Secreted variant of nucleoside diphosphate kinase from the intracellular parasitic nematode Trichinella spiralis.

The molecular components involved in the survival of the parasitic nematode Trichinella spiralis in an intracellular environment are poorly characterized. Here we demonstrate that infective larvae secrete a nucleoside diphosphate kinase when maintained in vitro. The secreted enzyme forms a phosphohistidine intermediate and shows broad specificity in that it readily accepts gamma-phosphate from both ATP and GTP and donates it to all nucleoside and deoxynucleoside diphosphate acceptors tested. The enzyme was partially purified from culture medium by ATP affinity chromatography and identified as a 17-kDa protein by autophosphorylation and reactivity with an antibody to a plant-derived homologue. Secreted nucleoside diphosphate kinases have previously been identified only in prokaryotic organisms, all of them bacterial pathogens. The identification of a secreted variant of this enzyme from a multicellular eukaryote is very unusual and is suggestive of a role in modulating host cell function.

Vaccinia virus blocks gamma interferon signal transduction: viral VH1 phosphatase reverses Stat1 activation.

We have analyzed the effects of vaccinia virus (VV) on gamma interferon (IFN-gamma) signal transduction. Infection of cells with VV 1 to 2 h prior to treatment with IFN-gamma inhibits phosphorylation and nuclear translocation of Stat1 and consequently blocks accumulation of mRNAs normally induced by IFN-gamma. While phosphorylation of other proteins in the IFN-gamma pathway was not affected, activation of Stat1 by other ligand-receptor systems was also blocked by VV. This block of Stat1 activation was dose dependent, and although viral protein synthesis was not required, entry and uncoating of viral cores appear to be needed to block the accumulation of phosphorylated Stat1. These results suggest that a virion component is responsible for the effect. VV virions contain a phosphatase (VH1) that is sensitive to the phosphatase inhibitor Na(3)VO(4) but not to okadaic acid. Addition of Na(3)VO(4) but not okadaic acid restored normal Stat1 phosphorylation levels in VV-infected cells. Moreover, virions containing reduced levels of VH1 were unable to block the IFN-gamma signaling pathway. In vitro studies show that the phosphatase can bind and dephosphorylate Stat1, indicating that this transcription factor can be a substrate for VH1. Our results reveal a novel mechanism by which VV interferes with the onset of host immune responses by blocking the IFN-gamma signal cascade through the dephosphorylating activity of the viral phosphatase VH1.

Inhibition of mitochondrial function by interferon.

We showed previously that type I interferon causes a down-regulation of mitochondrial gene expression. We show here that IFN treatment leads to functional impairment of mitochondria. Western blot analysis indicated that interferon treatment reduces the steady-state level of cytochrome b in murine L-929 cells. Interferon produced a reduction in cytochrome c oxidase and NADH-cytochrome c reductase activities of isolated mitochondria as well as inhibiting electron transport in isolated mitochondria and in intact cells. Several mitochondrial mRNAs are affected by interferon treatment in human Daudi lymphoblastoid cells, which are highly sensitive to the antiproliferative effects of interferon. Electron transport in Daudi cells was also inhibited by interferon both in intact cells and isolated mitochondria with a dose response identical to that for the antiproliferative response. In contrast, a Daudi strain resistant to the antiproliferative effects of interferon showed no down-regulation of mRNA expression and no inhibition of electron transport. Possibly as a consequence of the inhibitory effect on mitochondrial gene expression, treatment with interferon causes a reduction in cellular ATP levels. The inhibition of cellular growth by interferon may thus be partly a consequence of a reduction in cellular ATP levels.