Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, efficiently suppresses cellular innate immunity, represented by type I interferon (IFN), using its two unique nonstructural proteins, NS1 and NS2. In a search for their mechanism, NS1 was previously shown to decrease levels of TRAF3 and IKK, whereas NS2 interacted with RIG-I and decreased TRAF3 and STAT2. Here, we report on the interaction, cellular localization, and functional domains of these two proteins. We show that recombinant NS1 and NS2, expressed in lung epithelial A549 cells, can form homo-as well as heteromers. Interestingly, when expressed alone, substantial amounts of NS1 and NS2 localized to the nuclei and to the mitochondria, respectively. However, when coexpressed with NS2, as in RSV infection, NS1 could be detected in the mitochondria as well, suggesting that the NS1-NS2 heteromer localizes to the mitochondria. The C-terminal tetrapeptide sequence, DLNP, common to both NS1 and NS2, was required for some functions, but not all, whereas only the NS1 N-terminal region was important for IKK reduction. Finally, NS1 and NS2 both interacted specifically with host microtubule-associated protein 1B (MAP1B). The contribution of MAP1B in NS1 function was not tested, but in NS2 it was essential for STAT2 destruction, suggesting a role of the novel DLNP motif in protein-protein interaction and IFN suppression. Human respiratory syncytial virus (RSV) is a member of thePneumovirus genus in the Paramyxoviridae family (9, 25). RSV replicates primarily in the respiratory epithelium and is a leading cause of respiratory disease and asthma in the very young and the elderly (41, 42). The nonsegmented negative-strand RNA genome of RSV encodes 10 genes, of which the two most promoterproximal and abundantly transcribed genes code for nonstructural proteins 1 and 2 (NS1 and NS2), which are small proteins, 139 and 124 amino acid residues long, respectively (9, 25). The NS proteins circumvent the host innate immune system by preventing the induction of type I interferons (IFNs) as well as IFN-induced antiviral responses (6,12,20,22,23,29,(33)(34)(35)(36)39), thus allowing a more robust replication of the virus, leading to the severe respiratory disease that characterizes RSV infection.The IFN pathways of the cell can be divided into two functional ones, the IFN induction pathway, in which the cells produce IFN, and the IFN response pathway, in which the cells respond to exogenous IFN. One of the most proximal steps in the IFN induction pathway is the activation of the cytoplasmic RNA sensors of the RIG-I family (27,37,43,44). This triggers a cascade of signaling events whereby the CARD sequence of RIG-I interacts with and activates the CARD-like domaincontaining mitochondrial protein, MAVS (7, 18). Activated MAVS then activates TRAF3 (7, 31), which in turn activates two downstream kinases, IKKε and TBK1. The latter are Ser/ Thr kinases that phosphorylate the C-terminal domain of interferon regulatory factor 3 (IRF3) and IRF7, leading to...

The cyclic nucleotide phosphodiesterase10A (PDE10) has been mostly studied as a therapeutic target for certain psychiatric and neurological conditions, although a potential role in tumorigenesis has not been reported. Here we show that PDE10 is elevated in human colon tumor cell lines compared with normal colonocytes, as well as in colon tumors from human clinical specimens and intestinal tumors from ApcMin/+ mice compared with normal intestinal mucosa, respectively. An isozyme and tumor-selective role of PDE10 was evident by the ability of small molecule inhibitors and siRNA knockdown to suppress colon tumor cell growth with reduced sensitivity of normal colonocytes. Stable knockdown of PDE10 by shRNA also inhibits colony formation and increases doubling time of colon tumor cells. PDE10 inhibition selectively activates cGMP/PKG signaling to suppress β-catenin levels and T-cell factor (TCF) transcriptional activity in colon tumor cells. Conversely, ectopic expression of PDE10 in normal and precancerous colonocytes increases proliferation and activates TCF transcriptional activity. These observations suggest a novel role of PDE10 in colon tumorigenesis and that inhibitors may be useful for the treatment or prevention of colorectal cancer.

Phosphodiesterase 10A (PDE10) is a cGMP and cAMP degrading PDE isozyme that is highly expressed in the brain striatum where it appears to play an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntington's disease and are generally well tolerated, possibly because of low expression levels in most peripheral tissues. We recently reported high levels of PDE10 in colon tumors and that genetic silencing of PDE10 by siRNA or inhibition with small molecule inhibitors can suppress colon tumor cell growth with a high degree of selectivity over normal colonocytes (Li et al., Oncogene 2015). These observations suggest PDE10 may have an unrecognized role in tumorigenesis. Here we report that the concentration range by which the highly specific PDE10 inhibitor, Pf-2545920 (MP-10), inhibits colon tumor cell growth parallels the concentration range required to increase cGMP and cAMP levels, and activates PKG and PKA, respectively. Moreover, PDE10 knockdown by shRNA reduces the sensitivity of colon tumor cells to the growth inhibitory activity of Pf-2545920. Pf-2545920 also inhibits the translocation of β-catenin to the nucleus, thereby reducing β-catenin mediated transcription of survivin, resulting in caspase activation and apoptosis. PDE10 mRNA was also found to be elevated in colon tumors compared with normal tissues. These findings suggest that PDE10 can be targeted for cancer therapy or prevention whereby inhibition results in cGMP elevation and PKG activation to reduce β-catenin-mediated transcription of survival proteins leading to the selective apoptosis of cancer cells.