SUMMARY Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein had antiviral activity and mediated RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone marrow derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL; Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pulldown assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the nonneuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the proteinprotein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

MOV10 (Moloney leukemia virus 10, homolog) is an interferon-inducible RNA helicase, associated with small RNA-induced silencing. Here, we report that MOV10 exhibits antiviral activity, independent of its helicase function, against a number of positive and negative-strand RNA viruses by enhancing type I interferon (IFN) induction. Using a number of CRISPR/Cas9-mediated knockout human cells, we show that IRF3-mediated IFN induction and downstream IFN signaling through IFN receptor was necessary to inhibit virus replication by MOV10. MOV10 enhanced IRF3-mediated transcription of IFN. However, this IFN induction by MOV10 was unique and independent of the known RIG-I/MAVS-mediated RNA-sensing pathway. Upon virus infection, MOV10 specifically required IKKε not TBK1, for its antiviral activity. The important role of MOV10 in mediating antiviral signaling was further supported by the finding that viral proteases from picornavirus family specifically targeted MOV10 as a possible innate immune evasion mechanism. These results establish MOV10, an evolutionary conserved protein involved in RNA silencing, as an antiviral gene against RNA viruses that uses a RLR-independent pathway to enhance IFN response.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen. It is a common colonizer of the human respiratory track, where it utilizes cell-cell communication systems to coordinate population-level behaviors. We reasoned that secreted peptides that are highly expressed during infection are pivotal for virulence. Thus, we used in silico pattern searches to define a pneumococcal secretome, and analyzed the transcriptome of the clinically important PMEN1 lineage to identify which peptide-encoding genes are highly expressed in vivo. In this study, we characterized virulence peptide 1 (vp1), a highly expressed Gly-Gly peptide-encoding gene in chinchilla middle ear effusions. The vp1 gene is widely distributed across pneumococcus as well as encoded in related species. Studies in the chinchilla model of middle ear infection demonstrated that VP1 is a virulence determinant. The vp1 gene is positively regulated by a transcription factor from the Rgg family and its cognate SHP (short hydrophobic peptide). In vitro data indicated that VP1 promotes increased thickness and biomass for biofilms grown on chinchilla middle ear epithelial cells. Further, the wild-type biofilm is restored with the exogenous addition of synthetic VP1. We conclude that VP1 is a novel streptococcal regulatory peptide that controls biofilm development and pneumococcal pathogenesis.

Centrosome aberrations are a frequent finding in human tumors. However, very little is known about the molecular mechanisms leading to disruption of centrosome duplication control and the functional consequences of aberrant centrosome numbers. The high-risk human papillomavirus Type 16 (HPV-16) E6 and E7 oncoproteins are overexpressed in HPV-associated malignancies of the anogenital tract and have been instrumental in delineating different pathways of centrosome amplification. Whereas the E6 oncoprotein was found to provoke centrosome accumulation, the HPV-16 E7 oncoprotein triggers a genuine disruption of the centrosome duplication cycle. Importantly, the E7 oncoprotein can rapidly cause centrosome overduplication through a pathway that involves the concurrent formation of multiple daughters at single maternal centrioles (centriole flowers). Several lines of evidence suggest that cyclin E/CDK2 complexes and Polo-like kinase 4 (PLK4) are crucial players in this process. These findings underscore that the HPV-16 E7 oncoprotein is a unique tool to dissect normal and abnormal centriole biogenesis and the underlying molecular circuitry.

Abnormal centrosome and centriole numbers are frequently detected in tumor cells where they can contribute to mitotic aberrations that cause chromosome missegregation and aneuploidy. The molecular mechanisms of centriole overduplication in malignant cells, however, are poorly characterized. Here, we show that the core SKP1-cullin-F-box component cullin 1 (CUL1) localizes to maternal centrioles and that CUL1 is critical for suppressing centriole overduplication through multiplication, a recently discovered mechanism whereby multiple daughter centrioles form concurrently at single maternal centrioles. We found that this activity of CUL1 involves the degradation of Polo-like kinase 4 (PLK4) at maternal centrioles. PLK4 is required for centriole duplication and strongly stimulates centriole multiplication when aberrantly expressed. We found that CUL1 is critical for the degradation of active PLK4 following deregulation of cyclin E/cyclin-dependent kinase 2 activity, as is frequently observed in human cancer cells, as well as for baseline PLK4 protein stability. Collectively, our results suggest that CUL1 may function as a tumor suppressor by regulating PLK4 protein levels and thereby restraining excessive daughter centriole formation at maternal centrioles. [Cancer Res 2009;69(16):6668-75]

Most gastrointestinal stromal tumors (GIST) are caused by oncogenic KIT or platelet-derived growth factor receptor activation, and the small molecule kinase inhibitor imatinib mesylate is an effective first-line therapy for metastatic or unresectable GIST. However, complete remissions are rare and most patients ultimately develop resistance, mostly because of secondary mutations in the driver oncogenic kinase. Hence, there is a need for novel treatment options to delay failure of primary treatment and restore tumor control in patients who progress under therapy with targeted agents. Historic data suggest that GISTs do not respond to classical chemotherapy, but systematic unbiased screening has not been performed. In screening a compound library enriched for U.S. Food and Drug Administration (FDA)-approved chemotherapeutic agents (NCI Approved Oncology Drugs Set II), we discovered that GIST cells display high sensitivity to transcriptional inhibitors and topoisomerase II inhibitors. Mechanistically, these compounds exploited the cells' dependency on continuous KIT expression and/or intrinsic DNA damage response defects, explaining their activity in GIST. Mithramycin A, an indirect inhibitor of the SP1 transcription factor, and mitoxantrone, a topoisomerase II inhibitor, exerted significant antitumor effects in mouse xenograft models of human GIST. Moreover, these compounds were active in patient-derived imatinib-resistant primary GIST cells, achieving efficacy at clinically relevant concentrations. Taken together, our findings reveal that GIST cells have an unexpectedly high and specific sensitivity to certain types of FDAapproved chemotherapeutic agents, with immediate implications for encouraging their clinical exploration. Cancer Res; 74(4); 1200-13. Ó2014 AACR.

2=-5=-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein.Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. C ellular innate immunity against virus infection is primarily mediated by type I interferons (IFNs). In turn, the IFNs exert their pleiotropic effects through the induction of a variety of IFNstimulated genes (ISGs) (1-4). Although the general antiviral roles of several ISGs have been demonstrated, the roles of individual ISGs and their effect on specific viruses have remained largely unidentified (5-9). On the other hand, the coevolution of the host and the virus has resulted in viral strategies to evade the host IFN response by targeting ISGs and other IFN pathway proteins (10). Oligoadenylate synthetases (OAS) are a family of ISGs characterized by their ability to synthesize 2=-5=-oligoadenylate (2-5A), which induces RNA degradation by activating RNase L (11, 12). Human oligoadenylate synthetase-like protein (OASL) is related to the OAS family by its N-terminal OAS-like domain but is devoid of 2-5A synthetase activity. Additionally, OASL contains two tandem ubiquitin-like (UBL) domains in the C terminus, which are absent in any of the other members of the OAS family (12-15). OASL is directly and rapidly induced by virus infection via interferon regulatory factor 3 (IRF3) as well as by IFN signaling (1,12,16,17). Unlike in humans, two OASL isoforms have been identified in the mouse, Oasl1 and Oasl2. We have recently described the (18), were grown in monolayers on coverslips and infected with RSV Long at a multiplicity of infection of 3. At 18 h postinfection, cells were fixed and immunostained with mouse anti-RSV nucleoprotein (N) antibody (Abnova clone B023), followed by Alex Fluor 610-conjugated donkey anti-mouse IgG (Life Technologies). Images were captured in a Nikon AIRSI spectral confocal microscope system. (B) (Top) The same cell lines were infected as described above, and the total cell lysates were analyzed by immunoblotting using the same primary antibody described above and horseradish peroxidase (HRP)-conjugated secondary antibody, followed by ECL (enhanced chemiluminescence) detection. Actin is the loading control. (Bottom) Total RNA isolated from parallel cultures was subjected to quantitative reverse transcription-PCR (qRT-PCR), as described previously (38). The primers, synthesized by Integrated DNA Technologies (Coralville, IA), were as follows. RSV N gene, forward 5=-TGCAGGGCAAGTGATGTTAC-3=, and reverse, 5=-TTCCATTTCTGCTTGCACAC-3=; actin, forward, 5=-AGAAAATCTGGCACCACACC-3=, and reverse, 5=-GGGGTGTTGAAGGTCTCAAA-3=. A portion of the PCR sample was analyzed on 1.5% agarose...