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Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus

Translational readthrough—suppression of termination at a stop codon—is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading...

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Autores principales: Csibra, Eszter, Brierley, Ian, Irigoyen, Nerea
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178896/
https://www.ncbi.nlm.nih.gov/pubmed/24991001
http://dx.doi.org/10.1128/JVI.00898-14
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author Csibra, Eszter
Brierley, Ian
Irigoyen, Nerea
author_facet Csibra, Eszter
Brierley, Ian
Irigoyen, Nerea
author_sort Csibra, Eszter
collection PubMed
description Translational readthrough—suppression of termination at a stop codon—is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an ∼8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial. IMPORTANCE Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational “recoding” processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.
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spelling pubmed-41788962014-10-24 Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus Csibra, Eszter Brierley, Ian Irigoyen, Nerea J Virol Genome Replication and Regulation of Viral Gene Expression Translational readthrough—suppression of termination at a stop codon—is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an ∼8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial. IMPORTANCE Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational “recoding” processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses. American Society for Microbiology 2014-09 /pmc/articles/PMC4178896/ /pubmed/24991001 http://dx.doi.org/10.1128/JVI.00898-14 Text en Copyright © 2014 Csibra et al. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license (http://creativecommons.org/licenses/by/3.0/) .
spellingShingle Genome Replication and Regulation of Viral Gene Expression
Csibra, Eszter
Brierley, Ian
Irigoyen, Nerea
Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus
title Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus
title_full Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus
title_fullStr Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus
title_full_unstemmed Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus
title_short Modulation of Stop Codon Read-Through Efficiency and Its Effect on the Replication of Murine Leukemia Virus
title_sort modulation of stop codon read-through efficiency and its effect on the replication of murine leukemia virus
topic Genome Replication and Regulation of Viral Gene Expression
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178896/
https://www.ncbi.nlm.nih.gov/pubmed/24991001
http://dx.doi.org/10.1128/JVI.00898-14
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