scholarly journals Characterization of RNA Elements That Regulate Gag-Pol Ribosomal Frameshifting in Equine Infectious Anemia Virus

2003 ◽  
Vol 77 (19) ◽  
pp. 10280-10287 ◽  
Author(s):  
Chaoping Chen ◽  
Ronald C. Montelaro
Keyword(s):  
Viral Replication ◽  
Rna Structure ◽  
Equine Infectious Anemia Virus ◽  
Stop Codon ◽  
Structural Determinants ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Anemia Virus ◽  
Antiviral Targets ◽  
Slippery Sequence

ABSTRACT Synthesis of Gag-Pol polyproteins of retroviruses requires ribosomes to shift translational reading frame once or twice in a −1 direction to read through the stop codon in the gag reading frame. It is generally believed that a slippery sequence and a downstream RNA structure are required for the programmed −1 ribosomal frameshifting. However, the mechanism regulating the Gag-Pol frameshifting remains poorly understood. In this report, we have defined specific mRNA elements required for sufficient ribosomal frameshifting in equine anemia infectious virus (EIAV) by using full-length provirus replication and Gag/Gag-Pol expression systems. The results of these studies revealed that frameshifting efficiency and viral replication were dependent on a characteristic slippery sequence, a five-base-paired GC stretch, and a pseudoknot structure. Heterologous slippery sequences from human immunodeficiency virus type 1 and visna virus were able to substitute for the EIAV slippery sequence in supporting EIAV replication. Disruption of the GC-paired stretch abolished the frameshifting required for viral replication, and disruption of the pseudoknot reduced the frameshifting efficiency by 60%. Our data indicated that maintenance of the essential RNA signals (slippery sequences and structural elements) in this region of the genomic mRNA was critical for sufficient ribosomal frameshifting and EIAV replication, while concomitant alterations in the amino acids translated from the same region of the mRNA could be tolerated during replication. The data further indicated that proviral mutations that reduced frameshifting efficiency by as much as 50% continued to sustain viral replication and that greater reductions in frameshifting efficiency lead to replication defects. These studies define for the first time the RNA sequence and structural determinants of Gag-Pol frameshifting necessary for EIAV replication, reveal novel aspects relative to frameshifting elements described for other retroviruses, and provide new genetic determinants that can be evaluated as potential antiviral targets.

scholarly journals A Computational and Biochemical Study of -1 Ribosomal Frameshifting in Human mRNAs

2021 ◽  
Author(s):  
Xia Zhou ◽  
Xiaolan Huang ◽  
Zhihua Du
Keyword(s):  
Rna Structure ◽  
Stop Codon ◽  
Biochemical Study ◽  
Viral Origin ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Human Transcriptome ◽  
Cellular Genes ◽  
Cellular Mrnas ◽  
Ncbi Refseq

Abstract−1 programmed ribosomal frameshifting (−1 PRF) is a translational recoding mechanism used by many viral and cellular mRNAs. −1 PRF occurs at a heptanucleotide slippery sequence and is stimulated by a downstream RNA structure, most often in the form of a pseudoknot. The utilization of −1 PRF to produce proteins encoded by the −1 reading frame is wide-spread in RNA viruses, but relatively rare in cellular mRNAs. In human, only three such cases of −1 PRF events have been reported, all involving retroviral-like genes and protein products. To evaluate the extent of −1 PRF utilization in the human transcriptome, we have developed a computational scheme for identifying putative pseudoknot-dependent −1 PRF events and applied the method to a collection of 43,191 human mRNAs in the NCBI RefSeq database. In addition to the three reported cases, our study identified more than two dozen putative −1 PRF cases. The genes involved in these cases are genuine cellular genes without a viral origin. Moreover, in more than half of these cases, the frameshift site locates far upstream (>250 nt) from the stop codon of the 0 reading frame, which is nonviral-like. Using dual luciferase assays in HEK293T cells, we confirmed that the −1 PRF signals in the mRNAs of CDK5R2 and SEMA6C are functional in inducing efficient frameshifting. Our findings have significant implications in expanding the repertoire of the −1 PRF phenomenon and the protein-coding capacity of the human transcriptome.

scholarly journals Ribosomal Pausing at a Frameshifter RNA Pseudoknot Is Sensitive to Reading Phase but Shows Little Correlation with Frameshift Efficiency

2001 ◽  
Vol 21 (24) ◽  
pp. 8657-8670 ◽  
Author(s):  
Harry Kontos ◽  
Sawsan Napthine ◽  
Ian Brierley
Keyword(s):  
Rna Structure ◽  
Phase Dependence ◽  
Stem Loop ◽  
Reading Frame ◽  
Stem Loop Structure ◽  
Rna Pseudoknot ◽  
Ribosomal P ◽  
Slippery Sequence ◽  
The Impact ◽  
Ribosomal Pausing

ABSTRACT Here we investigated ribosomal pausing at sites of programmed −1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.

scholarly journals Thermodynamic control of −1 programmed ribosomal frameshifting

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lars V. Bock ◽  
Neva Caliskan ◽  
Natalia Korniy ◽  
Frank Peske ◽  
Marina V. Rodnina ◽  
...  
Keyword(s):  
Energy Difference ◽  
Mrna Sequence ◽  
Thermodynamic Control ◽  
Base Pairs ◽  
Free Energy Difference ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Slippery Sequence

Abstract mRNA contexts containing a ‘slippery’ sequence and a downstream secondary structure element stall the progression of the ribosome along the mRNA and induce its movement into the −1 reading frame. In this study we build a thermodynamic model based on Bayesian statistics to explain how −1 programmed ribosome frameshifting can work. As training sets for the model, we measured frameshifting efficiencies on 64 dnaX mRNA sequence variants in vitro and also used 21 published in vivo efficiencies. With the obtained free-energy difference between mRNA-tRNA base pairs in the 0 and −1 frames, the frameshifting efficiency of a given sequence can be reproduced and predicted from the tRNA−mRNA base pairing in the two frames. Our results further explain how modifications in the tRNA anticodon modulate frameshifting and show how the ribosome tunes the strength of the base-pair interactions.

scholarly journals The S2 Gene of Equine Infectious Anemia Virus Is a Highly Conserved Determinant of Viral Replication and Virulence Properties in Experimentally Infected Ponies

2000 ◽  
Vol 74 (1) ◽  
pp. 573-579 ◽  
Author(s):  
Feng Li ◽  
Caroline Leroux ◽  
Jodi K. Craigo ◽  
Sheila J. Cook ◽  
Charles J. Issel ◽  
...  
Keyword(s):  
Viral Replication ◽  
Equine Infectious Anemia Virus ◽  
Parental Virus ◽  
In Vivo Studies ◽  
Viral Gene ◽  
Viral Virulence ◽  
Equine Infectious Anemia ◽  
Anemia Virus

ABSTRACT Equine infectious anemia virus (EIAV) is genetically one of the simplest lentiviruses in that the viral genome encodes only three accessory genes, tat, rev, and S2. Although serological analyses demonstrate the expression of the S2 protein in persistently infected horses, the role of this viral gene remains undefined. We recently reported that the S2 gene is not essential for EIAV replication in primary equine macrophages, as EIAV mutants lacking the S2 gene replicate to levels similar to those of the parental virus (F. Li, B. A. Puffer, and R. C. Montelaro, J. Virol. 72:8344–8348, 1998). We now describe in vivo studies that examine the evolution and role of theS2 gene in ponies experimentally infected with EIAV. The results of these studies reveal for the first time that theS2 gene is highly conserved during persistent infection and that deletion of the S2 gene reduces viral virulence and virus replication levels compared to those of the parental virus containing a functional S2 gene. These data indicate that the EIAV S2 gene is in fact an important determinant of viral replication and pathogenic properties in vivo, despite the evident lack of S2 influence on viral replication levels in vitro. Thus, these observations suggest in vivo functions of EIAVS2 that are not adequately reflected in simple infections of cultured cells, including natural target macrophages.

scholarly journals Equine Infectious Anemia Virus Genomic Evolution in Progressor and Nonprogressor Ponies

2001 ◽  
Vol 75 (10) ◽  
pp. 4570-4583 ◽  
Author(s):  
Caroline Leroux ◽  
Jodi K. Craigo ◽  
Charles J. Issel ◽  
Ronald C. Montelaro
Keyword(s):  
Steady State ◽  
Viral Replication ◽  
Virus Replication ◽  
Equine Infectious Anemia Virus ◽  
Genomic Rna ◽  
Equine Infectious Anemia ◽  
Steady State Levels ◽  
Anemia Virus ◽  
First Time

ABSTRACT A primary mechanism of lentivirus persistence is the ability of these viruses to evolve in response to biological and immunological selective pressures with a remarkable array of genetic and antigenic variations that constitute a perpetual natural experiment in genetic engineering. A widely accepted paradigm of lentivirus evolution is that the rate of genetic variation is correlated directly with the levels of virus replication: the greater the viral replication, the more opportunities that exist for genetic modifications and selection of viral variants. To test this hypothesis directly, we examined the patterns of equine infectious anemia virus (EIAV) envelope variation during a 2.5-year period in experimentally infected ponies that differed markedly in clinical progression and in steady-state levels of viral replication as indicated by plasma virus genomic RNA assays. The results of these comprehensive studies revealed for the first time similar extents of envelope gp90 variation in persistently infected ponies regardless of the number of disease cycles (one to six) and viremia during chronic disease. The extent of envelope variation was also independent of the apparent steady-state levels of virus replication during long-term asymptomatic infection, varying from undetectable to 105 genomic RNA copies per ml of plasma. In addition, the data confirmed the evolution of distinct virus populations (genomic quasispecies) associated with sequential febrile episodes during acute and chronic EIA and demonstrated for the first time ongoing envelope variation during long-term asymptomatic infections. Finally, comparison of the rates of evolution of the previously defined EIAV gp90 variable domains demonstrated distinct differences in the rates of nucleotide and amino acid sequence variation, presumably reflecting differences in the ability of different envelope domains to respond to immune or other biological selection pressures. Thus, these data suggest that EIAV variation can be associated predominantly with ongoing low levels of virus replication and selection in target tissues, even in the absence of substantial levels of plasma viremia, and that envelope variation continues during all stages of persistent infection as the virus successfully avoids clearance by host defense mechanisms.

scholarly journals Equine Infectious Anemia Virus Is Found in Tissue Macrophages during Subclinical Infection

1998 ◽  
Vol 72 (9) ◽  
pp. 7263-7269 ◽  
Author(s):  
J. Lindsay Oaks ◽  
Travis C. McGuire ◽  
Catherine Ulibarri ◽  
Timothy B. Crawford
Keyword(s):  
Viral Load ◽  
Viral Replication ◽  
Equine Infectious Anemia Virus ◽  
Disease Status ◽  
Viral Rna ◽  
Subclinical Infection ◽  
Equine Infectious Anemia ◽  
Infected Cells ◽  
Anemia Virus

ABSTRACT The equine infectious anemia virus (EIAV) often results in lifelong subclinical infection following early episodes of clinical disease. To identify the cellular reservoirs of EIAV during subclinical infection, horses were infected with EIAV and allowed to develop subclinical infections. Horses with acute disease served as a basis for comparison. The tissue distribution, replication status, location of infected cells, and viral load were characterized by PCR for proviral DNA and reverse transcriptase PCR for viral RNA, in situ hybridization, and in situ PCR. Proviral DNA was widespread in tissues regardless of disease status. Viral gag and env RNAs were also detected in tissues of all horses regardless of disease status. Plasma viral RNA (viremia) could be detected in some, but not all, horses with subclinical infections. In situ assays determined that a primary cellular reservoir and site of viral replication during subclinical infection is the macrophage. During subclinical infection, viral load was decreased 4- to 733-fold and there was decreased viral RNA expression within infected cells. These data indicate that viral replication continues at all times, even in horses that are clinically quiescent. Moreover, restricted viral replication at the cellular level is associated with clinical remission.

Sign in / Sign up

Export Citation Format

Share Document