scholarly journals Effect of NS3 and NS5B proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation

2008 ◽  
Vol 89 (4) ◽  
pp. 994-999 ◽  
Author(s):  
Ming Xiao ◽  
Yan Bai ◽  
Hui Xu ◽  
Xiaolu Geng ◽  
Jun Chen ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Classical Swine Fever ◽  
Rna Helicase ◽  
Helicase Domain ◽  
Dependent Manner ◽  
Helicase Activity ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Cellular Translation

A full-length NS3 (NS3F) and a truncated NS3 protein (NS3H) with an RNA helicase domain possess RNA helicase activity. Using an in vitro system with a monocistronic reporter RNA or DNA, containing the CSFV 5′-UTR, we observed that both NS3F and NS3H enhanced internal ribosome entry site (IRES)-mediated and cellular translation in a dose-dependent manner, but NS3 protease (NS3P) that lacks a helicase domain did not. NS3F was stronger than NS3H in promoting both translations. These results showed that viral RNA helicase could promote viral and cellular translation, and higher RNA helicase activity might be more efficient. The NS5B protein, the viral replicase, did not significantly affect the IRES-directed or cellular translation alone. NS5B significantly enhanced the stimulative effect of NS3F on both IRES-mediated and cellular translation, but did not affect that of NS3H or NS3P. This suggests that NS5B and NS3 interact via the protease domain during the enhancement of translation.

scholarly journals Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Proteinase Inhibits Viral Internal Ribosome Entry Site-Mediated Translation

2008 ◽  
Vol 82 (19) ◽  
pp. 9389-9399 ◽  
Author(s):  
Jennifer M. Bonderoff ◽  
Jennifer L. LaRey ◽  
Richard E. Lloyd
Keyword(s):  
Internal Ribosome Entry Site ◽  
Binding Protein ◽  
Virus Protein ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
Polypyrimidine Tract Binding Protein ◽  
In Cells

ABSTRACT The two enteroviral proteinases, 2A proteinase (2Apro) and 3C proteinase (3Cpro), induce host cell translation shutoff in enterovirus-infected cells by cleaving canonical translation initiation factors. Cleavage of poly(A)-binding protein (PABP) by 3Cpro has been shown to be a necessary component for host translation shutoff. Here we show that 3Cpro inhibits cap-independent translation mediated by the poliovirus internal ribosome entry site (IRES) in a dose-dependent manner in HeLa translation extracts displaying cap-poly(A) synergy. This effect is independent of the stimulatory effect of 2Apro on IRES translation, and 3Cpro-induced translation inhibition can be partially rescued by addition of recombinant PABP in vitro. 3Cpro inhibits IRES translation on transcripts containing or lacking poly(A) tails, suggesting that cleavage of PABP and IRES trans-activating factors polypyrimidine tract-binding protein and poly r(C)-binding protein 2 may also be important for inhibition. Expression of 3Cpro cleavage-resistant PABP in cells increased translation of nonreplicating viral minigenome reporter RNAs during infection and also delayed and reduced virus protein synthesis from replicating RNA. Further, expression of cleavage-resistant PABP in cells reduced the accumulation of viral RNA and the output of infectious virus. These results suggest that cleavage of PABP contributes to viral translation shutoff that is required for the switch from translation to RNA replication.

scholarly journals Influence of NS5A protein of classical swine fever virus (CSFV) on CSFV internal ribosome entry site-dependent translation

2009 ◽  
Vol 90 (12) ◽  
pp. 2923-2928 ◽  
Author(s):  
Ming Xiao ◽  
Yujing Wang ◽  
Zailing Zhu ◽  
Jialin Yu ◽  
Lingzhu Wan ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Internal Ribosome Entry Site ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Border Disease Virus ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Diarrhea Virus ◽  
Border Disease

An internal ribosome entry site (IRES) present in the 5′ untranslated region (UTR) promotes translation of classical swine fever virus (CSFV) genomes. Using an in vitro system with monocistronic reporter RNA containing the CSFV 5′UTR, this study found that CSFV NS5A decreased CSFV IRES-mediated translation in a dose-dependent manner. Deletion analysis showed that the region responsible for repressing CSFV IRES activity might cover aa  390–414, located in the C-terminal half of CSFV NS5A. Triple and single alanine-scanning mutagenesis revealed that the inhibitory effect on CSFV IRES-directed translation mapped to the K399, T401, E406 and L413 residues of NS5A. These important amino acids were also found to be present in the NS5A proteins of bovine viral diarrhea virus (BVDV)-1, BVDV-2, border disease virus and hepatitis C virus, indicating that NS5A may play an important role in the switch from translation to replication in these viruses.

scholarly journals Resurrection of a Viral Internal Ribosome Entry Site from a 700 Year Old Ancient Northwest Territories Cripavirus

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 493
Author(s):  
Xinying Wang ◽  
Marli Vlok ◽  
Stephane Flibotte ◽  
Eric Jan
Keyword(s):  
Northwest Territories ◽  
Internal Ribosome Entry Site ◽  
Infectious Clone ◽  
Dependent Manner ◽  
Binding Assays ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Conserved Core ◽  
Partial Fragment

The dicistrovirus intergenic region internal ribosome entry site (IGR IRES) uses an unprecedented, streamlined mechanism whereby the IRES adopts a triple-pseudoknot (PK) structure to directly bind to the conserved core of the ribosome and drive translation from a non-AUG codon. The origin of this IRES mechanism is not known. Previously, a partial fragment of a divergent dicistrovirus RNA genome, named ancient Northwest territories cripavirus (aNCV), was extracted from 700-year-old caribou feces trapped in a subarctic ice patch. The aNCV IGR sequence adopts a secondary structure similar to contemporary IGR IRES structures, however, there are subtle differences including 105 nucleotides upstream of the IRES of unknown function. Using filter binding assays, we showed that the aNCV IRES could bind to purified ribosomes, and toeprinting analysis pinpointed the start site at a GCU alanine codon adjacent to PKI. Using a bicistronic reporter RNA, the aNCV IGR can direct translation in vitro in a PKI-dependent manner. Lastly, a chimeric infectious clone swapping in the aNCV IRES supported translation and virus infection. The characterization and resurrection of a functional IGR IRES from a divergent 700-year-old virus provides a historical framework for the importance of this viral translational mechanism.

scholarly journals Pestivirus Internal Ribosome Entry Site (IRES) Structure and Function: Elements in the 5′ Untranslated Region Important for IRES Function

2002 ◽  
Vol 76 (10) ◽  
pp. 5024-5033 ◽  
Author(s):  
Simon P. Fletcher ◽  
Richard J. Jackson
Keyword(s):  
Internal Ribosome Entry Site ◽  
Untranslated Region ◽  
Residual Activity ◽  
Classical Swine Fever ◽  
Structural Features ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT The importance of certain structural features of the 5′ untranslated region of classical swine fever virus (CSFV) RNA for the function of the internal ribosome entry site (IRES) was investigated by mutagenesis followed by in vitro transcription and translation. Deletions made from the 5′ end of the CSFV genome sequence showed that the IRES boundary was close to nucleotide 65: thus, the IRES includes the whole of domain II but no sequences upstream of this domain. Deletions which invaded domain II even to a small extent reduced activity to about 20% that of the full-length structure, and this 20% residual activity persisted with more extensive deletions until the whole of domain II had been removed and the deletions invaded the pseudoknot, whereupon IRES activity fell to zero. The importance of both stems of the pseudoknot was verified by making mutations in both sides of each stem; this severely reduced IRES activity, but the compensating mutations which restored base pairing caused almost full IRES function to be regained. The importance of the length of the loop linking the two stems of the pseudoknot was demonstrated by the finding that a reduction in length from the wild-type AUAAAAUU to AUU almost completely abrogated IRES activity. Random A→U substitutions in the wild-type sequence showed that IRES activity was fairly proportional to the number of A residues retained in this pseudoknot loop, with a preference for clustered neighboring A residues rather than dispersed As. Finally, it was found that the sequence of the highly conserved domain IIIa loop is, rather surprisingly, not important for the maintenance of full IRES activity, although amputation of the entire domain IIIa stem and loop was highly debilitating. These results are interpreted in the light of recent models, derived from cryo-electron microscopy, of the interaction of the closely related hepatitis C virus IRES with 40S ribosomal subunits.

scholarly journals In vivo footprint of a picornavirus internal ribosome entry site reveals differences in accessibility to specific RNA structural elements

2007 ◽  
Vol 88 (11) ◽  
pp. 3053-3062 ◽  
Author(s):  
Olga Fernández-Miragall ◽  
Encarnación Martínez-Salas
Keyword(s):  
Internal Ribosome Entry Site ◽  
Living Cells ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Essential Information ◽  
Mouth Disease Virus ◽  
Ires Element

Internal ribosome entry site (IRES) elements were described in picornaviruses as an essential region of the viral RNA. Understanding of IRES function requires a detailed knowledge of each step involved in the internal initiation process, from RNA folding and IRES–protein interaction to ribosome recruitment. Thus, deciphering IRES accessibility to external agents due to RNA structural features, as well as RNA–protein protection within living cells, is of primary importance. In this study, two chemical reagents, dimethylsulfate (DMS) and aminomethylpsoralen, have been used to footprint the entire IRES of foot-and-mouth disease virus (FMDV) in living cells; these reagents enter the cell membrane and interact with nucleic acids in a structure-dependent manner. For FMDV, as in other picornaviruses, viral infection is dependent on the correct function of the IRES; therefore, the IRES region itself constitutes a useful target of antiviral drugs. Here, the in vivo footprint of a picornavirus IRES element in the context of a biologically active mRNA is shown for the first time. The accessibility of unpaired adenosine and cytosine nucleotides in the entire FMDV IRES was first obtained in vitro by DMS probing; subsequently, this information was used to interpret the footprint data obtained in vivo for the mRNA encompassing the IRES element in the intercistronic space. The results of DMS accessibility and UV–psoralen cross-linking studies in the competitive cellular environment provided evidence for differences in RNA structure from data obtained in vitro, and provided essential information to identify appropriate targets within the FMDV IRES aimed at combating this important pathogen.

scholarly journals Dengue Virus Utilizes a Novel Strategy for Translation Initiation When Cap-Dependent Translation Is Inhibited

2006 ◽  
Vol 80 (6) ◽  
pp. 2976-2986 ◽  
Author(s):  
Dianna Edgil ◽  
Charlotta Polacek ◽  
Eva Harris
Keyword(s):  
Dengue Virus ◽  
Translation Initiation ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Eukaryotic Initiation Factor 4E ◽  
Cellular Translation ◽  
Cellular Mrnas ◽  
Novel Strategy

ABSTRACT Viruses have developed numerous mechanisms to usurp the host cell translation apparatus. Dengue virus (DEN) and other flaviviruses, such as West Nile and yellow fever viruses, contain a 5′ m7GpppN-capped positive-sense RNA genome with a nonpolyadenylated 3′ untranslated region (UTR) that has been presumed to undergo translation in a cap-dependent manner. However, the means by which the DEN genome is translated effectively in the presence of capped, polyadenylated cellular mRNAs is unknown. This report demonstrates that DEN replication and translation are not affected under conditions that inhibit cap-dependent translation by targeting the cap-binding protein eukaryotic initiation factor 4E, a key regulator of cellular translation. We further show that under cellular conditions in which translation factors are limiting, DEN can alternate between canonical cap-dependent translation initiation and a noncanonical mechanism that appears not to require a functional m7G cap. This DEN noncanonical translation is not mediated by an internal ribosome entry site but requires the interaction of the DEN 5′ and 3′ UTRs for activity, suggesting a novel strategy for translation of animal viruses.

scholarly journals La Autoantigen Is Necessary for Optimal Function of the Poliovirus and Hepatitis C Virus Internal Ribosome Entry Site In Vivo and In Vitro

2004 ◽  
Vol 24 (15) ◽  
pp. 6861-6870 ◽  
Author(s):  
Mauro Costa-Mattioli ◽  
Yuri Svitkin ◽  
Nahum Sonenberg
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Ribosomal Subunit ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5′ untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.

scholarly journals Novel Characteristics of the Biological Properties of the YeastSaccharomyces cerevisiaeEukaryotic Initiation Factor 2A

2005 ◽  
Vol 280 (16) ◽  
pp. 15601-15611 ◽  
Author(s):  
Anton A. Komar ◽  
Stephane R. Gross ◽  
Diane Barth-Baus ◽  
Ryan Strachan ◽  
Jack O. Hensold ◽  
...  
Keyword(s):  
Translational Control ◽  
Internal Ribosome Entry Site ◽  
Biological Properties ◽  
Yeast Cells ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Entry Site ◽  
Regulation Of Expression ◽  
Internal Ribosome Entry ◽  
Expression Of Genes

Eukaryotic initiation factor 2A (eIF2A) has been shown to direct binding of the initiator methionyl-tRNA (Met-tRNAi) to 40 S ribosomal subunits in a codon-dependent manner, in contrast to eIF2, which requires GTP but not the AUG codon to bind initiator tRNA to 40 S subunits. We show here that yeast eIF2A genetically interacts with initiation factor eIF4E, suggesting that both proteins function in the same pathway. The doubleeIF2A/eIF4E-tsmutant strain displays a severe slow growth phenotype, which correlated with the accumulation of 85% of the double mutant cells arrested at the G2/M border. These cells also exhibited a disorganized actin cytoskeleton and elevated actin levels, suggesting that eIF2A might be involved in controlling the expression of genes involved in morphogenic processes. Further insights into eIF2A function were gained from the studies of eIF2A distribution in ribosomal fractions obtained from either aneIF5BΔ (fun12Δ) strain or aeIF3b-ts(prt1-1) strain. It was found that the binding of eIF2A to 40 and 80 S ribosomes was not impaired in either strain. We also found that eIF2A functions as a suppressor of Ure2p internal ribosome entry site-mediated translation in yeast cells. The regulation of expression from theURE2internal ribosome entry site appears to be through the levels of eIF2A protein, which has been found to be inherently unstable with a half-life of ∼17 min. It was hypothesized that this instability allows for translational control through the level of eIF2A protein in yeast cells.

scholarly journals Translation Mediated by the Internal Ribosome Entry Site of thecat-1mRNA Is Regulated by Glucose Availability in a PERK Kinase-dependent Manner

2002 ◽  
Vol 277 (14) ◽  
pp. 11780-11787 ◽  
Author(s):  
James Fernandez ◽  
Barry Bode ◽  
Antonis Koromilas ◽  
J. Alan Diehl ◽  
Irene Krukovets ◽  
...  
Keyword(s):  
Internal Ribosome Entry Site ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Glucose Availability
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