All the data are normalized against mock infection. MI, mock infection without virus infection. MT, mock transfection transfection reagent control ; h. The intact and digested eIF4G were indicated by arrowheads. HEKT cells were transfected with the reporter vector and miRNAs, as indicated, and firefly luciferase activity measured 48 hr later. NC, 5 pmole of negative control miRNA. Various amounts of miR 25, Paralleling the previous results, miR negatively regulated V5-eIF4E with a wild-type 30 UTR in a dose-dependent manner, but not with the mutant one.
Interestingly, the expressions of miR were mutant miR, named miRM six point mutations in increased to The result showed that neither five miRs compared with MT control column 5 and 6 in left panel of Fig- nor miRM could decrease the expression of endogenous ure S2.
The upregulations of miR were To elucidate was gradually reduced in a time-dependent manner from 2 h. RD cells were mock infected MI or in- protein expression levels were measured at the indicated time fected with EV71 and pulse labeled with [35S] methionine. The points by western blot.
Figure S1H. These findings imply that the assayed Figure 2D. To vali- miR might play a crucial role. RD cells were transfected with antagomiR ure 2A. The pressed the luciferase activity of the reporter vector harboring protein expression of eIF4E and eIF4G was determined by the wild-type 30 UTR at all the three doses assayed, but mutant western blot.
To further explore the tion was almost eliminated in antagomiR transfected cells posttranscriptionally regulatory effect of miR on eIF4E, but not in antagomiR-negative control transfected cells or the two expression vectors encoding V5-eIF4E fusion proteins, in mock transfected cells at 4 h.
Figure 3A. These results which the open reading frame of eIF4E was followed by either clearly demonstrated that miR has an important role in the the wild-type or mutant type 30 UTR, were constructed and are suppression of eIF4E during virus infection. These stable We investigated whether virus-infection-induced miR, clones were then transfected with miR and assayed for which targets eIF4E, did contribute to translational switch.
RD cells were transfected with antago- miR followed by EV71 infection. B Shutoff of host protein synthesis was delayed by antagomiR RD cells were infected with EV71, and de novo protein synthesis was detected at the indicated time points using metabolic labeling. The [35S]methionine incorporation assay was quantified by densitometry and showed at the bottoms. The viral proteins were indicated by arrowheads. C miR specifically attenuated cap-depen- dent translation.
Luciferase is expressed through cap-dependent translation and EGFP is ex- pressed through cap-independent translation. Isotope metabolic labeling was performed to characterize the Silencing of miR Delays CPE Occurrence role of miR in the shutoff of host protein synthesis during and Reduces Virus Production virus infection.
The antagomiR, but not the anta- points, respectively. Figure 3B. Even at 12 h. Furthermore, the onset of cytopathic effects protein synthesis remained in antagomiR transfected cells. The blockage of Figure 4B. As shown in Figure S3B, CPE was imaged with miR expression with antagomiR could manifestly delay magnification and quantified at indicated time points. To the process of translation switching, with host protein synthesis address whether antagomiR can reduce viral replication, remained relatively stable at 4 h.
To investigate whether miR can directly cause antagomiR A fold reduction in viral propagation, protein synthesis switch, we construct a plasmid, which tran- accompanied by the complete digestion of eIF4G, was scribes a transcript containing fire luciferase gene and observed when the virus-induced miR was neutralized by enhanced green fluorescence protein EGFP driven by cap- antagomiR Figures 3A and 4C. On transfection Table S3B. The roles of eIF4E in enterovirus the other hand, we performed northern blot to check the intact- replication were further validated by an RNAi strategy.
The data eIF4E-pecific siRNAs were individually introduced into RD cells showed that the transcripts in miR transfectants kept in in the presence of antagomiR followed by virus infection. The antagomiRs transfected RD cells were infected with EV71, and the viral proteins VPs were immunoblotted with anti- EV71 antibody at the indicated time points in trip- licate. B CPE was attenuated by antagomiR Magnification, C Virus production was attenuated by antago- miR Virus yield was measured by plaque assay.
The results clearly demonstrated precursor was found to increase the luciferase activity up to that the effect of antagomiR on virus production was fold compared with the pGL vector control Figure 5B.
Using mainly, if not totally, via regulation of eIF4E. Consistent with the findings in RD 8 h. Table S2. Further matching the results from prediction cells, EV71 infection induced eIF4E suppression, which can of binding sites and the expression profile of transcription factors be rescued, accompanied with decrease EV71 replication, by identified the early growth response 1 EGR1. These data suggest that and protein expression levels of EGR1 in EVinfected cells the miRmediated regulatory mechanism might be a were determined.
Consistent with the microarray results, EGR1 general phenomenon for EVinfected cells and that miRNA- expression was dramatically induced by up to fold at 4 mediated eIF4E suppression might not only play a role in viral and 8 h. RNA and protein levels Figure 5C. Figure S4F. Next, a chromatin miRb, and miR has thus been elucidated Bracken immunoprecipitation ChIP assay was performed to pinpoint et al.
Although the transcription start site of miR whether EGR1 could directly bind onto the two predicted binding has been predicted to be bp upstream from miR, the sites. To with a V5 tag because commercial anti-EGR1 antibody yielded explore the regulatory mechanisms adopted by EV71 to induce nonspecific bands. RD cells were transfected with an expression miR expression, the regulatory element responsible for vector expressing V5 tag EGR1, and anti-V5 antibody was used miR induction was determined by luciferase reporter assays to perform ChIP assays.
Individual construct was trans- were essential for maximal expression of miR Figure 5B. After 16 hr, all transfectants were infected to miR regulation in nature context, the luciferase activity with EV71 at an moi of 10 and the luciferase activities of cell of reporter vector containing full-length regulatory fragment lysates were measured at 8 h.
Figures 5A and 5B. Regulation of miR A Schematic organization of miR B Identification of the regulatory elements of miR Transcriptional activity of miRtruncated upstream sequences with or without EGR1 binding site mutations was determined by luciferase activity assays. Ctrl-1 and ctrl-2 primers located at the 3 kb downstream of EGR1 binding site 1 and site 2 act as PCR primer controls. E EGR1 activated miR expression. The results from off-target effect, the suppressive activity of siEGR1 mutations at site 1 and site 2 attenuated the luciferase activity should be not able to rescue by sieIF4E.
It has been predicted that miRc and miR form a cluster and are located within In this study we investigated the role of miRNAs in viral replication. EGR1 should activate Up to now, only a few reports have shown that cellular or viral miR expression as well as miRc if miR shares miRNAs are involved in reciprocal interactions between virus a promoter with miRc. Thus, we examined whether miR- and host cells Gottwein et al. Picorna- We designed two sets of specific primers to amplify the virus infection is known to induce a dramatic shutoff of host pri-miRNA containing miR and miRc.
We observed protein synthesis that subsequently contributes to the pathogen- that both pairs of primers could specifically amplify the expected esis of virus infection. The results revealed that the expres- how viruses, particularly the picornaviruses, can specifically block sion of both miR and miRc was induced in the host protein synthesis, although poliovirus 2A protease-mediated V5-EGR1 transfectants but not in the mock transfection or vector cleavage of eIF4G, which shuts down host protein synthesis, has control transfectants Figures S4C and S4D.
These data imply been reported Belsham and Sonenberg, ; Goldstaub et al. We found that enterovirus expression is also regulated by EGR1. The nous EGR1. Next, the role of 4E-BPs Gingras et al. Sukarieh and coworkers found that the attenuated ranging from 3- to fold Figure 5F. Subsequently, nuclear relocalization of eIF4E corresponds with shutoff of host experiments were performed to rule out the possibility that the protein synthesis in response to poliovirus infection Sukarieh suppressive effect of EGR1 siRNAs on the virus production et al.
However, no study investigates whether picornavirus was due to a nonspecific interferon response induced by infection can directly regulate the expression of eIF4E itself.
The study, we demonstrated that picornavirus infection could repress levels of interferon a in the EGR1 siRNA and negative control eIF4E expression and that the knockdown of miR by a specific transfectants were measured by interferon a multisubtype inhibitor, antagomiR, is able to restore host eIF4E expression, ELISA; the interferon a expression levels were then compared delay the occurrence of CPE, and reduce viral propagation. The results showed that there was no Consistently, silencing of eIF4E was able to completely block induction of interferon a by the EGR1 siRNAs or the negative the effect of antagomiR on viral propagation.
We have also control compared with the cell controls Table S3B. We also demonstrated that the induction of miR is at least partially evaluated the potential off-target effects of EGR1 siRNAs using due to increased expression of EGR1 upon EV71 infection; sequence alignment to calculate whether the viral genome might furthermore, the silencing of EGR1 attenuated virus production. The alignment revealed that These evidences imply that enterovirus infection promotes the sequences with no three or fewer mismatches could be identi- expression of the host miRNA, miR, which, by suppression fied Table S3C , suggesting that the reduction of virus produc- of host cap-dependent translation, switches the host machinery tion caused by the EGR1 siRNAs was not due to a nonspecific to support viral replication through cap-independent translation.
Therefore, 2C against EV71 protease 2C has been successfully used to any disruption between the viral protein synthesis and release of inhibit EV71 replication through protease 2C suppression Tan infectious virus particles may contribute to a significant decrease et al. It implied that the reduction of virus production might the genome of EV These data as well as certain unidentified mechanisms might contribute to provide direct evidence that the suppressive activity of siEGR1 the virus titer reduction.
Recent reports have identified several of up to fold but that the neutralization of virus-infection- cellular miRNAs that contribute to viral replication. Another example is miR, which is specifically In conclusion, we have identified a virus-host interaction expressed in the liver and is able to stabilize HCV genomic pathway wherein virus infection results in EGR1-mediated RNA Jopling et al. Consequently, the suppression of miR induction and that this leads to the silencing of eIF4E, miR by a locked nucleic acid-modified oligonucleotide a switch from cap-dependent to cap-independent translation was found to attenuate HCV infection in the chimpanzee model in the host cells, augmentation of CPE, and increased virus Lanford et al.
A phase I clinical trial has been conducted production Figure 6. In addition, antagomiR infection was performed in serum-free condition. Importantly, the metabolic labeling data with magnification.
Details are given in the Supplemental Information. The downregulation of eIF4E is reagent Invitrogen. Second, in presence of eIF4E we can determine how much of the observed repression of host protein synthesis is Oligonucleotide Microarray Analysis due to the contribution of other translational factors including cRNA preparation and array hybridization were performed according to the eIF4G depletion, 4E-BP phosphorylation, and so on.
Proteins were resolved by Although we have demonstrated primary antibodies. Details are given in that miR and miRc were located within the same the Supplemental Information.
Thiouridine Incorporation Assay Recently Buck and coworkers demonstrated that CMV infection The thiouridine incorporation assay was performed as described previously specifically suppresses miRa expression but not miRa Norman and Sarnow, RD cells were infected with EV71 and the culture and miR located in the same genomic cluster through medium contained 4-thiouridine at mM. Total RNAs were collected at indi- posttranscriptional regulation Buck et al.
The biotinylated RNAs were captured with streptavidin beads Invitrogen. After 48 hr incubation the Dual-Glo luciferase substrate Promega simplex virus type 1 HSV-1 , and that this induction promotes was added and the luminescent signals were measured by Victor3 multilabel viral replication Chang et al. However, counter PerkinElmer The activity of Renilla luciferase was used as an internal we noticed that the suppression of virus-infection-induced control to normalize transfection efficiency.
Details are given in the Supple- miR by antagomiR led to a decrease in virus production mental Information. The reduction in eIF4E causes a protein synthesis switch from cap-dependent to cap-independent translation that might contribute viral pathogenesis and virus propaga- tion.
EGR1 s, s, and s; Applied Biosys- tems were transfected into RD cells by Lipofect- amine reagent Invitrogen and the transfected RD cells were infected with virus after 24 hr. Metabolic Labeling and Immunoprecipitation For characterizing the role of miR in the shutoff of host protein synthesis during virus infection, RD cells were transfected with antagomiRs 24 hr before metabolic labeling experiments.
Two paired primers F1 labeled with [35S] methionine. De novo-synthesized proteins were labeled by incubation in pulse seed region of miRbinding site by PCR-based mutagenesis method. Cells Both PCR fragments were cloned into pMIR-reporter luciferase vector were harvested in protein lysis buffer and total proteins were incubated with Ambion.
The supernatants were re- cells and cloned into pcDNA 3. The beads were resolved by A tenth of total proteins were analyzed by western blot. Details upstream fragments of the miRc precursor were constructed into pGL are given in the Supplemental Information.
Plaque Assay EV71 plaque assays were carried out in triplicate in 6-well plates. After 1 hr incubation the Transfections infected cells were washed and incubated for 3 days in 0. Cells were fixed with formaldehyde and stained with crystal violet. The significant miRNAs with greater than 2-fold Acad.
USA 95, — MicroRNAs as oncogenes. Holland, J. USA 49, 23— Hou, J. Huang, C. Neurologic complications in children with enterovirus 71 infection. We also Jopling, C. This MicroRNA. Science , — Krek, A. Revised: October 11, Kuyumcu-Martinez, N. Accepted: November 22, Cleavage of poly A -binding protein by poliovirus 3C protease inhibits host cell Published: January 19, translation: a novel mechanism for host translation shutoff.
Therapeutic silencing of Belsham, G. Science for cellular proteins. Trends Microbiol. Published online December 3, Bracken, C. A double-negative feedback loop between Viral and host proteins involved in picornavirus life cycle.
Cancer Res. Martinez-Salas, E. New Buck, A. Post-transcriptional expression. RNA 16, — Miranda, K. A pattern-based method for the identification of Schaefer, B. See page 8 and 9 for packaging information. Bold part numbers Mount Mount 13 Graphique 1. The indicated cables are recommended for the C B Fig. Bold part numbers represent Your most recent searches Delete.
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