Poliovirus neuropathogenicity depends on sequences within the 5 nontranslated region of the virus. Exchange of the poliovirus internal ribosomal entry site with its counterpart from human rhinovirus type 2 resulted in attenuation of neurovirulence in primates. Despite deficient virus propagation in cells of neuronal origin, nonpathogenic polio recombinants retain excellent growth characteristics in cell lines derived from glial neoplasms. Susceptibility of malignant glioma cells to poliovirus may be mediated by expression of a poliovirus receptor, CD155, in glial neoplasms. Intergeneric polio recombinants with heterologous internal ribosomal entry site elements unfolded strong oncolytic potential against experimentally induced gliomas in athymic mice. Our observations suggest that highly attenuated poliovirus recombinants may have applicability as biotherapeutic antineoplastic agents.M alignant gliomas are the most common primary tumors of the central nervous system (1). Available treatment is of limited utility for these tumors, and prognosis is therefore poor (1). The resistance of malignant gliomas to conventional therapies has inspired the search for novel strategies, and recently, these strategies have involved animal viruses, either as attenuated variants of pathogenic species with direct oncolytic properties (2, 3) or as delivery vehicles for foreign genetic material (4 -6).Poliovirus is a nonenveloped plus-stranded RNA virus belonging to Picornaviridae and is the causative agent of paralytic poliomyelitis. The vast majority of poliovirus infections remain asymptomatic, but 1-2% of cases result in neurologic complications (7). Restriction of poliovirus cell tropism to lower motor neurons resident within the spinal cord and brainstem gives rise to a highly characteristic clinical syndrome dominated by flaccid paralysis. Selective targeting of motor neurons by poliovirus is most likely determined by the distribution of its cellular receptor, the Ig superfamily molecule CD155. This assumption is supported by the observation that mice transgenic for CD155 develop a polio-like syndrome after poliovirus infection (8-10). In addition, cell-internal conditions favoring viral replication may contribute to poliovirus cell-type specificity.The neuropathogenicity of poliovirus depends on the celltype-specific function of its internal ribosomal entry site (IRES) element in cells of neuronal origin (10). The IRES is part of the 5Ј nontranslated region not only of picornaviruses (11) but also of hepatitis C virus (12, 13). IRES elements assure initiation of translation in a 5Ј end-independent, cap-independent manner (14-16). We have demonstrated that IRES elements encode strong cell-type-specific restrictions toward virus propagation.This restriction is illustrated by the highly attenuated phenotype of intergeneric recombinant polioviruses carrying IRES elements derived from human rhinovirus type 2 (10, 17). We have demonstrated that a prototype intergeneric poliovirus chimera [called PV1(RIPO)] is characterized by exce...

The mammalian protein kinase N (PKN) family of Serine/Threonine kinases comprises three isoforms, which are targets for Rho family GTPases. Small GTPases are major regulators of the cellular cytoskeleton, generating interest in the role(s) of specific PKN isoforms in processes such as cell migration and invasion. It has been reported that PKN3 is required for prostate tumour cell invasion but not PKN1 or 2. Here we employ a cell model, the 5637 bladder tumour cell line where PKN2 is relatively highly expressed, to assess the potential redundancy of these isoforms in migratory responses. It is established that PKN2 has a critical role in the migration and invasion of these cells. Furthermore, using a PKN wild-type and chimera rescue strategy, it is shown that PKN isoforms are not simply redundant in supporting migration, but appear to be linked through isoform specific regulatory domain properties to selective upstream signals. It is concluded that intervention in PKNs may need to be directed at multiple isoforms to be effective in different cell types.

SummaryIn animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality.

The multifunctional protein NS1 of minute virus of mice (MVMp) is posttranslationally modified and at least in part regulated by phosphorylation. The atypical lambda isoform of protein kinase C (PKC) phosphorylates residues T435 and S473 in vitro and in vivo, leading directly to an activation of NS1 helicase function, but it is insufficient to activate NS1 for rolling circle replication. The present study identifies an additional cellular protein kinase phosphorylating and regulating NS1 activities. We show in vitro that the recombinant novel PKC phosphorylates NS1 and in consequence is able to activate the viral polypeptide in concert with PKC for rolling circle replication. Moreover, this role of PKC was confirmed in vivo. We thereby created stably transfected A9 mouse fibroblasts, a typical MVMp-permissive host cell line with Flag-tagged constitutively active or inactive PKC mutants, in order to alter the activity of the NS1 regulating kinase. Indeed, tryptic phosphopeptide analyses of metabolically 32 P-labeled NS1 expressed in the presence of a dominant-negative mutant, PKCDN, showed a lack of distinct NS1 phosphorylation events. This correlates with impaired synthesis of viral DNA replication intermediates, as detected by Southern blotting at the level of the whole cell population and by BrdU incorporation at the single-cell level. Remarkably, MVM infection triggers an accumulation of endogenous PKC in the nuclear periphery, suggesting that besides being a target for PKC, parvovirus infections may also affect the regulation of this NS1 regulating kinase. Altogether, our results underline the tight interconnection between PKC-mediated signaling and the parvoviral life cycle.The regulation not only of cellular proteins but also of viral proteins by phosphorylation has attracted research interest for many years. Besides characterization of proteins which become phosphorylated and the identification of their regulatory kinases, much effort has been spent on the analysis of the signaling pathways involved and their functional consequences. We are particularly interested in understanding how the multifunctional nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVMp) is regulated. MVM consists of a small icosahedral capsid with a linear singlestranded DNA of negative polarity as a genome. The DNA of MVMp codes for the nonstructural proteins NS1 and NS2, of which the latter exists in three different isoforms, differing in their unique C termini only, as well as two capsid proteins, VP1 and VP2. NS1 is endowed with numerous biochemical activities, such as ATP binding and hydrolysis (12, 62), helicase (44, 62), site-specific binding to the cognate recognition motif [ACCA] 2-3 which is scattered throughout the viral genome (13, 19), and site-and strand-specific endonuclease (11,18,44). Furthermore, NS1 takes part in protein-protein interactions to form homo-oligomers (40, 52) or complexes with cellular partner proteins like the transcription factor SP1 (35), the cochaperone SGT (20, 58), or...