RNPS1, Acinus, and SAP18 form the apoptosis-and splicing-associated protein (ASAP) complex, which is also part of the exon junction complex. Whereas RNPS1 was originally identified as a general activator of mRNA processing, all three proteins have been found within functional spliceosomes. Both RNPS1 and Acinus contain typical motifs of splicing regulatory proteins including arginine/serine-rich domains. Due to the absence of such structural features, however, a function of SAP18 in splicing regulation is completely unknown. Here we have investigated splicing regulatory activities of the ASAP components. Whereas a full-length Acinus isoform displayed only limited splicing regulatory activity, both RNPS1 and, surprisingly, SAP18 strongly modulated splicing regulation. Detailed mutational analysis and three-dimensional modeling data revealed that the ubiquitinlike fold of SAP18 was required for efficient splicing regulatory activity. Coimmunoprecipitation and immunofluorescence experiments demonstrated that SAP18 assembles a nuclear speckle-localized splicing regulatory multiprotein complex including RNPS1 and Acinus via its ubiquitin-like fold. Our results therefore suggest a novel function of SAP18 in splicing regulation.
Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5'-triphosphate RNA (3pRNA) triggers anti-tumor immunity, which is dependent on natural killer (NK) cell activation and cytokine induction. However, to date, RIG-I expression and the functional consequences of RIG-I activation in NK cells have not been examined. Here, we show for the first time the expression of RIG-I in human NK cells and their activation upon RIG-I ligand (3pRNA) transfection. 3pRNA-activated NK cells killed melanoma cells more efficiently than NK cells activated by type I interferon. Stimulation of RIG-I in NK cells specifically increased the surface expression of membrane-bound TNF-related apoptosis-inducing ligand (TRAIL) on NK cells, while activated NK cell receptors were not affected. RIG-I-induced membrane-bound TRAIL initiated death-receptor-pathway-mediated apoptosis not only in allogeneic but also in autologous human leukocyte antigen (HLA) class I-positive and HLA class I-negative melanoma cells. These results identify the direct activation of RIG-I in NK cells as a novel mechanism for how RIG-I can trigger enhanced NK cell killing of tumor cells, underscoring the potential of RIG-I activation for tumor immunotherapy. This article is protected by copyright. All rights reserved.
The liver constitutes a prime site of cytomegalovirus (CMV) replication and latency. Hepatocytes produce, secrete, and recycle a chemically diverse set of bile acids, with the result that interactions between bile acids and cytomegalovirus inevitably occur. Here we determined the impact of naturally occurring bile acids on mouse CMV (MCMV) replication. In primary mouse hepatocytes, physiological concentrations of taurochenodeoxycholic acid (TCDC), glycochenodeoxycholic acid, and to a lesser extent taurocholic acid significantly reduced MCMV-induced gene expression and diminished the generation of virus progeny, while several other bile acids did not exert antiviral effects. The anticytomegalovirus activity required active import of bile acids via the sodium-taurocholate-cotransporting polypeptide (NTCP) and was consistently observed in hepatocytes but not in fibroblasts. Under conditions in which alpha interferon (IFN-␣) lacks antiviral activity, physiological TCDC concentrations were similarly effective as IFN-␥. A detailed investigation of distinct steps of the viral life cycle revealed that TCDC deregulates viral transcription and diminishes global translation in infected cells. IMPORTANCECytomegaloviruses are members of the Betaherpesvirinae subfamily. Primary infection leads to latency, from which cytomegaloviruses can reactivate under immunocompromised conditions and cause severe disease manifestations, including hepatitis. The present study describes an unanticipated antiviral activity of conjugated bile acids on MCMV replication in hepatocytes. Bile acids negatively influence viral transcription and exhibit a global effect on translation. Our data identify bile acids as site-specific soluble host restriction factors against MCMV, which may allow rational design of anticytomegalovirus drugs using bile acids as lead compounds. C ytomegaloviruses (CMV) are members of the Betaherpesvirinae subfamily and persist for life in infected individuals during alternating phases of latency and productive reactivation. Seroprevalence studies indicate that the large majority of the global human population is currently infected with human cytomegalovirus (HCMV) (human herpesvirus 5 [HHV-5]; taxonomy ID 10359). Although HCMV-related fatalities in apparently healthy individuals sporadically occur (1, 2), HCMV replication usually remains subclinical. This drastically changes under immunocompromising conditions, where untreated CMV infections often cause overt disease, including hepatitis (3) and dysfunction, bleeding, ulceration, and perforation of organs of the upper as well as lower gastrointestinal tract (4). HCMV hepatitis is particularly common in liver transplant recipients and is associated with organ dysfunction and graft failure (5-9).The species specificity of CMV precludes meaningful in vivo experimentation with HCMV in small animal models. Therefore, the homologous mouse CMV (MCMV) (Murid herpesvirus 1, taxonomy ID 10366), which infects Mus musculus (taxonomy ID 10090) as its natural host, has been estab...
Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using ⌬ie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611. IMPORTANCECytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using ⌬ie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication and delineated pIE611-dependent changes of the MCMV proteome. Our findings have fundamental implications for the interpretation of earlier studies on pIE3 functions and highlight the complex orchestration of MCMV gene regulation. Human cytomegalovirus (HCMV) is a ubiquitous humanpathogenic herpesvirus. Primary infection is mostly subclinical and leads to lifelong persistence of the viral genome in a latent state from which reactivation occurs under immune stress or immunosuppression. HCMV reactivation is a major cause of morbidity and mortality in AIDS patients and other immunocompromised individuals (1). Like HCMV, mouse cytomegalovirus (MCMV) represents a prototypical member of the family of betaherpesviruses, and it has been extensively used as a model system to study common aspects of CM...
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