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It is uncertain whether nonenveloped karyophilic virus particles may actively traffic from the nucleus outward. The unordered amino-terminal domain of the VP2 major structural protein (2Nt) of the icosahedral parvovirus minute virus of mice (MVM) is internal in empty capsids, but it is exposed outside of the shell through the fivefold axis of symmetry in virions with an encapsidated single-stranded DNA genome, as well as in empty capsids subjected to a heat-induced structural transition. In productive infections of transformed and normal fibroblasts, mature MVM virions were found to efficiently exit from the nucleus prior to cell lysis, in contrast to the extended nuclear accumulation of empty capsids. Newly formed mutant viruses lacking the three phosphorylated serine residues of 2Nt were hampered in their exit from the human transformed NB324K nucleus, in correspondence with the capacity of 2Nt to drive microinjected phosphorylated heated capsids out of the nucleus. However, in normal mouse A9 fibroblasts, in which the MVM capsid was phosphorylated at similar sites but with a much lower rate, the nuclear exit of virions and microinjected capsids harboring exposed 2Nt required the infection process and was highly sensitive to inhibition of the exportin CRM1 in the absence of a demonstrable interaction. Thus, the MVM virion exits the nucleus by accessing nonconventional export pathways relying on cell physiology that can be intensified by infection but in which the exposure of 2Nt remains essential for transport. The flexible 2Nt nuclear transport signal may illustrate a common structural solution used by nonenveloped spherical viruses to propagate in undamaged host tissues.Many eukaryotic viruses invade the nucleus to access the cellular replication and transcription machineries that are necessary for their multiplication. A central process in the life cycle of karyophilic viruses is the passage of viral macromolecules across the nuclear pore complex (NPC) (13, 58), a supramolecular structure regulating nuclear-cytoplasmic transport. For nuclear invasion, viruses use the cellular classical and nonclassical protein import routes (73), which are directed in the classical pathway by basic nuclear localization signals (NLS) (26, 57) recognizing soluble transport receptors of the importin family and other cofactors (31, 72; for a review, see reference 38). The exposure of an NLS to interact with importins (27, 44) and the size of the nuclear viruses, which are generally larger than the 25-to 39-nm functional diameter of the NPC central channel for nondeformable cargo (14, 49), imposes in most cases a drastic conformational change or a complete disassembly of the virus structure for genome delivery into the nucleus (53, 66; for a review, see reference 12).Late in infection, viruses maturing within the nucleus must egress from the infected cell, and it is generally believed that membrane disorganization and cellular lysis follow the nuclear accumulation of virus particles. Nevertheless, the large enveloped herpesvirus...

Minute virus of mice (MVM) enters the host cell via receptor-mediated endocytosis. Although endosomal processing is required, its role remains uncertain. In particular, the effect of low endosomal pH on capsid configuration and nuclear delivery of the viral genome is unclear. We have followed the progression and structural transitions of DNA full-virus capsids (FC) and empty capsids (EC) containing the VP1 and VP2 structural proteins and of VP2-only virus-like particles (VLP) during the endosomal trafficking. Three capsid rearrangements were detected in FC: externalization of the VP1 N-terminal sequence (N-VP1), cleavage of the exposed VP2 N-terminal sequence (N-VP2), and uncoating of the full-length genome. All three capsid modifications occurred simultaneously, starting as early as 30 min after internalization, and all of them were blocked by raising the endosomal pH. In particles lacking viral single-stranded DNA (EC and VLP), the N-VP2 was not exposed and thus it was not cleaved. However, the EC did externalize N-VP1 with kinetics similar to those of FC. The bulk of all the incoming particles (FC, EC, and VLP) accumulated in lysosomes without signs of lysosomal membrane destabilization. Inside lysosomes, capsid degradation was not detected, although the uncoated DNA of FC was slowly degraded. Interestingly, at any time postinfection, the amount of structural proteins of the incoming virions accumulating in the nuclear fraction was negligible. These results indicate that during the early endosomal trafficking, the MVM particles are structurally modified by low-pH-dependent mechanisms. Regardless of the structural transitions and protein composition, the majority of the entering viral particles and genomes end in lysosomes, limiting the efficiency of MVM nuclear translocation.

IntroductionThere is an increasing interest in the active vitamin D metabolite 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ] and its analogs as preventive and therapeutic anticancer agents, and a number of clinical trials are presently underway (1-4). Epidemiological and experimental data in cultured cells and in animal models indicate their beneficial effect against colon cancer through the inhibition of cell proliferation and invasion and the induction of pro-apoptotic and pro-differentiation activities (5-7). Global transcriptomic studies have led to the identification of a few 1α,25(OH) 2 D 3 target genes, including some that encode cell-cycle regulators (p21 WAF1/CIP1 , p27 KIP1 , several cyclins), the gene that encodes the crucial intercellular adhesion and invasion suppressor E-cadherin, and several that encode cytoskeletal proteins (8-11). In addition, 1α,25(OH) 2 D 3 exerts an indirect gene-regulatory effect through the antagonism of the Wnt/β-catenin signaling pathway, which is aberrantly activated in most human colon cancers. By inducing a rapid interaction of its receptor (vitamin D receptor [VDR]) and β-catenin and the subsequent nuclear export of β-catenin, 1α,25(OH) 2 D 3 opposes the regulation of β-catenin/TCF complexes over a large number of genes in colon carcinoma cells (12,13).

Vitamin D deficiency is associated with the high risk of colon cancer and a variety of other diseases. The active vitamin D metabolite 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) regulates gene transcription via its nuclear receptor (VDR), and posttranscriptional regulatory mechanisms of gene expression have also been proposed. We have identified microRNA-22 (miR-22) and several other miRNA species as 1,25(OH)(2)D(3) targets in human colon cancer cells. Remarkably, miR-22 is induced by 1,25(OH)(2)D(3) in a time-, dose- and VDR-dependent manner. In SW480-ADH and HCT116 cells, miR-22 loss-of-function by transfection of a miR-22 inhibitor suppresses the antiproliferative effect of 1,25(OH)(2)D(3). Additionally, miR-22 inhibition increases cell migration per se and decreases the antimigratory effect of 1,25(OH)(2)D(3) in both cell types. In silico analysis shows a significant overlap between genes suppressed by 1,25(OH)(2)D(3) and miR-22 putative target genes. Consistently, miR-22 inhibition abrogates the 1,25(OH)(2)D(3)-mediated suppression of NELL2, OGN, HNRPH1, RERE and NFAT5 genes. In 39 out of 50 (78%) human colon cancer patients, miR-22 expression was found lower in the tumour than in the matched normal tissue and correlated directly with that of VDR. Our results indicate that miR-22 is induced by 1,25(OH)(2)D(3) in human colon cancer cells and it may contribute to its antitumour action against this neoplasia.

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The central role of Raf protein kinase isoforms in human cancer demands specific anti-Raf therapeutic inhibitors. Parvoviruses are currently used in experimental cancer therapy due to their natural oncotropism and lytic life cycle. In searching for mechanisms underlying parvovirus oncolysis, we found that trimers of the major structural protein (VP) of the parvovirus minute virus of mice (MVM), which have to be imported into the nucleus for capsid assembly, undergo phosphorylation by the Raf-1 kinase. Purified Raf-1 phosphorylated the capsid subunits in vitro to the two-dimensional pattern found in natural MVM infections. VP trimers isolated from mammalian cells translocated into the nucleus of digitonin-permeabilized human cells. In contrast, VP trimers isolated from insect cells, which are devoid of Raf-1, were neither phosphorylated nor imported into the mammalian nucleus. However, the coexpression of a constitutively active Raf-1 kinase in insect cells restored VP trimer phosphorylation and nuclear transport competence. In MVM-infected normal and transformed cells, Raf-1 inhibition resulted in cytoplasmic retention of capsid proteins, preventing their nuclear assembly and progeny virus maturation. The level of Raf-1 activity in cancer cells was consistent with the extent of VP specific phosphorylation and with the permissiveness to MVM infection. Thus, Raf-1 control of nuclear translocation of MVM capsid assembly intermediates provides a novel target for viral oncolysis. MVM may reinforce specific therapies against frequent human cancers with deregulated Raf signaling.The Raf protein isoforms (A, B, C, or Raf-1) belong to the conserved Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling module. This signal cascade transduces mitogenic and other stimuli from the cell surface to the nucleus (reviewed in reference 41). Activation of the Raf kinases is primarily triggered by increased levels of the upstream regulator Ras-GTP protein, which directly binds and recruits cytosolic dimers of the Raf kinases to the plasma membrane (58). The membrane-associated Raf kinases are activated by phosphorylation and other events (48), assembling a MAPK signaling complex formed by the MEK and ERK kinases and scaffold proteins (8). Upon phosphorylation and dimerization (25), an active ERK kinase dissociates from the complex and translocates into the nucleus. Intranuclear ERK kinase promotes multiple protein phosphorylations and changes of gene expression that may be altered in oncogenic and deregulated signaling (50, 55). (40,67), which led to the use of lytic viruses dependent on this pathway (7) in ongoing preclinical and clinical trials (51). Molecular insights into the interactions between viral substrates and components of the MAPK pathway could thus increase specificity and therapeutic efficacy of oncolytic viruses.The replication of virus members of the Parvoviridae relies on functions provided by proliferative cells (24). Together with diverse factors that are linked to the neoplastic growth, the underlyi...

The Wnt/b-catenin signalling pathway is activated in 90% of human colon cancers by nuclear accumulation of b-catenin protein due to its own mutation or to that of adenomatous polyposis coli. In the nucleus, b-catenin regulates gene expression promoting cell proliferation, migration and invasiveness. 1a,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) inhibits b-catenin signalling by inducing its binding to vitamin D receptor (VDR) and by promoting b-catenin nuclear export. The transcription factor Snail1 represses VDR expression and we demonstrate here that Snail1 also abolishes the nuclear export of b-catenin induced by 1,25(OH) 2 D 3 in SW480-ADH cells. Accordingly, Snail1 relieves the inhibition exerted by 1,25(OH) 2 D 3 on genes whose expression is driven by b-catenin, such as c-MYC, ectodermal-neural cortex-1 (ENC-1) or ephrin receptor B2 (EPHB2). In addition, Snail1 abrogates the inhibitory effect of 1,25(OH) 2 D 3 on cell proliferation and migration. In xenografted mice, Snail1 impedes the nuclear export of b-catenin and the inhibition of ENC-1 expression induced by EB1089, a 1,25(OH) 2 D 3 analogue. The elevation of endogenous SNAIL1 protein levels reproduces the effect of an ectopic Snail1 gene. Remarkably, the expression of exogenous VDR in cells with high levels of Snail1 normalizes the transcriptional responses to 1,25(OH) 2 D 3 . However, this exogenous VDR failed to fully restore the blockage of the Wnt/b-catenin pathway by 1,25(OH) 2 D 3 . This suggests that the effects of Snail1 on this pathway are not merely due to the repression of VDR gene. We conclude that Snail1 is a positive regulator of the Wnt/b-catenin signalling pathway in part through the abrogation of the inhibitory action of 1,25(OH) 2 D 3 .