During egress from the nucleus, HSV capsids that contain DNA (termed C capsids) are preferentially enveloped at the inner nuclear membrane over capsid types lacking DNA. Using coimmunoprecipitation and biochemical analyses of wild-type and mutant capsids, we identify an interaction between a complex of pU L 17/ pU L 25, termed the C capsid-specific complex (CCSC), and pU L 31, a component of the nuclear egress complex (NEC). We also show that the interactions between these components are dependent on expression of all three proteins but occur independently of the pU L 31 interacting protein and NEC component pU L 34, as well as a kinase encoded by U S 3 that phosphorylates both pU L 31 and pU L 34. The interaction between the CCSC and pU L 31 in the NEC suggests a mechanism to conserve viral resources by promoting assembly of only those viral particles with the potential to become infectious. virus assembly | virus egress H erpesvirus nucleocapsids (type C capsids) are assembled in the nucleoplasm and are preferentially selected over other capsid types, such as type B that lack DNA, to undergo an initial or primary envelopment reaction at the inner nuclear membrane (INM) (reviewed in refs. 1 and 2). Primary envelopment requires the products of genes U L 31 and U L 34 in the HSV system (3-5). Orthologs of U L 31 and U L 34 are present in all known herpesviruses, and for those systems in which it has been studied, the requirement for these proteins in primary envelopment is conserved (6-9). U L 31 encodes a nucleoplasmic phosphoprotein, pU L 31 (10), that is maintained in close approximation to the INM by association with pU L 34, a type II integral membrane protein (5,(11)(12)(13). The bulk of pU L 34 is located within the nucleoplasm, with only five amino acids predicted to lie within the perinuclear space (14, 15). Although it has been established that pU L 31 and pU L 34 are incorporated into perinuclear virions (16,17), whether the capsid engages the pU L 31/pU L 34 complex (also known as the nuclear envelopment complex or NEC) directly or indirectly is not known.The U L 17 and U L 25 gene products interact, forming a stable complex (18). DNA-containing C capsids contain ≈75 copies of pU L 25, whereas B capsids contain ≈20 copies (10). Because of its enrichment in C capsids, the U L 25/U L 17 complex was named the C capsid-specific complex or CCSC (19). The CCSC bridges pentameric vertices to the adjacent 20 planar faces on the capsid surface (10,19,20). One hypothesis to explain how C capsids are selected for envelopment is that the products of U L 25 and U L 17 bind more efficiently to the surface of type C capsids after DNA packaging is complete (19), and these capsids subsequently engage the NEC complex either directly or indirectly. Consistent with this hypothesis is the observation that U L 17 and U L 25 null capsids do not become enveloped (21,22). However, CCSC components have additional functions that may contribute indirectly to envelopment. For example, U L 17 is necessary for DNA to be cleaved and...

Caudal-related homeobox transcription factor 2 (CDX2), an intestine-specific nuclear transcription factor, has been strongly implicated in the tumourigenesis of various human cancers. However, the functional role of CDX2 in the development and progression of colorectal cancer (CRC) is not well known. In this study, CDX2 knockdown in colon cancer cells promoted cell proliferation in vitro, accelerated tumor formation in vivo, and induced a cell cycle transition from G0/G1 to S phase, whereas CDX2 overexpression inhibited cell proliferation. TOP/FOP-Flash reporter assay showed that CDX2 knockdown or CDX2 overexpression significantly increased or decreased Wnt signaling activity. Western blot assay showed that downstream targets of Wnt signaling, including β-catenin, cyclin D1 and c-myc, were up-regulated or down-regulated in CDX2-knockdown or CDX2-overexpressing colon cancer cells. In addition, suppression of Wnt signaling by XAV-939 led to a marked suppression of the cell proliferation enhanced by CDX2 knockdown, whereas activation of this signaling by CHIR-99021 significantly enhanced the cell proliferation inhibited by CDX2 overexpression. Dual-luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays further confirmed that CDX2 transcriptionally activates glycogen synthase kinase-3β (GSK-3β) and axis inhibition protein 2 (Axin2) expression by directly binding to the promoter of GSK-3β and the upstream enhancer of Axin2. In conclusion, these results indicated that CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/β-catenin signaling.

Herpes simplex virus (HSV) terminase is an essential component of the molecular motor that translocatesHerpesvirus procapsids and concatameric viral DNA accumulate in the nuclei of infected cells. The procapsids consist of a roughly spherical proteinaceous shell surrounding an inner protein shell or scaffold (16,24,36). To initiate DNA packaging, an enzyme called the terminase is believed to scan the viral DNA in search of genomic ends, cleave the concatemer into single genomes, engage the procapsid at a portal vertex designed for the passage of the DNA, and drive the genome into capsids through the hydrolysis of ATP.Current evidence supports the hypotheses that the herpes simplex virus (HSV) terminase comprises the products of U L 15, U L 28, and U L 33 (pU L 15, pU L 28, and pU L 33, respectively), whereas the portal vertex consists of a dodecamer of the U L 6 protein (pU L 6). These hypotheses are supported by the observations that (i) pU L 6, pU L 15, pU L 28, and pU L 33 are each essential for DNA packaging (2,5,25,26,34,44); (ii) epitopes of these proteins are present on the external surface of viral capsids, and at least pU L 15 and pU L 28 are associated with procapsids (23, 31, 41); (iii) pU L 15 interacts with the pU L 28 moiety of a pU L 28/pU L 33 complex in infected cells (9,18,19,43); (iv) pU L 15 contains an ATPase-like motif that is essential for viral replication (13, 45); (v) pU L 28 binds DNA sequences known to be required for the formation of normal DNA termini (1, 17); and (iv) pU L 6 forms a dodecameric ring in vitro with a size and conformation that match the dimensions of capsid vertices and portal vertices of some bacteriophages (23, 37).The main focus of the current study concerns a key question that distinguishes two models of DNA packaging: specifically, whether the terminase engages the portal vertex in the cytoplasm or in the nucleus. If the terminase were to engage the portal in the cytoplasm, it follows that portal assembly into the procapsid would also incorporate the bound terminase. This would imply that the entire procapsid, with incorporated terminase, would then scan viral DNA in search of genomic ends. On the other hand, if the terminase were imported into the nucleus separately from the portal, it would be free to scan the DNA independently of the procapsid and, once bound to target DNA sequences, could engage the portal vertex for eventual DNA cleavage and translocation into the capsid. The latter mechanism is similar to that used by many bacteriophage terminases (4,6,11).Where the HSV terminase forms in the cell and where the portal and terminase interact have been addressed previously using transient expression assays. For example, transiently expressed pseudorabies virus pU L 28 localizes in the cytoplasm unless coexpressed with HSV type 1 (HSV-1) pU L 15, suggesting that pU L 15 is responsible for the import of the terminase complex (20). On the other hand, pU L 6 was also shown to import pU L 28 into the nucleus when the proteins were coexpressed, and mutations that ...

Viral terminases play essential roles as components of molecular motors that package viral DNA into capsids. Previous results indicated that the putative terminase subunits of herpes simplex virus 1 (HSV-1Late in infection with all herpesviruses, capsids lacking DNA and viral concatameric DNA accumulate in infected-cell nuclei. By analogy to double-stranded DNA bacteriophages, it is presumed that capsid assembly culminates when a viral terminase cleaves the concatameric DNA into genomic lengths and hydrolyzes ATP to drive the DNA through a unique structure within the capsid, termed the portal vertex. In the case of herpes simplex virus (HSV), the portal vertex is likely composed of a dodecameric ring of the U L 6 protein (pU L 6) (16,21).Terminases consist of at least two subunits in all viral systems studied to date (7). Although obtaining direct evidence for the identity of the terminase subunits in herpesviruses has been hampered by the lack of an in vitro packaging system, several lines of indirect evidence have implicated the products of U L 15 and U L 28 (pU L 15 and pU L 28, respectively) as terminase components as follows: (i) pU L 15 and pU L 28 interact in vitro and in vivo with one another and in vitro with the portal protein pU L 6 (1, 6, 11, 12, 22), (ii) pU L 28 has been shown to bind DNA sequences necessary for formation of genomic ends (2), (iii) pU L 15 contains a highly conserved Walker box motif that is essential for HSV DNA packaging and resembles motifs maintained in the ATPase domains of some bacteriophage terminases (9,15,23), and (iv) it is likely that the terminase functions are conserved, inasmuch as the homologs of pU L 15 and pU L 28 in human cytomegalovirus (hCMV), encoded by U L 89 and U L 56, respectively, which also interact, have been shown to form a complex with the hCMV portal protein and are required for DNA packaging (10, 13).The approximately 19,000-M r protein encoded by herpes simplex virus 1 (HSV-1) U L 33 has also been shown to interact with pU L 15 and pU L 28 by immunoprecipitation from lysates of HSV-infected cells (6). Although its exact function is not known, pU L 33, like pU L 15 and pU L 28, is required for DNA cleavage and packaging (3,8). Thus, engineered mutations in any of these genes can cause empty capsids lacking DNA to accumulate in infected cells (3,4,20). Small amounts of pU L 33, pU L 15, and pU L 28 have also been shown to associate with HSV-1 capsids, suggesting that they maintain their interaction during packaging (5,18,24).Because it would provide information about the HSV terminase, one goal of the present work was to characterize the roles of the individual proteins in the formation of the pU L 15/ pU L 33/pU L 28 complex. MATERIALS AND METHODS Cells and virus.Vero cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, 100 U of penicillin per ml, and 100 g of streptomycin per ml (growth medium). A U L 15 deletion virus was propagated on a cell line designated clone 17 as previously described (4). A pr...

Prp8 stands out among hundreds of splicing factors as a key regulator of spliceosome activation and a potential cofactor of the splicing reaction. We present here the crystal structure of a 274-residue domain (residues 1,822-2,095) near the C terminus of Saccharomyces cerevisiae Prp8. The most striking feature of this domain is a ␤-hairpin finger protruding out of the protein (hence, this domain will be referred to as the ␤-finger domain), resembling many globular ribosomal proteins with protruding extensions. Mutations throughout the ␤-finger change the conformational equilibrium between the first and the second catalytic step. Mutations at the base of the ␤-finger affect U4/U6 unwinding-mediated spliceosome activation. Prp8 may insert its ␤-finger into the first-step complex (U2/U5/U6/pre-mRNA) or U4/U6.U5 tri-snRNP and stabilize these complexes. Mutations on the ␤-finger likely alter these interactions, leading to the observed mutant phenotypes. Our results suggest a possible mechanism of how Prp8 regulates spliceosome activation. These results also demonstrate an analogy between a spliceosomal protein and ribosomal proteins that insert extensions into folded rRNAs and stabilize the ribosome. P re-mRNA splicing is a critical step for gene expression in all eukaryotes. In eukaryotes, DNA is first transcribed to premRNAs whose introns have to be accurately removed before mRNA export and translation. Introns are removed through two transesterification steps. In the first step, the 2Ј-OH group of a critical adenosine residue in the branch point sequence (BPS) attacks the 5Ј end of the intron and forms a lariat intermediate. In the second step, the newly freed 3Ј-OH group of the 5Ј-end exon attacks the 3Ј-end of the intron, releasing the lariat and ligating the two exons.Pre-mRNA splicing is catalyzed by the spliceosome, a large RNA/protein complex that contains five snRNAs (U1, U2, U4, U5, and U6) and over 100 different protein factors. The spliceosome appears to assemble on pre-mRNA in a stepwise manner (1), although evidence also exists that the spliceosome may preassemble before encountering a pre-mRNA substrate (2). During spliceosome assembly, the 5Ј splice site (ss), BPS, and 3Ј ss of pre-mRNA are first recognized by the U1 snRNP, SF1/BBP, and U2AF65/35, respectively. Next, U2 snRNP replaces SF1 and base-pairs with the BPS. Subsequently, the U4/U6.U5 tri-snRNP joins the spliceosome. Next, extensive structural rearrangements occur to form the catalytically active spliceosome complex (first-step complex), which contains U2, U5, U6, and the pre-mRNA (3). During this activation process, the base-pairing between the 5Ј ss and U1 snRNA is disrupted, and the 5Ј ss interacts with the ACAGA box of U6 instead, using largely the same nucleotides that base-paired with U1 snRNA. The base-pairing between U4 and U6 is also disrupted, and new interactions between U2 and U6, which are mutually exclusive with those in the original U4/U6 complex, are formed. In addition, the BPS interacts with U2 snRNA, and both exons interac...

Branchio-oto-renal syndrome (BOR) is an autosomal dominant developmental disorder characterized by hearing loss, branchial arch defects, and renal anomalies. Recently, eight mutations in the SIX1 homeobox gene were discovered in BOR patients. To characterize the effect of SIX1 BOR mutations on the EYA-SIX1-DNA complex, we expressed and purified six of the eight mutants in Escherichia coli. We demonstrate that only the most N-terminal mutation in SIX1 (V17E) completely abolishes SIX1-EYA complex formation, whereas all of the other mutants are able to form a stable complex with EYA. We further show that only the V17E mutant fails to localize EYA to the nucleus and cannot be stabilized by EYA in the cell. The remaining five SIX1 mutants are instead all deficient in DNA binding. In contrast, V17E alone has a DNA binding affinity similar to that of wild type SIX1 in complex with the EYA co-factor. Finally, we show that all SIX1 BOR mutants are defective in transcriptional activation using luciferase reporter assays. Taken together, our experiments demonstrate that the SIX1 BOR mutations contribute to the pathology of the disease through at least two different mechanisms that involve: 1) abolishing the formation of the SIX1-EYA complex or 2) diminishing the ability of SIX1 to bind DNA. Furthermore, our data demonstrate for the first time that EYA: 1) requires the N-terminal region of the SIX1 Six domain for its interaction, 2) increases the level of the SIX1 protein within the cell, and 3) increases the DNA binding affinity of SIX1. Branchio-oto-renal syndrome (BOR; Mendelian Inheritance in Man (MIM) 113650)5 is an autosomal dominant developmental disorder that is characterized by hearing loss, branchial fistulae, and renal anomalies. Although the penetrance of the syndrome is highly variable between and even within families (1), 70 -93% of BOR patients exhibit hearing loss (1). This hearing loss can be conductive, sensorineural, or mixed and ranges in severity. In total, BOR affects an estimated 1 in 40,000 children and accounts for 2% of profoundly deaf children (2).The most commonly mutated gene in BOR syndrome is EYA1 (3), with an estimated 40% of BOR patients exhibiting mutations in this gene (4). EYA1 belongs to the EYA gene family of transcriptional co-factors. There are four mammalian members (EYA1-4), each containing an N-terminal transactivation domain (5), and a highly conserved ϳ270-amino acid C-terminal Eya domain (ED), also referred to as the eya homologous region. The ED possesses phosphatase activity (6 -8) and is involved in protein-protein interactions with the SIX family of homeoproteins (9 -12). The SIX family of homeoproteins are characterized by a DNA-binding homeodomain (HD) and the protein-interaction Six domain (SD), which binds directly to the ED of EYA (9). As a complex, the SIX and EYA proteins are believed to form a bipartite transcription factor where SIX confers DNA binding and EYA confers transactivation activity.Recently, mutations in two SIX family members (SIX5 and SIX1) have also be...

Prp8 is a critical pre-mRNA splicing factor. Prp8 is proposed to help form and stabilize the spliceosome catalytic core and to be an important regulator of spliceosome activation. Mutations in human Prp8 (hPrp8) cause a severe form of the genetic disorder retinitis pigmentosa, RP13. Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been hindered by its large size (over 2000 amino acids) and remarkably low-sequence similarity with other proteins. Here we present the crystal structure of the C-terminal domain (the last 273 residues) of Caenorhabditis elegans Prp8 (cPrp8). The core of the C-terminal domain is an a/b structure that forms the MPN (Mpr1, Pad1 N-terminal) fold but without Zn 2+ coordination. We propose that the C-terminal domain is a protein interaction domain instead of a Zn 2+ -dependent metalloenzyme as proposed for some MPN proteins. Mapping of RP13 mutants on the Prp8 structure suggests that these residues constitute a binding surface between Prp8 and other partner(s), and the disruption of this interaction provides a plausible molecular mechanism for RP13.Keywords: Prp8; retinitis pigmentosa; MPN domain; crystal structure Supplemental material: see www.proteinscience.org Pre-mRNA splicing is carried out by the spliceosome, a huge RNA/protein complex made of five snRNAs and over 100 protein factors. Prp8 is a key factor for premRNA splicing. Prp8 was proposed to help form/stabilize the catalytic core and to be an important regulator in spliceosome activation (Collins and Guthrie 2000; Grainger and Beggs 2005). Prp8 is the only spliceosome protein that extensively cross-links with the 59 splice site, the 39 splice site, the branch point sequence, and the U5 and U6 snRNAs, all of which are considered components of the catalytic core (Grainger and Beggs 2005 and references therein). In addition, Prp8 interacts with a number of protein partners required for spliceosome activation (Grainger and Beggs 2005 and references therein; Brenner and Guthrie 2006;Liu et al. 2006;Small et al. 2006). Many Prp8 mutations suppress or enhance splicing defects caused by splice-site mutations and other mutations affecting spliceosome activation or the second step of splicing (Grainger and Beggs 2005 and references therein). Several mutations in the C terminus of Prp8 are associated with RP13, a severe form of human genetic disorder retinitis pigmentosa, which leads to progressive photoreceptor degeneration in the retina and eventual blindness (McKie et al. 2001;van Lith-Verhoeven et al. 2002;Kondo et al. 2003;Martinez-Gimeno et al. 2003).Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been difficult due to its large size and low-sequence similarity 3 These authors contributed equally to this work. Reprint requests to: Rui Zhao, M.S. 8101, P.O. Box 6511, Aurora, CO 80045, USA; e-mail: rui.zhao@uchsc.edu; fax: (303) 724-3215.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/...

The herpes simplex virus 1 (HSV-1) UL51 gene encodes a 244-amino-acid (aa) palmitoylated protein that is conserved in all herpesviruses. The alphaherpesvirus UL51 (pUL51) protein has been reported to function in nuclear egress and cytoplasmic envelopment. No complete deletion has been generated because of the overlap of the UL51 coding sequence 5= end with the UL52 promoter sequences, but partial deletions generated in HSV and pseudorabies virus (PrV) suggest an additional function in epithelial cell-to-cell spread. Here we show partial uncoupling of the replication, release, and cell-to-cell spread functions of HSV-1 pUL51 in two ways. Viruses in which aa 73 to 244 were deleted from pUL51 or in which a conserved YXX⌽ motif near the N terminus was altered showed cell-specific defects in spread that cannot be accounted for by defects in replication and virus release. Also, a cell line that expresses C-terminally enhanced green fluorescent protein (EGFP)-tagged pUL51 supported normal virus replication and release into the medium but the formation of only small plaques. This cell line also failed to support normal localization of gE to cell junctions. gE and pUL51 partially colocalized in infected cells, and these two proteins could be coimmunoprecipitated from infected cells, suggesting that they can form a complex during infection. The cell-to-cell spread defect associated with the pUL51 mutation was more severe than that associated with gE-null virus, suggesting that pUL51 has gE-independent functions in epithelial cell spread. IMPORTANCEHerpesviruses establish and reactivate from lifelong latency in their hosts. When they reactivate, they are able to spread within their hosts despite the presence of a potent immune response that includes neutralizing antibody. This ability is derived in part from a specialized mechanism for virus spread between cells. Cell-to-cell spread is a conserved property of herpesviruses that likely relies on conserved viral genes. An understanding of their function may aid in the design of vaccines and therapeutics. Here we show that one of the conserved viral genes, UL51, has an important role in cell-to-cell spread in addition to its previously demonstrated role in virus assembly. We find that its function depends on the type of cell that is infected, and we show that it interacts with and modulates the function of another viral spread factor, gE.