scholarly journals In vitro comparison of the internal ribosomal entry site activity from rodent hepacivirus and pegivirus

2019 ◽  
Author(s):  
Stuart Sims ◽  
Kevin Michaelsen ◽  
Sara Burkhard ◽  
Cornel Fraefel
Keyword(s):  
Untranslated Region ◽  
Internal Ribosomal Entry Site ◽  
Surface Expression ◽  
Rna Polymerase I ◽  
Cell Surface Expression ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Type Iv ◽  
Polymerase I

AbstractThe 5’ untranslated region (5’ UTR) of rodent hepacivirus (RHV) and pegivirus (RPgV) contain sequence homology to the HCV type IV internal ribosome entry sites (IRES). Utilizing a monocistronic expression vector with an RNA polymerase I promoter to drive transcription we show cell-specific IRES translation and regions within the IRES required for full functionality. Focusing on RHV we further pseudotyped lentivirus with RHV and showed cell surface expression of the envelope proteins and transduction of murine hepatocytes.

scholarly journals In Vitro Comparison of the Internal Ribosomal Entry Site Activity from Rodent Hepacivirus and Pegivirus and Construction of Pseudoparticles

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Stuart Sims ◽  
Kevin Michaelsen ◽  
Sara Burkhard ◽  
Cornel Fraefel
Keyword(s):  
Internal Ribosomal Entry Site ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Entry Site ◽  
Antiviral Signaling ◽  
Internal Ribosome Entry ◽  
Mitochondrial Antiviral Signaling ◽  
Polymerase I ◽  
Mitochondrial Antiviral Signaling Protein

The 5′ untranslated region (5′ UTR) of rodent hepacivirus (RHV) and pegivirus (RPgV) contains sequence homology to the HCV type III internal ribosome entry sites (IRES). Utilizing a monocistronic expression vector with an RNA polymerase I promoter to drive transcription, we show cell-specific IRES translation and regions within the IRES required for full functionality. Focusing on RHV, we further pseudotyped lentivirus with RHV and showed cell surface expression of the envelope proteins and transduction of murine hepatocytes and we then constructed full-length RHV and RPgV replicons with reporter genes. Using the replicon system, we show that the RHV NS3-4A protease cleaves a mitochondrial antiviral signaling protein reporter. However, liver-derived cells did not readily support the complete viral life cycle.

scholarly journals Identification of minimal sequences of the Rhopalosiphum padi virus 5′ untranslated region required for internal initiation of protein synthesis in mammalian, plant and insect translation systems

2007 ◽  
Vol 88 (5) ◽  
pp. 1583-1588 ◽  
Author(s):  
Elisabetta Groppelli ◽  
Graham J. Belsham ◽  
Lisa O. Roberts
Keyword(s):  
Untranslated Region ◽  
Rhopalosiphum Padi ◽  
Open Reading Frames ◽  
Distinct Region ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Internal Initiation ◽  
Minimal Sequences ◽  
Translation Systems

Rhopalosiphum padi virus (RhPV) is a member of the family Dicistroviridae. The genomes of viruses in this family contain two open reading frames, each preceded by distinct internal ribosome entry site (IRES) elements. The RhPV 5′ IRES is functional in mammalian, insect and plant translation systems and can form 48S initiation complexes in vitro with just the mammalian initiation factors eIF2, eIF3 and eIF1. Large regions of the 5′ untranslated region (UTR) can be deleted without affecting initiation-complex formation. The minimal sequences required for directing internal initiation in mammalian (rabbit reticulocyte lysate), plant (wheatgerm extract) and insect (Sf21 cells) translation systems have now been defined. A fragment (nt 426–579) from the 3′ portion of the 5′ UTR can direct translation in each of these translation systems. In addition, a distinct region (nt 300–429) is also active. Thus, unstructured regions within the 5′ UTR seem to be critical for IRES function.

scholarly journals Sequence analysis of the 5′ untranslated region of swine vesicular disease virus reveals block deletions between the end of the internal ribosomal entry site and the initiation codon

2005 ◽  
Vol 86 (10) ◽  
pp. 2753-2761 ◽  
Author(s):  
Andrew E. Shaw ◽  
Scott M. Reid ◽  
Nick J. Knowles ◽  
Geoffrey H. Hutchings ◽  
Ginette Wilsden ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Disease Virus ◽  
Untranslated Region ◽  
Internal Ribosomal Entry Site ◽  
Initiation Codon ◽  
Entry Site ◽  
Swine Vesicular Disease Virus ◽  
Ribosomal Entry ◽  
Vesicular Disease

Swine vesicular disease virus (SVDV) is a picornavirus closely related to the human pathogen coxsackievirus B5. In common with other picornaviruses, the 5′ untranslated region (5′ UTR) of SVDV contains an internal ribosomal entry site (IRES) that plays an important role in cap-independent translation. The aim of this study was to use RT-PCR and sequencing to characterize a fragment of the 5′ UTR encompassing the entire IRES. Sequence analysis demonstrated high nucleotide identities within the IRES between 33 representative SVDV isolates. These data support the choice of this region as a diagnostic target and provide information for the improvement of laboratory-based molecular assays to detect SVDV. In contrast to the relative conservation of the IRES element, there was considerable nucleotide variability in the spacer region located between the cryptic AUG at the 3′ end of the IRES and the initiation codon of the polyprotein. Interestingly, 11 SVDV isolates had block deletions of between 6 and 125 nt in this region. Nine of these isolates were of recent European origin and were phylogenetically closely related. In vitro growth studies showed that selected isolates with these deletions had a significantly reduced plaque diameter and grew to a significantly lower titre relative to an isolate with a full-length 5′ UTR. Further work is required to define the significance of these deletions and to assess whether they impact on the pathogenesis of SVD.

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 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 AB0024 IN VITRO CHARACTERIZATION OF CENERIMOD, A POTENT AND SELECTIVE SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 (S1P1) MODULATOR IN PREVENTING MIGRATION OF NON-ACTIVATED AND ACTIVATED PRIMARY HUMAN B CELLS IN THE PRESENCE OR ABSENCE OF GLUCOCORTICOIDS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1046.1-1046
Author(s):  
L. Schlicher ◽  
P. Kulig ◽  
M. Murphy ◽  
M. Keller
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Activation ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
B Cell Activation ◽  
Human B Cells ◽  
Circulating Lymphocytes

Background:Cenerimod is a potent, selective, and orally active sphingosine 1-phosphate receptor 1 (S1P1) modulator that is currently being evaluated in a Phase 2b study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including B lymphocytes) in the blood stream and in inflamed tissues. Extensive clinical experience has become available for the nonselective S1P receptor modulator fingolimod in relapsing forms of multiple sclerosis, supporting this therapeutic concept for the treatment of autoimmune disorders.Objectives:Although the effect of S1P-receptor modulators in reducing peripheral B cells is well documented1,2, the role of the S1P1 receptor on this cell type is only incompletely understood. In this study, the mode of action of cenerimod on primary human B cells was investigated in a series of in vitro experiments, including S1P1 receptor cell surface expression and chemotaxis towards S1P. Moreover, S1P1 expression following B cell activation in vitro was studied. As glucocorticoids (GC) are frequently used in the treatment of patients with autoimmune disorders including SLE, the potential influence of GC on the mode of action of cenerimod was evaluated.Methods:Primary human B lymphocytes from healthy donors were isolated from whole blood. In one set of experiments, cells were treated with different concentrations of cenerimod to measure S1P1 receptor internalization by flow cytometry. In a second set of experiments, isolated B cells were activated using different stimuli or left untreated. Cells were then analysed for S1P1 and CD69 cell surface expression and tested in a novel real-time S1P-mediated migration assay. In addition, the effect of physiological concentrations of GCs (prednisolone and prednisone) on cenerimod activity in preventing S1P mediated migration was tested.Results:In vitro, cenerimod led to a dose-dependent internalization of the S1P1 receptor on primary human B lymphocytes. Cenerimod also blocked migration of nonactivated and activated B lymphocytes towards S1P in a concentration-dependent manner, which is in line with the retention of lymphocytes in the lymph node and the reduction of circulating lymphocytes observed in the clinical setting. Upon B cell activation, which was monitored by CD69 upregulation, a simultaneous downregulation of S1P1 expression was detected, leading to less efficient S1P-directed cell migration. Importantly, physiological concentrations of GC did not affect the inhibitory activity of cenerimod on B cell migration.Conclusion:These results show that cenerimod, by modulating S1P1, blocks B lymphocyte migration towards its natural chemoattractant S1P and demonstrate compatibility of cenerimod with GC. These results are consistent with results of comparable experiments done previously using primary human T lymphocytes.References:[1]Nakamura M et al., Mult Scler. 2014 Sep; 20(10):1371-80.[2]Strasser DS et al., RMD Open 2020;6:e001261.Disclosure of Interests:None declared

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