Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is involved in pre-mRNA splicing in the nucleus and translational regulation in the cytoplasm. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here we demonstrate that hnRNP A1 not only is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5 untranslated region (UTR) of enterovirus 71 (EV71) and regulates IRES-dependent translation but also binds to the 5 UTR of Sindbis virus (SV) and facilitates its translation. The cytoplasmic relocalization of hnRNP A1 in EV71-infected cells leads to the enhancement of EV71 IRES-mediated translation, and its function can be substituted by hnRNP A2, whereas the cytoplasmic relocalization of hnRNP A1 following SV infection enhances the SV translation, but this function cannot be replaced by hnRNP A2. Our study provides the first direct evidence that the cytoplasmic relocalization of hnRNP A1 controls not only the IRES-dependent but also non-IRES-dependent translation initiations of RNA viruses.hnRNP A1, an RNA binding protein that shuttles between the nucleus and the cytoplasm, belongs to a large group of RNA binding proteins (hnRNPs) that are classified into several families and subfamilies based on conserved structural and functional motifs (13,25). Several hnRNPs, such as hnRNPs A1, C1/C2, E1/E2, I (polypyrimidine tract binding protein [PTB]), and L, are involved in the translational control of mRNAs containing internal ribosome entry sites (IRESs) through a cap-independent mechanism. These hnRNP proteins constitute IRES trans-acting factors (ITAFs) that modulate the activity of IRES sequences present in the 5Ј untranslated region (UTR) of viral or cellular mRNAs (7,29,44).hnRNP A1 is involved in several RNA metabolic processes such as pre-mRNA splicing and trafficking (13, 50). The hnRNP A1 protein is composed of 320 amino acids and contains two RNA binding domains and a glycine-rich domain that is responsible for protein-protein interactions. The signal that mediates shuttling between the nucleus and cytoplasm has been identified as the C-terminal 38 amino acids termed M9 (43, 49). Cytoplasmic hnRNP A1 and nuclear hnRNP A1 have different RNA binding profiles. Cytoplasmic hnRNP A1 has a high affinity for AU-rich elements (18, 20), whereas nuclear hnRNP A1 has a high affinity for a polypyrimidine stretch bordered by AG at the 3Ј ends of introns (34, 46). Interestingly, hnRNP A1 has been shown to be involved in the replication of many viruses such as mouse hepatitis virus (MHV), hepatitis C virus (HCV), dengue virus, and human papillomavirus type 16 through an interaction with the transcription-regulatory region of viral mRNA (9,23,36,39,52). hnRNP A1 has also been reported to interact with the human cytomegalovirus immediate-early gene 2 protein, which plays an important role in the regulation of virus replication (48). Recently, hnRNP A1 was reported to relocalize from the nucleus to the cytoplasm after vesicular stomatitis ...
Major biological effects of estrogen in the uterus are thought to be primarily mediated by nuclear estrogen receptors, ERalpha and ERbeta. We show here that estrogen in an ER-independent manner rapidly up-regulates the expression of Wnt4 and Wnt5a of the Wnt family and frizzled-2 of the Wnt receptor family in the mouse uterus. One of the mechanisms by which Wnts mediate canonical signaling involves stabilization of intracellular beta-catenin. We observed that estrogen treatment prompts nuclear localization of active beta-catenin in the uterine epithelium. We also found that adenovirus mediated in vivo delivery of SFRP-2, a Wnt antagonist, down-regulates estrogen-dependent beta-catenin activity without affecting some of the early effects (water imbibition and angiogenic markers) and inhibits uterine epithelial cell growth, suggesting that canonical Wnt signaling is critical to estrogen-induced uterine growth. Our present results provide evidence for a novel role of estrogen that targets early Wnt/beta-catenin signaling in an ER-independent manner to regulate the late uterine growth response that is ER dependent.
Enterovirus 71 (EV71) is a picornavirus that can cause severe neurological complications in children. Like other picornaviruses, the genomic RNA of EV71 contains a long 59 untranslated region (UTR). Cellular proteins interact with the EV71 59 UTR, and these interactions are important for virus replication. Using an RNA pull-down assay and proteomics approaches, this study identified the heterogeneous nuclear ribonucleoprotein K (hnRNP K) as one of the EV71 59 UTR-associated proteins. The interaction between hnRNP K and the 59 UTR was further confirmed by mapping the interaction regions to stem-loops I-II and IV in the 59 UTR. During EV71 infection, hnRNP K was enriched in the cytoplasm where virus replication occurs, whereas hnRNP K was localized in the nucleus in mock-infected cells. Viral yields were found to be significantly lower in hnRNP K knockdown cells and viral RNA synthesis was delayed in hnRNP K knockdown cells in comparison with negative-control cells treated with small interfering RNA. These results suggest that hnRNP K interacts with the EV71 59 UTR and participates in virus replication.
An internal ribosomal entry site (IRES) that directs the initiation of viral protein translation is a potential drug target for enterovirus 71 (EV71). Regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the internal ribosomal entry site. Biotinylated RNA-affinity chromatography and proteomic approaches were employed to identify far upstream element (FUSE) binding protein 2 (FBP2) as an ITAF for EV71. The interactions of FBP2 with EV71 IRES were confirmed by competition assay and by mapping the association sites in both viral IRES and FBP2 protein. During EV71 infection, FBP2 was enriched in cytoplasm where viral replication occurs, whereas FBP2 was localized in the nucleus in mock-infected cells. The synthesis of viral proteins increased in FBP2-knockdown cells that were infected by EV71. IRES activity in FBP2-knockdown cells exceeded that in the negative control (NC) siRNA-treated cells. On the other hand, IRES activity decreased when FBP2 was over-expressed in the cells. Results of this study suggest that FBP2 is a novel ITAF that interacts with EV71 IRES and negatively regulates viral translation.
Enterovirus 71 (EV71) is associated with severe neurological disorders in children, and has been implicated as the infectious agent in several large-scale outbreaks with mortalities. Upon infection, the viral RNA is translated in a cap-independent manner to yield a large polyprotein precursor. This mechanism relies on the presence of an internal ribosome entry site (IRES) element within the 5′-untranslated region. Virus–host interactions in EV71-infected cells are crucial in assisting this process. We identified a novel positive IRES trans-acting factor, far upstream element binding protein 1 (FBP1). Using binding assays, we mapped the RNA determinants within the EV71 IRES responsible for FBP1 binding and mapped the protein domains involved in this interaction. We also demonstrated that during EV71 infection, the nuclear protein FBP1 is enriched in cytoplasm where viral replication occurs. Moreover, we showed that FBP1 acts as a positive regulator of EV71 replication by competing with negative ITAF for EV71 IRES binding. These new findings may provide a route to new anti-viral therapy.
AU-rich element binding factor 1 (AUF1) has a role in the replication cycles of different viruses. Here we demonstrate that AUF1 binds the internal ribosome entry site (IRES) of enterovirus 71 (EV71) and negatively regulates IRES-dependent translation. During EV71 infection, AUF1 accumulates in the cytoplasm where viral replication occurs, whereas AUF1 localizes predominantly in the nucleus in mock-infected cells. AUF1 knockdown in infected cells increases IRES activity and synthesis of viral proteins. Taken together, the results suggest that AUF1 interacts with the EV71 IRES to negatively regulate viral translation and replication.
<b><i>Background:</i></b> Combined therapy with tyrosine kinase inhibitors (TKIs) and anti-PD-1 antibodies has shown high tumor response rates for patients with unresectable hepatocellular carcinoma (HCC). However, using this treatment strategy to convert initially unresectable HCC to resectable HCC was not reported. <b><i>Methods:</i></b> Consecutive patients with unresectable HCC who received first-line therapy with combined TKI/anti-PD-1 antibodies were analyzed. Tumor response and resectability were evaluated via imaging every 2 months (±2 weeks) using RECIST v1.1. Resectability criteria were (1) R0 resection could be achieved with sufficient remnant liver volume and function; (2) intrahepatic lesions were evaluated as partial responses or stable disease for at least 2 months; (3) no severe or persistent adverse effects occurred; and (4) hepatectomy was not contraindicated. <b><i>Results:</i></b> Sixty-three consecutive patients were enrolled. Of them, 10 (15.9%) underwent R0 resection in 3.2 months (range: 2.4–8.3 months) after the initiation of combination therapy. At baseline, these 10 patients had a median largest tumor diameter of 9.3 cm, 7 had Barcelona Clinic Liver Cancer stage C (vascular invasion) disease, 2 had stage B, and 1 had stage A. Before surgery, 6 patients were evaluated as a partial response, 3 stable disease, and 1 partial response in the intrahepatic lesion but a new metastatic lesion in the right adrenal gland. Six patients (60%) achieved a pathological complete response. One patient died from immune-related adverse effects 2.4 months after hepatectomy. After a median follow-up of 11.2 months (range: 7.8–15.9 months) for other 9 patients, 8 survived without disease recurrence, and 1 experienced tumor recurrence. <b><i>Conclusions:</i></b> Combination of TKI/anti-PD-1 antibodies is a feasible conversion therapy for patients with unresectable HCC to become resectable. This study represents the largest patient cohort on downstaging role of combinational systemic therapy on TKI and PD-1 antibody for HCC.
Enteroviruses use a type I IRES structure to facilitate protein synthesis and promote genome replication. Type I IRES elements require auxiliary host proteins to organize RNA structure for 40S ribosomal subunit assembly. Heterogeneous nuclear ribonucleoprotein A1 stimulates Enterovirus 71 (EV71) translation in part through specific interactions with its stem loop II (SLII) IRES domain. Here, we determined a conjoined NMR-SAXS structure of the EV71 SLII domain and a mutant that significantly attenuates viral replication by abrogating hnRNP A1 interactions. Native SLII adopts a locally compact structure wherein stacking interactions in a conserved 5′-AUAGC-3′ bulge preorganize the adjacent helices at nearly orthogonal orientations. Mutating the bulge sequence to 5′-ACCCC-3′ ablates base stacking in the loop and globally reorients the SLII structure. Biophysical titrations reveal that the 5′-AUAGC-3′ bulge undergoes a conformational change to assemble a functional hnRNP A1-RNA complex. Importantly, IRES mutations that delete the bulge impair viral translation and completely inhibit replication. Thus, this work provides key details into how an EV71 IRES structure adapts to hijack a cellular protein and it suggests the SLII domain is a potential target for antiviral therapy.
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