scholarly journals hnRNP A1 Interacts with the 5′ Untranslated Regions of Enterovirus 71 and Sindbis Virus RNA and Is Required for Viral Replication

2009 ◽  
Vol 83 (12) ◽  
pp. 6106-6114 ◽  
Author(s):  
Jing-Yi Lin ◽  
Shin-Ru Shih ◽  
Manjing Pan ◽  
Carol Li ◽  
Chia-Fang Lue ◽  
...  
Keyword(s):  
Translational Regulation ◽  
Sindbis Virus ◽  
Enterovirus 71 ◽  
Untranslated Regions ◽  
Hnrnp A1 ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Virus Rna ◽  
Infected Cells ◽  
Nuclear Ribonucleoprotein

ABSTRACT 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.

scholarly journals Phosphomimetic Substitution of Heterogeneous Nuclear Ribonucleoprotein A1 at Serine 199 Abolishes AKT-dependent Internal Ribosome Entry Site-transacting Factor (ITAF) Function via Effects on Strand Annealing and Results in Mammalian Target of Rapamycin Complex 1 (mTORC1) Inhibitor Sensitivity

2011 ◽  
Vol 286 (18) ◽  
pp. 16402-16413 ◽  
Author(s):  
Jheralyn Martin ◽  
Janine Masri ◽  
Cheri Cloninger ◽  
Brent Holmes ◽  
Nicholas Artinian ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Mammalian Target Of Rapamycin ◽  
Target Of Rapamycin ◽  
Hnrnp A1 ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Mtor Complex 1

The relative activity of the AKT kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mammalian target of rapamycin (mTOR) complex 1 inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates a salvage pathway facilitating internal ribosome entry site (IRES)-dependent mRNA translation of critical cellular determinants in an AKT-dependent manner following mTOR inhibitor exposure. This pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent AKT, resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated AKT activity, rendering them sensitive to rapamycin-induced G1 arrest as a result of the inhibition of global eIF-4E-mediated translation. AKT phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) that is capable of binding both the cyclin D1 and c-MYC IRES RNAs in vitro but lacks nucleic acid annealing activity, resulting in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which AKT is conditionally active, we demonstrate that overexpression of this mutant renders quiescent AKT-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated AKT is strongly correlated with elevated Ser(P)199-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 serine 199 regulates the AKT-dependent sensitivity of cells to rapamycin and functionally links IRES-transacting factor annealing activity to cellular responses to mTOR complex 1 inhibition.

scholarly journals Cytoplasmic Relocalization of Heterogeneous Nuclear Ribonucleoprotein A1 Controls Translation Initiation of Specific mRNAs

2007 ◽  
Vol 18 (12) ◽  
pp. 5048-5059 ◽  
Author(s):  
Anne Cammas ◽  
Frédéric Pileur ◽  
Sophie Bonnal ◽  
Stephen M. Lewis ◽  
Nicolas Lévêque ◽  
...  
Keyword(s):  
Hnrnp A1 ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Mrna Metabolism ◽  
Direct Demonstration ◽  
Specific Mrnas ◽  
Regulate Protein ◽  
Nuclear Ribonucleoprotein ◽  
Mrna Binding

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a nucleocytoplasmic shuttling protein that regulates gene expression through its action on mRNA metabolism and translation. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here, we show that hnRNP A1 is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5′ untranslated region of both the human rhinovirus-2 and the human apoptotic peptidase activating factor 1 (apaf-1) mRNAs, thereby regulating their translation. Furthermore, the cytoplasmic redistribution of hnRNP A1 after rhinovirus infection leads to enhanced rhinovirus IRES-mediated translation, whereas the cytoplasmic relocalization of hnRNP A1 after UVC irradiation limits the UVC-triggered translational activation of the apaf-1 IRES. Therefore, this study provides a direct demonstration that IRESs behave as translational enhancer elements regulated by specific trans-acting mRNA binding proteins in given physiological conditions. Our data highlight a new way to regulate protein synthesis in eukaryotes through the subcellular relocalization of a nuclear mRNA-binding protein.

scholarly journals Hsp27 Responds to and Facilitates Enterovirus A71 Replication by Enhancing Viral Internal Ribosome Entry Site-Mediated Translation

2019 ◽  
Vol 93 (9) ◽  
Author(s):  
Xuelian Dan ◽  
Qianya Wan ◽  
Lina Yi ◽  
Jing Lu ◽  
Yang Jiao ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Chinese Herb ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Initiation Factor ◽  
Hnrnp A1 ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Foot And Mouth

ABSTRACTEnterovirus 71 (EV-A71) is a human pathogen that causes hand, foot, and mouth disease (HFMD) and fatal neurological diseases, and no effective treatment is available. Characterization of key host factors is important for understanding its pathogenesis and developing antiviral drugs. Here we report that Hsp27 is one of the most upregulated proteins in response to EV-A71 infection, as revealed by two-dimensional gel electrophoresis-based proteomics studies. Depletion of Hsp27 by small interfering RNA or CRISPR/Cas9-mediated knockout significantly inhibited viral replication, protein expression, and reproduction, while restoration of Hsp27 restored such virus activities. Furthermore, we show that Hsp27 plays a crucial role in regulating viral internal ribosome entry site (IRES) activities by two different mechanisms. Hsp27 markedly promoted 2Apro-mediated eukaryotic initiation factor 4G cleavage, an important process for selecting and initiating IRES-mediated translation. hnRNP A1 is a key IREStrans-acting factor (ITAF) for enhancing IRES-mediated translation. Surprisingly, knockout of Hsp27 differentially blocked hnRNP A1 but not FBP1 translocation from the nucleus to the cytoplasm and therefore abolished the hnRNP A1 interaction with IRES. Most importantly, the Hsp27 inhibitor 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b] xanthone (TDP), a compound isolated from a traditional Chinese herb, significantly protected against cytopathic effects and inhibited EV-A71 infection. Collectively, our results demonstrate new functions of Hsp27 in facilitating virus infection and provide novel options for combating EV-A71 infection by targeting Hsp27.IMPORTANCEOutbreaks of infections with EV-A71, which causes hand, foot, and mouth disease, severe neurological disorders, and even death, have been repeatedly reported worldwide in recent decades and are a great public health problem for which no approved treatments are available. We show that Hsp27, a heat shock protein, supports EV-A71 infection in two distinct ways to promote viral IRES-dependent translation. A small-molecule Hsp27 inhibitor isolated from a traditional Chinese medicinal herb effectively reduces virus yields. Together, our findings demonstrate that Hsp27 plays an important role in EV-A71 infection and may serve as an antiviral target.

scholarly journals The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos.

1991 ◽  
Vol 11 (12) ◽  
pp. 5848-5859 ◽  
Author(s):  
I R Ghattas ◽  
J R Sanes ◽  
J E Majors
Keyword(s):  
Rous Sarcoma Virus ◽  
Cultured Cells ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Internal Promoter ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Infected Cells ◽  
Virus Internal Ribosome Entry

Rous sarcoma virus-based retroviral vectors were constructed to compare three different approaches for coexpressing two genes in individual infected cells. All vectors expressed the upstream gene (lacZ) from the Rous sarcoma virus long terminal repeat, while the downstream gene (the chloramphenicol acetyltransferase gene [cat] or v-src) was expressed in one of three ways: from a subgenomic mRNA generated by regulated splicing, from a strong internal promoter, or from the encephalomyocarditis virus internal ribosome entry site (IRES). Both biochemical and immunohistochemical assays of cultured cells showed that the encephalomyocarditis virus IRES provided the most efficient means for coexpressing two genes from a single provirus. Most importantly, most cells infected by a LacZ-IRES-CAT virus expressed both LacZ and CAT, whereas most cells infected by internal promoter or regulated splicing vectors expressed either LacZ or CAT but not both. In addition, viral titers were highest with IRES vectors. Presumably, use of the IRES avoids transcriptional controls and RNA processing steps that differentially affect expression of multiple genes from internal promoter and regulated splicing vectors. Finally, we injected a LacZ-IRES-v-Src virus into chicken embryos and then identified the progeny of infected cells with a histochemical stain for LacZ. LacZ-positive cells in both skin and mesenchyme displayed morphological abnormalities attributable to expression of v-src. Thus, IRES vectors can be used to coexpress a reporter gene and a bioactive gene in vivo.

scholarly journals Human Cytomegalovirus Induces the Endoplasmic Reticulum Chaperone BiP through Increased Transcription and Activation of Translation by Using the BiP Internal Ribosome Entry Site

2010 ◽  
Vol 84 (21) ◽  
pp. 11479-11486 ◽  
Author(s):  
Nicholas J. Buchkovich ◽  
Yongjun Yu ◽  
Francis J. Pierciey ◽  
James C. Alwine
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcriptional Activation ◽  
Internal Ribosome Entry Site ◽  
Hcmv Infection ◽  
Mrna Translation ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Unfolded Protein ◽  
Infected Cells ◽  
Protein Response

ABSTRACT The endoplasmic reticulum (ER) chaperone BiP (immunoglobulin binding protein) plays a major role in the control of the unfolded protein response. We have previously shown that BiP levels are dramatically increased during human cytomegalovirus (HCMV) infection, where BiP performs unique roles in viral assembly and egress. We show that BiP mRNA levels increase during infection due to activation of the BiP promoter by the major immediate-early (MIE) proteins. The BiP promoter, like other ER stress-activated promoters, contains endoplasmic reticulum stress elements (ERSEs), which are activated by unfolded protein response (UPR)-induced transcription factors. However, these elements are not needed for MIE protein-mediated transcriptional activation; thus, a virus-specific transcriptional activation mechanism is used. Transcriptional activation results in only a 3- to 4-fold increase in BiP mRNA, suggesting that additional mechanisms for BiP production are utilized. The BiP mRNA contains an internal ribosome entry site (IRES) which increases the level of BiP mRNA translation. We show that utilization of the BiP IRES is dramatically increased in HCMV-infected cells. Utilization of the BiP IRES can be activated by the La autoantigen, also called Sjögren's syndrome antigen B (SSB). We show that SSB/La levels are significantly increased during HCMV infection, and SSB/La depletion causes the loss of BiP IRES utilization and lowers endogenous BiP levels in infected cells. Our data show that BiP levels increase in HCMV-infected cells through the combination of increased BiP gene transcription mediated by the MIE proteins and increased BiP mRNA translation due to SSB/La-induced utilization of the BiP IRES.

scholarly journals Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation

2015 ◽  
Vol 89 (19) ◽  
pp. 10031-10043 ◽  
Author(s):  
Hua Zhang ◽  
Lei Song ◽  
Haolong Cong ◽  
Po Tien
Keyword(s):  
Life Cycle ◽  
Internal Ribosome Entry Site ◽  
Viral Protein ◽  
Nuclear Protein ◽  
Binding Protein ◽  
Enterovirus 71 ◽  
Protein Translation ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Cellular Factors

ABSTRACTEnterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES)trans-acting factor (ITAF) that binds specifically to the EV71 5′ untranslated region (5′UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation.IMPORTANCEThe nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection.

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