simplex virus type 1 (HSV-1) are hypothesized to contain the functional information required for the budding or envelopment process proposed to occur at cytoplasmic compartments of the host cell. One of the most abundant tegument proteins of HSV-1 is the U(L)49 gene product, VP22, a 38-kDa protein of unknown function. To study its subcellular localization, a VP22-green fluorescent protein chimera was expressed in transfected human melanoma (A7) cells. In the absence of other HSV-1 proteins, VP22 localizes to acidic compartments of the cell that may include the trans-Golgi network (TGN), suggesting that this protein is membrane associated. Membrane pelleting and membrane flotation assays confirmed that VP22 partitions with the cellular membrane fraction. Through truncation mutagenesis, we determined that the membrane association of VP22 is a property attributed to amino acids 120 to 225 of this 301-amino-acid protein. The above results demonstrate that VP22 contains specific information required for targeting to membranes of acidic compartments of the cell which may be derived from the TGN, suggesting a potential role for VP22 during tegumentation and/or final envelopment.     

A protein in rat prostatic interacting with androgen regulated gene

2M NaCl-insoluble fraction of rat ventral prostatechromatin(residual proteins)contain proteins able tointeract specifically with androgen-receptor complex andis,therefore,a part of the acceptor complex.Amongresidual proteins,a 97 KDa protein has been found whichbinds signifieantly to a genomic fragment containingan androgen-regulated gene coding for a 22 KDa protein.The biological significance of this binding in androgenaction need to be further studied. A mini-plasmid clone containing 22 KDa proteincoding sequence was cloned into Charon 4A genomiclibrary from which a 5.7 Kb genomic fragment wasisolated,identified by hybridization with a 5' and a 3'cDNA probes,and shown to contain the 5' flankingsequence.Restriction enzyme treatment of this fragmentyielded a 4.7 Kb restriction fragment representingthe 5' upstream region and a 1.0 Kb containing part ofthe coding sequence.Deletion studies indicated that the97 KDa protein bound only to a subclone of about 300 bpsegment.Furthermore,gel shifting experiment supportedits DNA-prptein binding.     

Herpes simplex virus tegument protein VP22 contains overlapping domains for cytoplasmic localization, microtubule interaction, and chromatin binding

We have previously shown that the 301-amino-acid herpes simplex virus tegument protein VP22 exhibits a range of subcellular localization patterns when expressed in isolation from other virus proteins. By using live-cell analysis of cells expressing green fluorescent protein (GFP)-tagged VP22 we have shown that when VP22 is first expressed in the cell it localizes to the cytoplasm, where, when present at high enough concentrations, it can assemble onto microtubules, causing them to bundle and become highly stabilized. In addition we have shown that when a cell expressing VP22 enters mitosis, the cytoplasmic population of VP22 translocates to the nucleus, where it efficiently binds mitotic chromatin. Here we have investigated the specific regions of the VP22 open reading frame required for these properties. Using GFP-VP22 as our starting molecule, we have constructed a range of N- and C-terminal truncations and analyzed their localization patterns in live cells. We show that the C-terminal 242 residues of VP22 are sufficient to induce microtubule bundling. Within this subregion, the C-terminal 89 residues contain a signal for cytoplasmic localization of the protein, while a larger region comprising the C-terminal 128 residues of the VP22 protein is required for mitotic chromatin binding. Furthermore, a central 100-residue domain of VP22 maintains the ability to bind microtubules without inducing bundling, suggesting that additional regions flanking this microtubule binding domain may be required to alter the microtubule network. Hence, the signals involved in dictating the complex localization patterns of VP22 are present in overlapping regions of the protein.     

Nuclear and mitochondrial localization signals overlap within bovine herpesvirus 1 tegument protein VP22

VP22, a tegument protein of bovine herpesvirus 1, accumulates in the nucleus of infected and transiently transfected cells. Previous studies indicated a possible regulatory function of VP22 within nuclei, but how VP22 enters nuclei is unknown. Despite the abundance of basic residues within this protein, no classic nuclear localization signal (NLS) motif has been identified. To identify the signal directing nuclear accumulation, a series of truncations, internal deletions, and point mutations were constructed. Fluorescence microscopy of cells transfected with VP22 constructs indicated that a sequence of 103 residues is necessary and sufficient for nuclear localization. This NLS sequence is conformation-sensitive in contrast to a classical sequential NLS. Energy depletion assays and co-immunoprecipitation suggested that this NLS sequence also binds histone H4, resulting in nuclear retention of VP22. In addition, a mitochondrial targeting sequence was identified at the C-terminal 49 amino acids, which overlapped the sequence required for nuclear targeting. Our findings demonstrate the diversity of VP22 protein to localize within the cell and provide the opportunity for VP22 to direct cargo specifically to different subcellular compartments.     

Bovine herpesvirus 1 tegument protein VP22 interacts with histones, and the carboxyl terminus of VP22 is required for nuclear localization

The bovine herpesvirus 1 (BHV-1) UL49 gene encodes a viral tegument protein termed VP22. UL49 homologs are conserved among alphaherpesviruses. Interestingly, the BHV-1 VP22 deletion mutant virus is asymptomatic and avirulent in infected cattle but produces only a slight reduction in titer in vitro. Attenuation of the BHV-1 VP22 deletion mutant virus in vivo suggests that VP22 plays a functional role in BHV-1 replication. In herpes simplex virus type 1, the VP22 homolog was previously shown to interact with another tegument protein,VP16, the alpha-transinducing factor in vitro. In this report, we show that (i) the nuclear targeting of VP22 is independent of other viral factors, (ii) the carboxyl terminus of VP22 is required for its nuclear localization, (iii) VP22 associates with histones and nucleosomes, (iv) an antihistone monoclonal antibody cross-reacts with VP22, and (v) acetylation of histone H4 is decreased in VP22-expressing cells as well as virus-infected cells. Our data suggest that VP22 may have a modulatory function during BHV-1 infection.     

Expression of largest RNA segment and synthesis of VP1 protein of bluetongue virus in insect cells by recombinant baculovirus: association of VP1 protein with RNA polymerase activity.

The bluetongue virus core particles have been shown to contain an RNA-directed RNA polymerase (1). To identify the protein responsible for the virion RNA polymerase activity, the complete 3.9 Kb DNA clone representing the largest RNA segment 1 (L1) of bluetongue virus (BTV-10) was placed under control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The derived recombinant virus was used to infect Spodoptera frugiperda cells. As demonstrated by stained polyacrylamide gel electrophoresis and by the use of bluetongue virus antibody, infected insect cells synthesized the largest protein of BTV-10 (VP1, 150 k Da). Antibody raised in rabbit to recombinant VP1 protein recognized bluetongue virus VP1 protein. The recombinant virus infected cell lysate had significantly inducible levels of RNA polymerase enzymatic activity as determined by a poly (U)-oligo (A) polymerase assay. The availability of enzymatically active bluetongue virus RNA polymerase provides a system in which we can precisely delineate the role this protein plays in the regulation of bluetongue replication.     

Distinctions between bovine herpesvirus 1 and herpes simplex virus type 1 VP22 tegument protein subcellular associations

The alphaherpesvirus tegument protein VP22 has been characterized with multiple traits including microtubule reorganization, nuclear localization, and nonclassical intercellular trafficking. However, all these data were derived from studies using herpes simplex virus type 1 (HSV-1) and may not apply to VP22 homologs of other alphaherpesviruses. We compared subcellular attributes of HSV-1 VP22 (HVP22) with bovine herpesvirus 1 (BHV-1) VP22 (BVP22) using green fluorescent protein (GFP)-fused VP22 expression vectors. Fluorescence microscopy of cell lines transfected with these constructs revealed differences as well as similarities between the two VP22 homologs. Compared to that of HVP22, the BVP22 microtubule interaction was much less pronounced. The VP22 nuclear interaction varied, with a marbled or halo appearance for BVP22 and a speckled or nucleolus-bound appearance for HVP22. Both VP22 homologs associated with chromatin at various stages of mitosis and could traffic from expressing cells to the nuclei of nonexpressing cells. However, distinct qualitative differences in microtubule, nuclear, and chromatin association as well as trafficking were observed. The differences in VP22 homolog characteristics revealed in this study will help define VP22 function within HSV-1 and BHV-1 infection.     

VP22 does not significantly enhance enzyme prodrug cancer gene therapy as a part of a VP22-HSVTk-GFP triple fusion construct

BACKGROUND: VP22 is a herpes simplex virus type 1 (HSV-1) tegument protein that has been suggested to spread from cell to cell, alone or as a part of fusion proteins. Creating controversy, some reports indicate that VP22 cannot facilitate significant intercellular spreading. To study the capacity of VP22 to cause spreading and enhance thymidine kinase/ganciclovir cancer gene therapy, we constructed a novel triple fusion protein containing VP22, HSV thymidine kinase and green fluorescent protein (VP22-Tk-GFP). This fusion protein has three functional domains in the same polypeptide, thus making it possible to reliably compare the causality between transduction rate and cell killing efficiency in vitro and in vivo. METHODS: VP22-Tk-GFP was cloned into lenti- and adenoviral vectors and used for expression studies, analyses for VP22-mediated protein spreading, and to study the effect of VP22 to thymidine kinase/ganciclovir-mediated cytotoxicity. The function of VP22-Tk-GFP was also investigated in vivo. RESULTS: The triple fusion protein was expressed correctly in vitro, but intercellular trafficking was not observed in any of the studied cell lines. However, under certain conditions, VP22-Tk-GFP sensitized cells more efficiently to ganciclovir than Tk-GFP. In vivo there was a trend for increased inhibition of tumor growth with VP22-Tk-GFP when ganciclovir was present, but the difference with Tk-GFP was not statistically significant. CONCLUSIONS: Based on our results, VP22 fusion proteins do not seem to traffic intercellularly at detectable levels in most tumor cell types. Even though VP22 enhanced cytotoxicity in one cell line in vitro, the effect in vivo was modest. Therefore, our results do not support the utility of VP22 as an enhancer of enzyme prodrug cancer gene therapy.     

Tyrosine phosphorylation of bovine herpesvirus 1 tegument protein VP22 correlates with the incorporation of VP22 into virions

Tyrosine phosphorylation has been shown to play a role in the replication of several herpesviruses. In this report, we demonstrate that bovine herpesvirus 1 infection triggered tyrosine phosphorylation of proteins with molecular masses similar to those of phosphorylated viral structural proteins. One of the tyrosine-phosphorylated viral structural proteins was the tegument protein VP22. A tyrosine 38-to-phenylalanine mutation totally abolished the phosphorylation of VP22 in transfected cells. However, construction of a VP22 tyrosine 38-to-phenylalanine mutant virus demonstrated that VP22 was still phosphorylated but that the phosphorylation site may change to the C terminus rather than be in the N terminus as in wild-type VP22. In addition, the loss of VP22 tyrosine phosphorylation correlated with reduced incorporation of VP22 compared to that of envelope glycoprotein D in the mutant viruses but not with the amount of VP22 produced during virus infection. Our data suggest that tyrosine phosphorylation of VP22 plays a role in virion assembly.     

Particle formation by a conserved domain of the herpes simplex virus protein VP22 facilitating protein and nucleic acid delivery

VP22, a structural protein of herpes simplex virus, exhibits unusual trafficking properties which we proposed might be exploited in gene and protein delivery applications. To pursue the use of the protein itself for cargo delivery into cells, we developed an expression system for the C-terminal half of VP22, residues 159-301 (VP22.C1), and purified the protein in high yields. Addition of short oligonucleotides (ODNs) induced the assembly of novel particles, which were regular spheres with a size range of 0.3 to 1.0 microm in diameter, incorporating both protein and ODN. Following the particles in living cells using fluorescently tagged ODNs, we show that they enter efficiently within 2-4 h, and reside stably in the cell cytoplasm for up to several days. Remarkably, however, light activation induced particle disruption and release of the protein and ODN to the nucleus and cytoplasm within seconds, a process that we have captured by time lapse microscopy. In addition to delivering antisense ODNs, ribozymes, and RNA/DNA hybrids, the VP22.C1 protein could also be modified to include peptides or proteins. These particles have the potential for delivery of a wide range of therapeutic agents in gene therapy and vaccine development.     

VP22 of herpes simplex virus 1 promotes protein synthesis at late times in infection and accumulation of a subset of viral mRNAs at early times in infection

VP22, encoded by the U_L49 gene, is one of the most abundant proteins of the herpes simplex virus 1 (HSV-1) tegument. In the present study we show VP22 is required for optimal protein synthesis at late times in infection. Specifically, in the absence of VP22, viral proteins accumulated to wild-type levels until ~6 h postinfection. At that time, ongoing synthesis of most viral proteins dramatically decreased in the absence of VP22, whereas protein stability was not affected. Of the individual proteins we assayed, VP22 was required for optimal synthesis of the late viral proteins gE and gD and the immediate-early protein ICP0 but did not have discernible effects on accumulation of the immediate-early proteins ICP4 or ICP27. In addition, we found VP22 is required for the accumulation of a subset of mRNAs to wild-type levels at early, but not late, times in infection. Specifically, the presence of VP22 enhanced the accumulation of gE and gD mRNAs until ~9 h postinfection, but it had no discernible effect at later times in infection. Also, VP22 did not significantly affect ICP0 mRNA at any time in infection. Thus, the protein synthesis and mRNA phenotypes observed with the U_L49-null virus are separable with regard to both timing during infection and the genes affected and suggest separate roles for VP22 in enhancing the accumulation of viral proteins and mRNAs. Finally, we show that VP22's effects on protein synthesis and mRNA accumulation occur independently of mutations in genes encoding the VP22-interacting partners VP16 and vhs.     

Herpes simplex virus tegument protein VP22 contains an internal VP16 interaction domain and a C-terminal domain that are both required for VP22 assembly into the virus particle

Many steps along the herpesvirus assembly and maturation pathway remain unclear. In particular, the acquisition of the virus tegument is a poorly understood process, and the molecular interactions involved in tegument assembly have not yet been defined. Previously we have shown that the two major herpes simplex virus tegument proteins VP22 and VP16 are able to interact, although the relevance of this to virus assembly is not clear. Here we have constructed a number of recombinant viruses expressing N- and C-terminal truncations of VP22 and have used them to identify regions of the protein involved in its assembly into the virus structure. Analysis of the packaging of these VP22 variants into extracellular virions revealed that the C terminus of VP22 is absolutely required for this process, with removal of the C-terminal 89 residues abrogating its incorporation. However, while these 89 residues alone were sufficient for specific incorporation of small amounts of VP22 into the tegument, efficient packaging of VP22 to the levels of full-length protein required an additional 52 residues of the protein. Coimmunoprecipitation assays indicated that these 52 residues also contained the interaction domain for VP16. Furthermore, analysis of the subcellular localization of the mutant forms of VP22 revealed that only those truncations that were efficiently assembled formed characteristic cytoplasmic trafficking complexes, suggesting that these complexes may represent the cellular location for VP22 assembly into the virus. Taken together, these results suggest that there are two determinants involved in the packaging of VP22-a C-terminal domain and an internal VP16 interaction domain, both of which are required for the efficient recruitment of VP22 to sites of virus assembly.     

Phosphorylation of the herpes simplex virus tegument protein VP22 has no effect on incorporation of VP22 into the virus but is involved in optimal expression and virion packaging of ICP0

Herpes simplex virus VP22 is a major tegument protein of unknown function. Very recently, we reported that the predominant effect of deleting the VP22 gene was on the expression, localization, and virion incorporation of ICP0. In addition, the Delta22 virus replicated poorly in epithelial MDBK cells. We have also previously shown that VP22 interacts with the tegument protein VP16 and the cellular microtubule network. While the majority of VP22 in infected cells is highly phosphorylated, the nonphosphorylated form of VP22 is the predominant species in the virion, suggesting a differential requirement for phosphorylation through virus replication. Hence, to study the significance of VP22 phosphorylation, we have now constructed two recombinant viruses expressing green fluorescent protein-VP22 (G22) in which the previously identified serine phosphorylation sites have been mutated either to alanine to abolish the phosphorylation status of VP22 (G22P-) or to glutamic acid to mimic permanent phosphorylation (G22P+). Localization studies indicated that the G22P- protein associated tightly with microtubules in some infected cells, suggesting that VP22 phosphorylation may control its interaction with the microtubule network. By contrast, VP22 phosphorylation had no effect on its ability to interact with VP16 and, importantly, had no effect on the relative packaging of VP22. Intriguingly, virion packaging of ICP0 was reduced in the G22P+ virus while ICP0 expression was reduced in the G22P- virus, suggesting that these two ICP0 defects, previously observed in the Delta22 virus, were attributable to different forms of VP22. Furthermore, the Delta22 virus replication defect in MDBK cells correlated with the expression of constitutively charged VP22 in the G22P+ virus. Taken together, these results suggest an important role for VP22 phosphorylation in its relationship with ICP0.     

Fluorescent tagging of herpes simplex virus tegument protein VP13/14 in virus infection

The cellular site of herpesvirus tegument assembly has yet to be defined. We have previously used a recombinant herpes simplex virus type 1 expressing a green fluorescent protein (GFP)-tagged tegument protein, namely VP22, to show that VP22 is localized exclusively to the cytoplasm during infection. Here we have constructed a similar virus expressing another fluorescent tegument protein, YFP-VP13/14, and have visualized the intracellular localization of this second tegument protein in live infected cells. In contrast to VP22, VP13/14 is targeted predominantly to the nuclei of infected cells at both early and late times in infection. More specifically, YFP-13/14 localizes initially to the nuclear replication compartments and then progresses into intense punctate domains that appear at around 12 h postinfection. At even later times this intranuclear punctate fluorescence is gradually replaced by perinuclear micropunctate and membranous fluorescence. While the vast majority of YFP-13/14 seems to be targeted to the nucleus, a minor subpopulation also appears in a vesicular pattern in the cytoplasm that closely resembles the pattern previously observed for GFP-22. Moreover, at late times weak fluorescence appears at the cell periphery and in extracellular virus particles, confirming that YFP-13/14 is assembled into virions. This predominantly nuclear targeting of YFP-13/14 together with the cytoplasmic targeting of VP22 may imply that there are multiple sites of tegument protein incorporation along the virus maturation pathway. Thus, our YFP-13/14-expressing virus has revealed the complexity of the intracellular targeting of VP13/14 and provides a novel insight into the mechanism of tegument, and hence virus, assembly.     

Intratumoral spread and increased efficacy of a p53-VP22 fusion protein expressed by a recombinant adenovirus

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.