Vesicular Egress of Non-Enveloped Lytic Parvoviruses Depends on Gelsolin Functioning

The autonomous parvovirus Minute Virus of Mice (MVM) induces specific changes in the cytoskeleton filaments of infected permissive cells, causing in particular the degradation of actin fibers and the generation of “actin patches.” This is attributed to a virus-induced imbalance between the polymerization factor N-WASP (Wiscott-Aldrich syndrome protein) and gelsolin, a multifunctional protein cleaving actin filaments. Here, the focus is on the involvement of gelsolin in parvovirus propagation and virus-induced actin processing. Gelsolin activity was knocked-down, and consequences thereof were determined for virus replication and egress and for actin network integrity. Though not required for virus replication or progeny particle assembly, gelsolin was found to control MVM (and related H1-PV) transport from the nucleus to the cell periphery and release into the culture medium. Gelsolin-dependent actin degradation and progeny virus release were both controlled by (NS1)/CKIIα, a recently identified complex between a cellular protein kinase and a MVM non-structural protein. Furthermore, the export of newly synthesized virions through the cytoplasm appeared to be mediated by (virus-modified) lysomal/late endosomal vesicles. By showing that MVM release, like entry, is guided by the cytoskeleton and mediated by vesicles, these results challenge the current view that egress of non-enveloped lytic viruses is a passive process

Replicating parvoviruses that target colon cancer cells.

The wnt signaling pathway is constitutively activated in colon tumors by mutations in the adenomatous polyposis coli and beta-catenin genes. We have modified the minute virus of mice (MVM) P4 promoter to make it responsive to wnt signaling by inserting binding sites for the heterodimeric beta-catenin/Tcf transcription factor. In luciferase assays we can see up to 20-fold selectivity of Tcf mutant P4 promoters for cells with activated wnt signaling. Hybrid MVM/H-1 viruses containing Tcf mutant promoters were tested for NS1 expression, viral DNA replication, virus replication, and cytopathic effect on colon, lung, kidney, and cervical cancer cell lines. Activation of the wnt pathway by expression of Delta N-beta-catenin increased NS1 expression and viral burst size in 293T and H1299 lung cancer cells, showing that the Tcf mutant P4 promoter can respond to wnt signals in the context of the virus. Compared to the parental virus, the burst size of the Tcf mutant viruses was reduced at least 1,000-fold in H1299, 293T, NB324K, and HeLa cells, which have inactive wnt signaling pathways. The burst size and cytopathic effect of the Tcf viruses was near wild-type levels in SW480 and Isreco1 colon cancer cell lines, which have high Tcf activity. The high specificity of these viruses should permit the development of H-1 virus-based vectors which combine high safety and greater efficacy in cancer therapy

Replicating parvoviruses that target colon cancer cells

The wnt signaling pathway is constitutively activated in colon tumors by mutations in the adenomatous polyposis coli and beta-catenin genes. We have modified the minute virus of mice (NWM) P4 promoter to make it responsive to wnt signaling by inserting binding sites for the heterodimeric beta-catenin/Tcf transcription factor. In luciferase assays we can see up to 20-fold selectivity of Tcf mutant P4 promoters for cells with activated wnt signaling. Hybrid MVM/H-1 viruses containing Tcf mutant promoters were tested for NS1 expression, viral DNA replication, virus replication, and cytopathic effect on colon, lung, kidney, and cervical cancer cell lines. Activation of the wnt pathway by expression of DeltaN-beta-catenin increased NS1 expression and viral burst size in 293T and H1299 lung cancer cells, showing that the Tcf mutant P4 promoter can respond to wnt signals in the context of the virus. Compared to the parental virus, the burst size of the Tcf mutant viruses was reduced at least 1,000-fold in H1299, 293T, NB324K, and HeLa cells, which have inactive wnt signaling pathways. The burst size and cytopathic effect of the Tcf viruses was near wild-type levels in SW480 and Isreco1 colon cancer cell lines, which have high Tcf activity. The high specificity of these viruses should permit the development of H-1 virus-based vectors which combine high safety and greater efficacy in cancer therapy.</p

The M2 muscarinic receptor antagonist methoctramine activates mast cells via pertussis toxin-sensitive G proteins

Methoctramine, a selective M2 muscarinic cholinergic receptor antagonist, has been reported to activate phosphoinositide breakdown at high concentrations. Its polyamine structure suggests a putative activation of guanine nucleotide-binding proteins (G proteins). Incubation of methoctramine with rat peritoneal mast cells resulted in a dose-dependent noncytotoxic histamine release, with an EC50 of 20 microM and a maximum effect at 1 mM. Atropine, pirenzepine and HHSiD neither inhibited methoctramine-induced histamine release nor stimulated histamine release. Histamine release and inositol phosphates generation induced by methoctramine were both inhibited by pertussis toxin pretreatment. Benzalkonium chloride, a selective inhibitor of histamine secretion induced by basic secretagogues, inhibited the secretory response to methoctramine. [p-Glu5, D-Trp7,9,l0]-SPs5-11 (GPAnt-2), a well-characterized antagonist of G proteins, blocked the methoctramine-induced histamine release when the antagonist was allowed to reach its intracellular target by streptolysin O-permeabilization. The response to methoctramine was prevented by the hydrolysis of sialic acid residues of the cell surface by neuraminidase. The response of mast cells was restored by permeabilization of the plasma membrane. These results demonstrate that methoctramine, following its entry into the cell and the involvement of pertussis toxin-sensitive G proteins, activates phosphoinositide hydrolysis leading to mast cell exocytosis

The Kinase IKKβ Regulates a STING-and NF-κB-Dependent Antiviral Response Pathway in Drosophila

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