A point mutation, E95D, in the mumps virus V protein disengages STAT3 targeting from STAT1 targeting.

Mumps virus, like other paramyxoviruses in the Rubulavirus genus, encodes a V protein that can assemble a ubiquitin ligase complex from cellular components, leading to the destruction of cellular signal transducer and activator of transcription (STAT) proteins. While many V proteins target the interferon-activated STAT1 or STAT2 protein, mumps virus V protein is unique in its ability to also target STAT3 for ubiquitin modification and proteasome-mediated degradation. Here we report that a single amino acid substitution in the mumps virus V protein, E95D, results in defective STAT3 targeting while maintaining the ability to target STAT1. Results indicate that the E95D mutation disrupts the ability of the V protein to associate with STAT3. A recombinant mumps virus carrying the E95D mutation in its P and V proteins replicates normally in cultured cells but fails to induce targeting of STAT3. Infection with the recombinant virus results in the differential regulation of a number of cellular genes compared to wild-type mumps virus and increases cell death in infected cells, producing a large-plaque phenotype.

Select paramyxoviral V proteins inhibit IRF3 activation by acting as alternative substrates for inhibitor of kappaB kinase epsilon (IKKe)/TBK1.

V accessory proteins from Paramyxoviruses are important in viral evasion of the innate immune response. Here, using a cell survival assay that identifies both inhibitors and activators of interferon regulatory factor 3 (IRF3)-mediated gene induction, we identified select paramyxoviral V proteins that inhibited double-stranded RNA-mediated signaling; these are encoded by mumps virus (MuV), human parainfluenza virus 2 (hPIV2), and parainfluenza virus 5 (PIV5), all members of the genus Rubulavirus. We showed that interaction between V and the IRF3/7 kinases, TRAF family member-associated NFkappaB activator (TANK)-binding kinase 1 (TBK1)/inhibitor of kappaB kinase epsilon (IKKe), was essential for this inhibition. Indeed, V proteins were phosphorylated directly by TBK1/IKKe, and this, intriguingly, resulted in lowering of the cellular level of V. Thus, it appears that V mimics IRF3 in both its phosphorylation by TBK1/IKKe and its subsequent degradation. Finally, a PIV5 mutant encoding a V protein that could not inhibit IKKe was much more susceptible to the antiviral effects of double-stranded RNA than the wild-type virus. Because many innate immune response signaling pathways, including those initiated by TLR3, TLR4, RIG-I, MDA5, and DNA-dependent activator of IRFs (DAI), use TBK1/IKKe as the terminal kinases to activate IRFs, rubulaviral V proteins have the potential to inhibit all of them.

Building proteomic pathways using Drosophila ventral furrow formation as a model.

Ventral furrow formation is the first morphogenetic movement to occur during Drosophila gastrulation causing the internalization of mesodermal precursors. A previous proteomic screen for ventral-specific proteome changes identified a set of about forty "difference-proteins" that spanned many cellular functions. To understand the connections between these disparate proteins, we initiated a pathway-building scheme using cycles of protein expression manipulation and proteome analysis. This pathway-building exercise started with the proteasomal subunit, Pros35, one of three proteasome subunits found to be ventral-specific difference-proteins. Here we show that Pros35 is a key regulator in ventral furrow formation. Altering the level of Pros35 led to ventral furrow defects. Proteome analysis of the changes induced by Pros35 RNAi showed extensive overlap with the original set of ventral-specific difference-proteins. One of the most prominent changes was in the extracellular iron carrier, Transferrin (Tsf1). Tsf1 is normally less abundant in ventral cells relative to lateral cells; however, RNAi of Pros35 in ventralized embryos negated this ventral-specific difference. Increasing Tsf1 in wild-type embryos blocked ventral furrow formation and caused proteome changes that were similar to the previously seen ventral-specific difference-proteins, including Pros35, which indicates the existence of an unprecedented regulatory loop between the proteasome and iron homeostasis. Additionally, we show that the iron regulatory protein, Irp-1A, also plays an important role in ventral furrow formation. Together these three proteins are part of a regulatory loop that coordinately controls a large number of ventral-specific protein changes.

Drosophila ventral furrow morphogenesis: a proteomic analysis.

Ventral furrow formation is a key morphogenetic event during Drosophila gastrulation that leads to the internalization of mesodermal precursors. While genetic analysis has revealed the genes involved in the specification of ventral furrow cells, few of the structural proteins that act as mediators of ventral cell behavior have been identified. A comparative proteomics approach employing difference gel electrophoresis was used to identify more than fifty proteins with altered abundance levels or isoform changes in ventralized versus lateralized embryos. Curiously, the majority of protein differences between these embryos appeared well before gastrulation, only a few protein changes coincided with gastrulation, suggesting that the ventral cells are primed for cell shape change. Three proteasome subunits were found to differ between ventralized and lateralized embryos. RNAi knockdown of these proteasome subunits and time-dependent difference-proteins caused ventral furrow defects, validating the role of these proteins in ventral furrow morphogenesis.