scholarly journals Interactions of Viral Protein 3CD and Poly(rC) Binding Protein with the 5′ Untranslated Region of the Poliovirus Genome

2000 ◽  
Vol 74 (5) ◽  
pp. 2219-2226 ◽  
Author(s):  
Andrea V. Gamarnik ◽  
Raul Andino
Keyword(s):  
Viral Protein ◽  
Untranslated Region ◽  
Rna Binding ◽  
Binding Protein ◽  
Cellular Protein ◽  
Site Directed Mutagenesis ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Cloverleaf Structure

ABSTRACT The poly(rC) binding protein (PCBP) is a cellular protein required for poliovirus replication. PCBP specifically interacts with two domains of the poliovirus 5′ untranslated region (5′UTR), the 5′ cloverleaf structure, and the stem-loop IV of the internal ribosome entry site (IRES). Using footprinting analysis and site-directed mutagenesis, we have mapped the RNA binding site for this cellular protein within the stem-loop IV domain. A C-rich sequence in a loop at the top of this large domain is required for PCBP binding and is crucial for viral translation. PCBP binds to stem-loop IV RNA with six-times-higher affinity than to the 5′ cloverleaf structure. However, the binding of the viral protein 3CD (precursor of the viral protease 3C and the viral polymerase 3D) to the cloverleaf RNA dramatically increases the affinity of PCBP for this RNA element. The viral protein 3CD binds to the cloverleaf RNA but does not interact directly with stem-loop IV nor with other RNA elements of the viral IRES. Our results indicate that the interactions of PCBP with the poliovirus 5′UTR are modulated by the viral protein 3CD.

scholarly journals In vitro and in vivo identification of structural and sequence elements in the 5′ untranslated region of Ectropis obliqua picorna-like virus required for internal initiation

2006 ◽  
Vol 87 (12) ◽  
pp. 3667-3677 ◽  
Author(s):  
Jie Lu ◽  
Jiamin Zhang ◽  
Xiaochun Wang ◽  
Hong Jiang ◽  
Chuanfeng Liu ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Site Directed Mutagenesis ◽  
Polypyrimidine Tract ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Sequence Elements ◽  
Internal Initiation

Ectropis obliqua picorna-like virus (EoPV) is a newly described insect virus that is classified as a putative member of the genus Iflavirus. The virus possesses a large, positive-sense RNA genome encoding a single polyprotein that shares physicochemical properties with those of members of the family Picornaviridae. The 5′ untranslated region (5′ UTR) plays an important role in picornavirus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation. To investigate translation in EoPV, an extensive range of mutations were engineered within the 5′ UTR and the effects of these changes were examined in vitro and in vivo by using a bicistronic construct. Results showed that deletions within the first 63 nt had little impact on IRES activity, whilst core IRES function was contained within stem–loops C and D, as their removal abrogated IRES activity significantly. In contrast to these findings, removal of stem–loop G containing two cryptic AUGs caused a remarkable increase in IRES activity, which was further investigated by site-directed mutagenesis at these two positions. It was also confirmed that initiation of protein synthesis occurs at AUG6 (position 391–394) and not at the AUG immediately downstream of the polypyrimidine tract. Mutation of the polypyrimidine tract (CCTTTC) had a slight effect on EoPV IRES activity. Furthermore, mutations of the RAAA motif led to a decrease in IRES activity of approximately 40 % in vitro, but these results were not supported by in vivo experiments. In conclusion, this study reveals that the EoPV IRES element is unique, although it has features in common with the type II IRESs.

scholarly journals Efficient Translation Initiation Directed by the 900-Nucleotide-Long and GC-Rich 5′ Untranslated Region of the Human Retrotransposon LINE-1 mRNA Is Strictly Cap Dependent Rather than Internal Ribosome Entry Site Mediated

2007 ◽  
Vol 27 (13) ◽  
pp. 4685-4697 ◽  
Author(s):  
Sergey E. Dmitriev ◽  
Dmitri E. Andreev ◽  
Ilya M. Terenin ◽  
Ivan A. Olovnikov ◽  
Vladimir S. Prassolov ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Untranslated Region ◽  
High Efficiency ◽  
Rna Binding ◽  
Cultured Cells ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Cellular Mrnas ◽  
Internal Initiation

ABSTRACT Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5′ untranslated region (5′UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5′UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5′UTR-Fluc) or bicistronic (Rluc-L1 5′UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5′UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5′UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5′UTR. Nevertheless, this cap-dependent initiation activity of the L1 5′UTR was unexpectedly high and resembles that of the beta-actin 5′UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5′UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5′UTRs and call into question the conception that every long GC-rich 5′UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.

scholarly journals RNA-binding Protein HuR Interacts with Thrombomodulin 5′Untranslated Region and Represses Internal Ribosome Entry Site–mediated Translation under IL-1β Treatment

2008 ◽  
Vol 19 (9) ◽  
pp. 3812-3822 ◽  
Author(s):  
Chiu-Hung Yeh ◽  
Liang-Yi Hung ◽  
Chin Hsu ◽  
Shu-Yun Le ◽  
Pin-Tse Lee ◽  
...  
Keyword(s):  
Protein Expression ◽  
Internal Ribosome Entry Site ◽  
Untranslated Region ◽  
Protein C ◽  
Rna Binding ◽  
Activated Protein C ◽  
Critical Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Sepsis And Septic Shock

Reduction in host-activated protein C levels and resultant microvascular thrombosis highlight the important functional role of protein C anticoagulant system in the pathogenesis of sepsis and septic shock. Thrombomodulin (TM) is a critical factor to activate protein C in mediating the anticoagulation and anti-inflammation effects. However, TM protein content is decreased in inflammation and sepsis, and the mechanism is still not well defined. In this report, we identified that the TM 5′ untranslated region (UTR) bearing the internal ribosome entry site (IRES) element controls TM protein expression. Using RNA probe pulldown assay, HuR was demonstrated to interact with the TM 5′UTR. Overexpression of HuR protein inhibited the activity of TM IRES, whereas on the other hand, reducing the HuR protein level reversed this effect. When cells were treated with IL-1β, the IRES activity was suppressed and accompanied by an increased interaction between HuR and TM 5′UTR. In the animal model of sepsis, we found the TM protein expression level to be decreased while concurrently observing the increased interaction between HuR and TM mRNA in liver tissue. In summary, HuR plays an important role in suppression of TM protein synthesis in IL-1β treatment and sepsis.

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.

scholarly journals Functional features of the bovine enterovirus 5′-non-translated region

1999 ◽  
Vol 80 (9) ◽  
pp. 2299-2309 ◽  
Author(s):  
Roland Zell ◽  
Karim Sidigi ◽  
Andreas Henke ◽  
Joachim Schmidt-Brauns ◽  
Elizabeth Hoey ◽  
...  
Keyword(s):  
Luciferase Expression ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Bovine Enterovirus ◽  
Genome Region ◽  
Functional Features ◽  
Domain I ◽  
Cloverleaf Structure ◽  
Structure Domain

The bovine enterovirus (BEV) serotypes exhibit unique features of the non-translated regions (NTRs) which separate them from the other enteroviruses. Their most remarkable property is an additional genome region of 110 nt located between the 5′-cloverleaf and the internal ribosome entry site (IRES). This genome region has the potential to form an additional cloverleaf structure (domain I*) separated from the 5′-cloverleaf (domain I) by a small stem–loop (domain I**). Other characteristics involve the putative IRES domains III and VI. In order to investigate the features of the 5′-NTR, several full-length coxsackievirus B3 (CVB3) cDNA plasmids with hybrid 5′-NTRs were engineered. After exchange of the CVB3 cloverleaf with the BEV1 genome region representing both cloverleafs, a viable virus chimera was generated. Deletion of domain I** within the exchanged region also yielded viable virus albeit with reduced growth capacity. Deletion of sequences encoding either the first or the second BEV cloverleaf resulted in non-infectious constructs. Hybrid plasmids with exchanges of the IRES-encoding sequence or the complete 5′-NTR were non-infectious. Transfection experiments with SP6 transcripts containing 5′-NTRs fused to the luciferase message indicated that IRES-driven translation is enhanced by the presence of the CVB3 cloverleaf and both BEV1 cloverleaf structures, respectively. Deletion of either the first or the second BEV cloverleaf domain reduced but did not abolish enhanced luciferase expression. These results suggest that the substitution of two putative BEV cloverleaf structures for the putative coxsackieviral cloverleaf yields viable virus, while BEV sequences encoding the IRES fail to functionally replace CVB3 IRES-encoding sequences.

scholarly journals Structural Elements in the 5′-Untranslated Region of Giardiavirus Transcript Essential for Internal Ribosome Entry Site-Mediated Translation Initiation

2005 ◽  
Vol 4 (4) ◽  
pp. 742-754 ◽  
Author(s):  
Srinivas Garlapati ◽  
Ching C. Wang
Keyword(s):  
Internal Ribosome Entry Site ◽  
Untranslated Region ◽  
Site Directed Mutagenesis ◽  
Appreciable Effect ◽  
Structural Elements ◽  
Coding Region ◽  
Drastic Reduction ◽  
Entry Site ◽  
Viral Transcript ◽  
Internal Ribosome Entry

ABSTRACT Translation of uncapped giardiavirus (GLV) mRNA in Giardia lamblia requires the presence of a 5′-untranslated region (5′-UTR) and a viral capsid coding region. We used dicistronic viral constructs to show that the downstream 253 nucleotides (nt) of the 5′-UTR plus the initial 264-nt capsid coding region constitute an internal ribosome entry site (IRES). Predicted secondary structures in the 253-nt 5′-UTR include stem-loops U3, U4a, U4b, U4c, and U5. Chemical and enzymatic probing analysis confirmed the presence of all predicted stem-loops except U4a. Disruption of stem-loop structures U3 and U5 by site-directed mutagenesis resulted in a drastic reduction in translation of a monocistronic viral transcript, which could be restored by compensatory sequence changes. Mutations disrupting stem-loops U4b and U4c do not exert an appreciable effect on translation, but certain sequences in the U4a region and in U4b do appear to play important roles in the IRES. Structural analysis also suggests that an 8-nt U3 loop sequence (nt 147 to 154) pairs with an 8-nt downstream sequence (nt 168 to 175) to form a pseudoknot. Disruption of this pseudoknot by mutagenesis resulted in a drastic reduction in translation, which could be restored by compensatory sequence changes. This study has defined the secondary structure in the 5′-UTR of the IRES. Together with the previous results, we have now completed analysis of the entire structure of GLV IRES and fully defined the functionally essential structural elements in it.

scholarly journals Functional Insights into the Adjacent Stem-Loop in Honey Bee Dicistroviruses That Promotes Internal Ribosome Entry Site-Mediated Translation and Viral Infection

2017 ◽  
Vol 92 (2) ◽  
Author(s):  
Hilda H. T. Au ◽  
Valentina M. Elspass ◽  
Eric Jan
Keyword(s):  
Protein Synthesis ◽  
Virus Infection ◽  
Honey Bee ◽  
Internal Ribosome Entry Site ◽  
Viral Protein ◽  
Entry Site ◽  
Viral Protein Synthesis ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Paralysis Virus

ABSTRACTAll viruses must successfully harness the host translational apparatus and divert it toward viral protein synthesis. Dicistroviruses use an unusual internal ribosome entry site (IRES) mechanism whereby the IRES adopts a three-pseudoknot structure that accesses the ribosome tRNA binding sites to directly recruit the ribosome and initiate translation from a non-AUG start site. A subset of dicistroviruses, including the honey bee Israeli acute paralysis virus (IAPV), encode an extra stem-loop (stem-loop VI [SLVI]) 5′ adjacent to the intergenic region (IGR) IRES. Previously, the function of this additional stem-loop was unknown. Here, we provide mechanistic and functional insights into the role of SLVI in IGR IRES translation and in virus infection. Biochemical analyses of a series of mutant IRESs demonstrated that SLVI does not function in ribosome recruitment but is required for proper ribosome positioning on the IRES to direct translation. Using a chimeric infectious clone derived from the related cricket paralysis virus, we showed that the integrity of SLVI is important for optimal viral translation and viral yield. Based on structural models of ribosome-IGR IRES complexes, SLVI is predicted to be in the vicinity of the ribosome E site. We propose that SLVI of IAPV IGR IRES functionally mimics interactions of an E-site tRNA with the ribosome to direct positioning of the tRNA-like domain of the IRES in the A site.IMPORTANCEViral internal ribosome entry sites are RNA elements and structures that allow some positive-sense monopartite RNA viruses to hijack the host ribosome to start viral protein synthesis. We demonstrate that a unique stem-loop structure is essential for optimal viral protein synthesis and for virus infection. Biochemical evidence shows that this viral stem-loop RNA structure impacts a fundamental property of the ribosome to start protein synthesis.

scholarly journals Influence of the 5′-proximal elements of the 5′-untranslated region of classical swine fever virus on translation and replication

2011 ◽  
Vol 92 (5) ◽  
pp. 1087-1096 ◽  
Author(s):  
Ming Xiao ◽  
Yujing Wang ◽  
Zailing Zhu ◽  
Chengli Ding ◽  
Jialin Yu ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Untranslated Region ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Loop Structure ◽  
Terminal Sequence ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Stem Loop Structure

The 5′-terminal sequence spanning nt 1–29 of the 5′-untranslated region of classical swine fever virus (CSFV) forms a 5′-proximal stem–loop structure known as domain Ia. Deletions and replacement mutations were performed to examine the role of this domain. Deletion of the 5′-proximal nucleotides and disruption of the stem–loop structure greatly increased internal ribosome entry site-mediated translation but abolished the replication of the replicons. Internal deletions resulting in a change in the size of the loop of domain Ia, and even removal of the entire domain, did not substantially change the translation activity, but reduced the replication of CSFV replicons provided the replicons contained the extreme 5′-GUAU terminal sequence. Internal replacements leading to a change in the nucleotide sequence of the loop did not alter the translation and replication activities of the CSFV RNA replicon, and did not influence the rescue of viruses and growth characteristics of new viruses. These results may be important for our understanding of the regulation of translation, replication and encapsidation in CSFV and other positive-sense RNA viruses.

scholarly journals Replication of Poliovirus Requires Binding of the Poly(rC) Binding Protein to the Cloverleaf as Well as to the Adjacent C-Rich Spacer Sequence between the Cloverleaf and the Internal Ribosomal Entry Site

2007 ◽  
Vol 81 (18) ◽  
pp. 10017-10028 ◽  
Author(s):  
Hidemi Toyoda ◽  
David Franco ◽  
Kentaro Fujita ◽  
Aniko V. Paul ◽  
Eckard Wimmer
Keyword(s):  
Binding Protein ◽  
Position Effect ◽  
Internal Ribosomal Entry Site ◽  
Rna Replication ◽  
Cellular Protein ◽  
Entry Site ◽  
Stem Loop ◽  
Nontranslated Region ◽  
Ribosomal Entry

ABSTRACT The 5′ nontranslated region of poliovirus RNA contains two highly structured regions, the cloverleaf (CL) and the internal ribosomal entry site (IRES). A cellular protein, the poly(rC) binding protein (PCBP), has been reported to interact with the CL either alone or in combination with viral protein 3CDpro. The formation of the ternary complex is essential for RNA replication and, hence, viral proliferation. PCBP also interacts with stem-loop IV of the IRES, an event critical for the initiation of cap-independent translation. Until recently, no special function was assigned to a spacer region (nucleotides [nt] 89 to 123) located between the CL and the IRES. However, on the basis of our discovery that this region strongly affects the neurovirulent phenotype of poliovirus, we have embarked upon genetic and biochemical analyses of the spacer region, focusing on two clusters of C residues (C93-95 and C98-100) that are highly conserved among entero- and rhinoviruses. Replacement of all six C residues with A residues had no effect on translation in vitro but abolished RNA replication, leading to a lethal growth phenotype of the virus in HeLa cells. Mutation of the first group of C residues (C93-95) resulted in slower viral growth, whereas the C98-100A change had no significant effect on viability. Genetic analyses of the C-rich region by extensive mutagenesis and analyses of revertants revealed that two consecutive C residues (C94-95) were sufficient to promote normal growth of the virus. However, there was a distinct position effect of the preferred C residues. A 142-nt-long 5′-terminal RNA fragment including the CL and spacer sequences efficiently bound PCBP, whereas no PCBP binding was observed with the CL (nt 1 to 88) alone. Binding of PCBP to the 142-nt fragment was completely ablated after the two C clusters in the spacer were mutated to A clusters. In contrast, the same mutations had no effect on the binding of 3CDpro to the 142-nt RNA fragment. Stepwise replacement of the C residues with A residues resulted in impaired replication that covaried with weaker binding of PCBP in vitro. We conclude that PCBP has little, if any, binding affinity for the CL itself (nt 1 to 88) but requires additional nucleotides downstream of the CL for its function as an essential cofactor in poliovirus RNA replication. These data reveal a new essential function of the spacer between the CL and the IRES in poliovirus proliferation.

Sign in / Sign up

Export Citation Format

Share Document