Characterization of RNA elements that regulate gag-pol ribosomal frameshifting in equine infectious anemia virus.
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.
Publication Date: 2003-09-13 PubMed ID: 12970412PubMed Central: PMC228510DOI: 10.1128/jvi.77.19.10280-10287.2003Google Scholar: Lookup
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- Journal Article
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- U.S. Gov't
- P.H.S.
Summary
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The research examines the role of specific mRNA elements in the synthesis of Gag-Pol polyproteins of the equine infectious anemia virus (EIAV) through a process called ribosomal frameshifting. Interestingly, the paper defines the necessary RNA sequence and structural requirements for frameshifting and reveals how disruptions and alterations can impact EIAV’s ability to replicate.
Process of Synthesis of Gag-Pol Polyproteins
- In a retrovirus like EIAV, ribosomes have to shift their reading frame in a -1 direction one or two times. This allows them to bypass the stop codon in the gag reading frame and facilitate the synthesis of Gag-Pol polyproteins.
- This study suggests that a slippery sequence – a stretch of RNA that allows the ribosome to realign itself on the mRNA – and a downstream RNA structure are usually needed for this programmed -1 ribosomal frameshifting.
Specific mRNA Elements Required for Ribosomal Frameshifting
- The researchers identified some specific mRNA elements—an indicative slippery sequence, a five-base-paired GC stretch, and a pseudoknot structure—as key to efficient ribosomal frameshifting in EIAV.
- A pseudoknot structure is a three-dimensional folding arrangement that RNA molecules can form, which contributes to their function.
- The frameshifting efficiency and viral replication were found to be dependent on these elements.
Testing the Role of These Elements
- The researchers tested the role of these elements by using slippery sequences obtained from human immunodeficiency virus type 1 and visna virus to replace the EIAV slippery sequence. The substitutions supported EIAV replication, suggesting the importance of a general slippery sequence in the process wherever it came from.
- However, disrupting the GC-paired stretch stopped the frameshifting vital for viral replication.
- Meanwhile, disruption of the pseudoknot structure reduced frameshifting efficiency by 60%, showing its role in the process, albeit not entirely crucial.
Implications of the Research
- The researchers suggest that the maintenance of these essential RNA signals in this part of the genomic mRNA is crucial for sufficient ribosomal frameshifting and EIAV replication.
- Meanwhile, alterations in the amino acids translated from the same area of the mRNA can be adapted to during replication.
- The findings have implications for understanding the replication strategy of retroviruses, which can be beneficial for the development of potential antiviral targets.
Cite This Article
APA
Chen C, Montelaro RC.
(2003).
Characterization of RNA elements that regulate gag-pol ribosomal frameshifting in equine infectious anemia virus.
J Virol, 77(19), 10280-10287.
https://doi.org/10.1128/jvi.77.19.10280-10287.2003 Publication
Researcher Affiliations
- Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
MeSH Terms
- Animals
- Base Sequence
- Frameshifting, Ribosomal
- Fusion Proteins, gag-pol / genetics
- Horses
- Infectious Anemia Virus, Equine / genetics
- Molecular Sequence Data
- RNA, Viral / chemistry
- RNA, Viral / physiology
Grant Funding
- R01 CA049296 / NCI NIH HHS
- T32 AI049820 / NIAID NIH HHS
- R01 CA49296 / NCI NIH HHS
- T32 AI49820 / NIAID NIH HHS
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Citations
This article has been cited 7 times.- Huang L, Li L, Tien C, LaBarbera DV, Chen C. Targeting HIV-1 Protease Autoprocessing for High-throughput Drug Discovery and Drug Resistance Assessment.. Sci Rep 2019 Jan 22;9(1):301.
- Atkins JF, Loughran G, Bhatt PR, Firth AE, Baranov PV. Ribosomal frameshifting and transcriptional slippage: From genetic steganography and cryptography to adventitious use.. Nucleic Acids Res 2016 Sep 6;44(15):7007-78.
- Counts CJ, Ho PS, Donlin MJ, Tavis JE, Chen C. A Functional Interplay between Human Immunodeficiency Virus Type 1 Protease Residues 77 and 93 Involved in Differential Regulation of Precursor Autoprocessing and Mature Protease Activity.. PLoS One 2015;10(4):e0123561.
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