Canonical Wnt/␤-catenin signaling regulates the activation of the myogenic determination gene Myf5 at the onset of myogenesis, but the underlying molecular mechanism is unknown. Here, we report that the Wnt signal is transduced in muscle progenitor cells by at least two Frizzled (Fz) receptors (Fz1 and/or Fz6), through the canonical ␤-catenin pathway, in the epaxial domain of newly formed somites. We show that Myf5 activation is dramatically reduced by blocking the Wnt/␤-catenin pathway in somite progenitor cells, whereas expression of activated ␤-catenin is sufficient to activate Myf5 in somites but not in the presomitic mesoderm. In addition, we identified Tcf/Lef sequences immediately 5Ј to the Myf5 early epaxial enhancer. These sites determine the correct spatiotemporal expression of Myf5 in the epaxial domain of the somite, mediating the synergistic action of the Wnt/␤-catenin and the Shh/Gli pathways. Taken together, these results demonstrate that Myf5 is a direct target of Wnt/␤-catenin, and that its full activation requires a cooperative interaction between the canonical Wnt and the Shh/Gli pathways in muscle progenitor cells.

Deletions and point mutations in the dystrophin gene cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy, depending on whether the translational reading frame is lost or maintained. Because internal in-frame deletions in the protein produce only mild myopathic symptoms, it should be possible, by preventing the inclusion of specific mutated exon(s) in the mature dystrophin mRNA, to restore a partially corrected phenotype. Such control has been previously accomplished by the use of synthetic oligonucleotides; nevertheless, a significant drawback to this approach is caused by the fact that oligonucleotides would require periodic administrations. To circumvent this problem, we have produced several constructs able to express in vivo, in a stable fashion, large amounts of chimeric RNAs containing antisense sequences. In this paper we show that antisense molecules against exon 51 splice junctions are able to direct skipping of this exon in the human DMD deletion 48 -50 and to rescue dystrophin synthesis. We also show that the highest skipping activity was found when antisense constructs against the 5 and 3 splice sites are coexpressed in the same cell. D uchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects 1 in every 3,500 males. It is characterized by the absence of the cytoskeletal dystrophin (427-kDa protein) that in turn produces a severe and progressive muscle deterioration. Most of the DMD mutations consist in deletions and point mutations in the 2.5-Mb dystrophin gene that introduce stop codons and consequently premature translation termination. A milder myopathy is the Becker muscular dystrophy; in this case, deletions inside the gene produce in frame mRNAs and consequently shorter but semifunctional dystrophin proteins (1). A third of DMD cases are the results of a de novo mutation (2, 3), therefore the disease can never be eliminated through genetic screening and counseling. For this reason, many efforts are now being devoted to the development of a treatment for this disorder, and several strategies have been designed that might provide an insight into finding a cure. One of these strategies involves the transplantation of normal myoblasts into the muscle tissues that lack this protein (4, 5), whereas the other strategy tries to restore correct expression of the dystrophin through a gene therapy approach. In this direction, several groups have tried to deliver full-length or mini cDNA copies of dystrophin into cells with the mutated gene (6-8). Even if this approach is very promising, several problems still remain to be solved, such as size capacity and transducing activity of the vector and immune response to the ''therapeutic'' gene (9).Another powerful approach of gene therapy is based on the fact that internal in-frame deletions in the protein produce only mild myopathic symptoms; therefore, it should be possible, by preventing the inclusion of specific mutated exon(s) in the mature dystrophin mRNA, to restore ...

Varicella-zoster virus (VZV) glycoprotein E (gE) is a multifunctional protein important for cell-cell spread, envelopment, and possibly entry. In contrast to other alphaherpesviruses, gE is essential for VZV replication. Interestingly, the N-terminal region of gE, comprised of amino acids 1 to 188, was shown not to be conserved in the other alphaherpesviruses by bioinformatics analysis. Mutational analysis was performed to investigate the functions associated with this unique gE N-terminal region. Linker insertions, serine-to-alanine mutations, and deletions were introduced in the gE N-terminal region in the VZV genome, and the effects of these mutations on virus replication and cell-cell spread, gE trafficking and localization, virion formation, and replication in vivo in the skin were analyzed. In summary, mutagenesis of the gE N-terminal region identified a new functional region in the VZV gE ectodomain essential for cell-cell spread and the pathogenesis of VZV skin tropism and demonstrated that different subdomains of the unique N-terminal region had specific roles in viral replication, cell-cell spread, and secondary envelopment.Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes two clinically different diseases: varicella (or chickenpox), during the primary infection, and zoster (or shingles), during virus reactivation from latency (3). The VZV genome is about 125 kb, the smallest among the human herpesviruses, and encodes at least 70 unique open reading frames (ORFs) (10,17,22). Although VZV and herpes simplex virus (HSV) types 1 and 2, the other two human alphaherpesviruses, show similarities in genome organization and share a number of homologous genes, VZV has unique mechanisms of pathogenesis that must be explained by its genetic differences from HSV. These mechanisms include T-cell tropism, which results in cell-associated viremia during primary VZV infection, and the characteristic formation of large polykaryocytes due to cell-cell fusion during skin infection (29).The VZV genome encodes nine putative glycoproteins that are known or presumed to be involved in different steps during the viral replication cycle: attachment and entry into the target cell, envelopment of the viral particles, cell-cell spread, and egress. The glycoprotein E (gE) is a 623-amino-acid (aa) typical type I membrane glycoprotein encoded by ORF68. gE is the most abundant glycoprotein expressed on the plasma membrane and in the cytoplasm of infected cells, and it is present on the virion envelope (10,20). gE is a multifunctional protein that has been shown to be involved in cell fusion and to localize to the trans-Golgi network (TGN), where the virus undergoes secondary envelopment (13,33,34,51).VZV gE moves from the endoplasmic reticulum to the Golgi membrane for its complete maturation (20). gE is recycled from the plasma membrane and targeted to the TGN through signal sequences contained in the C terminus (1, 63), which have been identified as the endocytosis motif YAGL (aa 582 to 585), and the TGN localizat...

Varicella-zoster virus (VZV) glycoprotein E (gE) is the most abundant glycoprotein in infected cells and, incontrast to those of other alphaherpesviruses, is essential for viral replication. The gE ectodomain contains a unique N-terminal region required for viral replication, cell-cell spread, and secondary envelopment; this region also binds to the insulin-degrading enzyme (IDE), a proposed VZV receptor. To identify new functional domains of the gE ectodomain, the effect of mutagenesis of the first cysteine-rich region of the gE ectodomain (amino acids 208 to 236) was assessed using VZV cosmids. Deletion of this region was compatible with VZV replication in vitro, but cell-cell spread of the rOka-⌬Cys mutant was reduced significantly. Deletion of the cysteine-rich region abolished the binding of the mutant gE to gI but not to IDE. Preventing gE binding to gI altered the pattern of gE expression at the plasma membrane of infected cells and the posttranslational maturation of gI and its incorporation into viral particles. In contrast, deletion of the first cysteine-rich region did not affect viral entry into human tonsil T cells in vitro or into melanoma cells infected with cell-free VZV. These experiments demonstrate that gE/gI heterodimer formation is essential for efficient cell-cell spread and incorporation of gI into viral particles but that it is dispensable for infectious varicella-zoster virion formation and entry into target cells. Blocking gE binding to gI resulted in severe impairment of VZV infection of human skin xenografts in SCIDhu mice in vivo, documenting the importance of cell fusion mediated by this complex for VZV virulence in skin.Varicella-zoster virus (VZV) is a human alphaherpesvirus and the causative agent of varicella (chicken pox). VZV infects the sensory ganglia, where it establishes lifelong latency, and causes herpes zoster (shingles) upon reactivation (8). VZV exhibits tropism for T cells (28,29), which appear to transport the virus from the site of inoculation to the skin during the primary infection through a cell-associated viremia; cell fusion during skin infection results in the formation of characteristic large polykaryocytes and vesicular lesions (8,27).The VZV genome (ϳ125 kb) encodes nine putative glycoproteins, which are known or presumed to contribute to the different steps of VZV replication: attachment and entry into the target cell, envelopment of the viral particles, cell-cell spread, and egress (8). Glycoprotein E (gE), the product of open reading frame 68 (ORF68), is a 623-amino-acid (aa) type I membrane protein that is essential for viral replication (34, 40) and involved in cell-cell fusion and secondary envelopment (3,9,35,36,50,53). gE, which is conserved among the alphaherpesviruses, is the most abundant glycoprotein expressed in VZV-infected cells (19). The cytosolic C terminus of gE (aa 562 to 623) contains sequences important for gE trafficking between the plasma membrane and the trans-Golgi network (TGN) of infected cells (1,25,49,62,65,66). Alteration of t...

Varicella-zoster virus (VZV) is a neurotropic alphaherpesvirus. VZV infection of human dorsal root ganglion (DRG) xenografts in immunodeficient mice models the infection of sensory ganglia. We examined DRG infection with recombinant VZV (recombinant Oka [rOka]) and the following gE mutants: gE⌬27-90, gE⌬Cys, gE-AYRV, and gE-SSTT. gE⌬27-90, which lacks the gE domain that interacts with a putative receptor insulin-degrading enzyme (IDE), replicated as extensively as rOka, producing infectious virions and significant cytopathic effects within 14 days of inoculation. Since neural cells express IDE, the gE/IDE interaction was dispensable for VZV neurotropism. In contrast, gE⌬Cys, which lacks gE/gI heterodimer formation, was significantly impaired at early times postinfection; viral genome copy numbers increased slowly, and infectious virus production was not detected until day 28. Delayed replication was associated with impaired cell-cell spread in ganglia, similar to the phenotype of a gI deletion mutant (rOka⌬gI). However, at later time points, infection of satellite cells and other supportive nonneuronal cells resulted in extensive DRG tissue damage and cell loss such that cytopathic changes observed at day 70 were more severe than those for rOka-infected DRG. The replication of gE-AYRV, which is impaired for trans-Golgi network (TGN) localization, and the replication of gE-SSTT, which contains mutations in an acidic cluster, were equivalent to that of rOka, causing significant cytopathic effects and infectious virus production by day 14; genome copy numbers were equivalent to those of rOka. These experiments suggest that the gE interaction with cellular IDE, gE targeting to TGN sites of virion envelopment, and phosphorylation at SSTT are dispensable for VZV DRG infection, whereas the gE/gI interaction is critical for VZV neurovirulence.Varicella-zoster virus (VZV) is a lymphotropic and neurotropic alphaherpesvirus and the etiological agent of varicella and herpes zoster. During primary infection, VZV virions gain access to sensory ganglion nerve cell bodies from either virion entry at nerve endings within cutaneous vesicular lesions or virus released by circulating infected T lymphocytes infiltrating ganglia (5). After the resolution of primary infection, VZV establishes a life-long latent infection within sensory nerve ganglia, from which it may reactivate to reinfect cutaneous sites. Thus, unlike infection of skin cells, VZV infection of neuronal ganglia requires neuronal cell survival to preserve host sites for the long-term establishment of latency.Experimental systems for examining early stages of VZV neurotropism are limited by a marked human host cell restriction that precludes the productive infection of sensory nerve ganglia in small-animal models (5). Experimental infection of human dorsal root ganglion (DRG) xenografts maintained in severe combined immunodeficient (SCID) mice permits the examination of the interactions between VZV and human neurons, satellite cells, and other neural cells in intact gan...