GO:0075525 viral translational termination-reinitiation: Viral Gene Expression Strategy, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0075525 describes a viral translation strategy in which ribosomes that terminate at an upstream open reading frame (ORF) remain tethered to the mRNA and reinitiate at a downstream ORF, enabling expression of multiple proteins from a single dicistronic mRNA.
• This mechanism is used by diverse RNA viruses, including influenza B virus, hepatitis B virus, and bovine norovirus, to express essential proteins such as BM2, polymerase, and capsid without producing subgenomic RNAs.
• The efficiency of termination-reinitiation depends on the length and sequence context of the upstream ORF, the spacing between ORFs, and RNA structures that interact with the ribosome.
• A conserved class of viral RNA structures dynamically interacts with the ribosome to regulate reinitiation, providing a potential target for antiviral intervention.
• Studying GO:0075525 requires specialized techniques such as Ribo-seq, dual-luciferase reporter assays, and CRISPR-based editing of viral or host genes to dissect the molecular requirements.
• Understanding this process illuminates fundamental translation control mechanisms and offers insights for vaccine design, antiviral development, and synthetic biology applications.
Description
Viral translational termination-reinitiation (GO:0075525) is a specialized translation mechanism that allows certain RNA viruses to express multiple proteins from a single dicistronic mRNA. In this process, ribosomes translate an upstream open reading frame (ORF), terminate at its stop codon, and then a proportion of 40S subunits remain associated with the mRNA and reinitiate translation at a downstream ORF. This strategy is particularly important for viruses with compact genomes, as it enables the production of essential proteins without the need for separate subgenomic RNAs. The mechanism was first characterized in influenza B virus, where the BM2 protein is expressed via termination-reinitiation of the segment 7 mRNA. Since then, similar strategies have been identified in hepatitis B virus, bovine norovirus, and other RNA viruses. Understanding GO:0075525 is crucial for researchers studying viral gene expression, as it reveals how viruses maximize coding capacity and regulate protein stoichiometry. Moreover, this process represents a potential target for antiviral therapies, as disrupting the interaction between viral RNA structures and the ribosome could inhibit viral replication. The study of termination-reinitiation also provides insights into fundamental translation mechanisms, including ribosome recycling and reinitiation, which are conserved across eukaryotes.
viral translational termination-reinitiation At A Glance
| GO ID | GO:0075525 |
|---|---|
| GO term | viral translational termination-reinitiation |
| Ontology | biological_process |
| Synonym | termination reinitiation involved in viral translation; viral translation involving termination re-initiation; viral translation involving termination-reinitiation; viral translation involving translational stop-start |
| Major function | Allows expression of a downstream ORF from a dicistronic viral mRNA by retaining 40S subunits after termination and reinitiating translation. |
| Organisms | RNA viruses including influenza B virus, hepatitis B virus, bovine norovirus, and others. |
| Key factors | Ribosomal subunits, viral mRNA structures, upstream ORF length, and termination-reinitiation signals. |
| Related processes | Translation termination, translation reinitiation, ribosome recycling, and non-canonical translation initiation. |
What Is GO:0075525?
GO:0075525, viral translational termination-reinitiation, is a biological process that occurs during viral mRNA translation. It allows the expression of a downstream open reading frame (ORF) from a dicistronic mRNA. In this process, ribosomes translate the upstream ORF and terminate at its stop codon. However, instead of fully dissociating from the mRNA, a proportion of 40S ribosomal subunits remain tethered to the mRNA and go on to reinitiate translation at the start codon of the downstream ORF. This mechanism is distinct from canonical translation termination because it involves the retention and reuse of ribosomal subunits on the same mRNA molecule. It is also known as termination reinitiation involved in viral translation, viral translation involving termination re-initiation, viral translation involving termination-reinitiation, and viral translation involving translational stop-start.
Why Is viral translational termination-reinitiation Important in Cell Biology?
Viral translational termination-reinitiation is important because it enables viruses with compact genomes to express multiple proteins from a single mRNA, thereby maximizing coding capacity and regulating protein stoichiometry. This mechanism is essential for the replication of several medically relevant viruses, including influenza B virus and hepatitis B virus. Disrupting termination-reinitiation could provide a novel antiviral strategy, as it would prevent the production of essential viral proteins. Furthermore, studying this process enhances our understanding of fundamental translation mechanisms, such as ribosome recycling and reinitiation, which have broad implications for cell biology and gene expression.
• Enables expression of multiple proteins from a single dicistronic viral mRNA, conserving genomic space.
• Critical for the replication of influenza B virus, where BM2 is expressed via termination-reinitiation.
• Required for hepatitis B virus polymerase expression from an upstream minicistron.
• Facilitates capsid expression in bovine norovirus without subgenomic RNA synthesis.
• Represents a potential target for antiviral drugs that disrupt RNA-ribosome interactions.
• Provides a model for studying ribosome recycling and reinitiation in eukaryotes.
• Influences viral protein stoichiometry, which is important for virion assembly and fitness.
• Offers insights for synthetic biology and mRNA vaccine design.
• Highlights the diversity of non-canonical translation initiation mechanisms in viruses.
• Can be studied using CRISPR-based editing to identify host factors involved in the process.
What Happens During viral translational termination-reinitiation?
Translation of the upstream ORF
In simple terms: The ribosome first reads the first gene on the viral mRNA.
In the initial step, a ribosome binds to the viral mRNA and translates the upstream open reading frame (ORF). This ORF typically encodes a short peptide or a full protein, depending on the virus. For example, in influenza B virus segment 7 mRNA, the upstream ORF encodes the M1 protein, while the downstream ORF encodes the BM2 protein. The length and sequence of the upstream ORF influence the efficiency of subsequent reinitiation.
Termination at the upstream stop codon
In simple terms: The ribosome stops at the end of the first gene but does not completely fall off.
When the ribosome reaches the stop codon of the upstream ORF, it undergoes termination. However, in termination-reinitiation, a proportion of 40S ribosomal subunits remain tethered to the mRNA instead of dissociating completely. This retention is facilitated by specific viral RNA structures and possibly by interactions with translation factors. The efficiency of this step is modulated by the distance between the upstream ORF and the downstream ORF, as well as by the sequence context of the stop codon.
Retention of 40S subunits on the mRNA
In simple terms: Part of the ribosome stays on the mRNA after finishing the first gene.
After termination, the 40S subunit, along with initiator tRNA and possibly other factors, remains associated with the mRNA. This retained 40S subunit is then capable of scanning or being positioned at the start codon of the downstream ORF. The retention is dependent on cis-acting RNA elements, such as those found in the influenza B virus segment 7 mRNA, which form specific structures that interact with the ribosome. These structures are conserved among related viruses and are critical for efficient reinitiation.
Reinitiation at the downstream ORF
In simple terms: The ribosome starts translating the second gene.
The retained 40S subunit, together with initiation factors and the 60S subunit, reinitiates translation at the start codon of the downstream ORF. This leads to the synthesis of the downstream protein, such as BM2 in influenza B virus or the polymerase in hepatitis B virus. The efficiency of reinitiation is influenced by the length of the upstream ORF, the spacing between ORFs, and the presence of RNA structures that promote ribosome retention. In some viruses, such as bovine norovirus, termination-reinitiation between ORF1 and ORF2 enables capsid expression without subgenomic RNA.
Regulation by viral RNA structures
In simple terms: Special RNA shapes help control how often the second gene is made.
Viral RNA structures play a key role in regulating termination-reinitiation. A conserved class of RNA structures, exemplified by the influenza B virus segment 7 mRNA, dynamically interacts with the ribosome to modulate reinitiation efficiency. These structures can affect the stability of the 40S subunit on the mRNA and its ability to reinitiate. Mutations that disrupt these structures reduce reinitiation, highlighting their functional importance. Understanding these RNA elements provides opportunities for antiviral intervention.
Key Genes Involved in GO:0075525 viral translational termination-reinitiation
The following genes and proteins are involved in or regulated by viral translational termination-reinitiation, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| BM2 (influenza B virus) | Downstream ORF product expressed via termination-reinitiation | Model for studying termination-reinitiation efficiency and RNA structure requirements. |
| M1 (influenza B virus) | Upstream ORF product; its translation precedes reinitiation | Length and sequence influence reinitiation efficiency. |
| HBV polymerase | Downstream ORF product expressed via termination-reinitiation | Demonstrates length-dependent regulation of reinitiation. |
| HBV core protein | Upstream minicistron product | Its translation is linked to polymerase expression via termination-reinitiation. |
| Bovine norovirus ORF1 | Upstream ORF encoding nonstructural proteins | Termination-reinitiation between ORF1 and ORF2 enables capsid expression. |
| Bovine norovirus ORF2 | Downstream ORF encoding capsid protein | Expressed via termination-reinitiation without subgenomic RNA. |
| RPS3 (40S subunit) | Component of the 40S ribosomal subunit retained after termination | Potential target for disrupting reinitiation. |
| RACK1 | Ribosome-associated protein involved in translation regulation | May modulate termination-reinitiation efficiency. |
| eIF3 | Translation initiation factor involved in reinitiation | Required for reinitiation at downstream ORF. |
| eIF2 | Delivers initiator tRNA during reinitiation | Its activity affects reinitiation efficiency. |
| eIF4F | Cap-binding complex; may be involved in reinitiation | Potential role in ribosome recruitment. |
| DAP5 | Alternative initiation factor implicated in reinitiation | May support non-canonical reinitiation. |
| PTB | RNA-binding protein that interacts with viral IRES-like structures | May facilitate ribosome retention. |
| La autoantigen | RNA-binding protein involved in translation enhancement | Potential role in termination-reinitiation. |
| hnRNP A1 | RNA-binding protein affecting translation | May modulate viral RNA structure and reinitiation. |
| Nucleolin | RNA-binding protein with roles in translation | Potential involvement in ribosome recruitment. |
How Is viral translational termination-reinitiation Regulated?
The regulation of viral translational termination-reinitiation is primarily mediated by cis-acting RNA elements within the viral mRNA and by trans-acting host factors. The length of the upstream ORF and the spacing between ORFs are critical determinants of reinitiation efficiency. Specific RNA structures, such as those in the influenza B virus segment 7 mRNA, dynamically interact with the ribosome to promote 40S subunit retention and reinitiation. Host translation initiation factors, including eIF3 and eIF2, are required for reinitiation. Additionally, cellular stress responses and mTOR signaling may influence the availability of these factors, thereby modulating termination-reinitiation. However, the exact regulatory mechanisms remain an active area of research.
viral translational termination-reinitiation and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| BM2 (influenza B virus) | Influenza B virus replication | Influenza B virus infection in cell culture; BM2 knockout virus. |
| HBV polymerase | Hepatitis B virus replication and liver disease | HBV-infected hepatoma cells; polymerase expression reporters. |
| Bovine norovirus ORF2 | Norovirus capsid production | Bovine norovirus replicon or infected cells. |
| eIF3 | Host translation initiation; viral replication | CRISPR knockout of eIF3 subunits in permissive cells. |
| RPS3 | Ribosome function; viral translation | Point mutations in RPS3 to test ribosome retention. |
Influenza B virus infection
Influenza B virus relies on termination-reinitiation to express the BM2 protein from its segment 7 mRNA. BM2 is essential for viral replication and is a component of the viral envelope. Disrupting this process could attenuate the virus, making it a potential target for antiviral drugs.
Hepatitis B virus infection
Hepatitis B virus uses termination-reinitiation to express its polymerase from an upstream minicistron. The efficiency of this process is length-dependent, and mutations that alter the upstream ORF affect polymerase expression and viral replication. This mechanism is critical for HBV persistence and liver disease progression.
Norovirus infection
Bovine norovirus employs termination-reinitiation between ORF1 and ORF2 to express its capsid protein without producing a subgenomic RNA. This strategy is essential for capsid production and virion assembly. Understanding this process may inform the development of antivirals against noroviruses, which cause gastroenteritis in humans.
Broad antiviral strategies
Because termination-reinitiation is used by multiple RNA viruses, targeting this mechanism could provide broad-spectrum antiviral effects. Small molecules or antisense oligonucleotides that disrupt the RNA structures or ribosome interactions required for reinitiation could inhibit viral replication. However, specificity and potential host toxicity must be carefully evaluated.
From viral translational termination-reinitiation-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does a candidate host gene regulate termination-reinitiation? | CRISPR knockout of the gene in cells expressing a dual-luciferase reporter with viral termination-reinitiation elements. |
| What is the role of a specific RNA structure in reinitiation? | Point mutations in the viral RNA structure, followed by reporter assays. |
| Can a drug inhibit termination-reinitiation? | High-throughput screening using viral reporter cells and small molecule libraries. |
| How does upstream ORF length affect reinitiation efficiency? | Knock-in of varying upstream ORF lengths into a reporter construct. |
| What host proteins interact with the viral RNA during reinitiation? | Tagged knock-in of RNA-binding proteins followed by RNA pulldown and proteomics. |
| Does overexpression of a translation factor enhance reinitiation? | Overexpression of eIF3 or eIF2 in reporter cells. |
How to Study the viral translational termination-reinitiation Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Ribo-seq | Ribosome occupancy and positioning on mRNA | Detecting reinitiation events in viral infection. |
| Dual-luciferase reporter assay | Efficiency of termination-reinitiation | Testing viral RNA elements and host factors. |
| SHAPE-MaP | RNA secondary structure | Mapping conserved RNA structures in viral mRNAs. |
| CRISPR knockout screen | Host genes affecting termination-reinitiation | Identifying antiviral targets. |
| Western blot | Protein expression levels of upstream and downstream ORFs | Validating reinitiation efficiency. |
| qRT-PCR | mRNA levels of viral transcripts | Controlling for transcription effects. |
| Immunofluorescence | Subcellular localization of viral proteins | Confirming expression of downstream ORF products. |
| Mass spectrometry | Proteomic changes upon infection or perturbation | Identifying host factors involved in reinitiation. |
Ribosome profiling (Ribo-seq)
Ribo-seq provides a genome-wide snapshot of ribosome positions on mRNAs at codon resolution. It can be used to detect ribosome accumulation at upstream ORFs and reinitiation at downstream ORFs in viral infections. By comparing wild-type and mutant viruses, researchers can identify RNA elements required for termination-reinitiation.
Dual-luciferase reporter assays
Dual-luciferase reporters containing viral upstream and downstream ORFs are widely used to measure termination-reinitiation efficiency. The upstream ORF is fused to one luciferase and the downstream ORF to another, allowing quantification of reinitiation. This assay is amenable to high-throughput screening for inhibitors or host factors.
RNA structure probing
Techniques such as SHAPE-MaP or DMS-seq can map RNA structures within viral mRNAs. These methods reveal conserved structural elements that interact with the ribosome and regulate termination-reinitiation. Combining structure probing with mutagenesis can pinpoint functional RNA motifs.
CRISPR-based genetic screens
Genome-wide CRISPR knockout or activation screens can identify host genes that modulate termination-reinitiation. Cells expressing a viral reporter are infected with a CRISPR library, and changes in reporter activity are measured. This approach has the potential to uncover novel therapeutic targets.
How CRISPR Can Be Used to Study GO:0075525 viral translational termination-reinitiation
Knockout
CRISPR knockout of candidate host genes can reveal their requirement for viral termination-reinitiation. For example, knocking out eIF3 subunits or ribosomal proteins followed by a dual-luciferase reporter assay can determine if they are essential for reinitiation. This approach is scalable for genome-wide screens.
Point Mutation
Point mutations can be introduced into viral RNA structures or host genes to dissect their role in termination-reinitiation. For instance, mutating conserved nucleotides in the influenza B virus segment 7 RNA structure can abolish reinitiation, as shown by reporter assays. Similarly, point mutations in ribosomal protein genes can test their function in ribosome retention.
Knock-in
Knock-in of reporter cassettes or epitope tags into viral genomes or host genes allows precise measurement of termination-reinitiation. For example, inserting a luciferase gene downstream of a viral ORF can create a reporter virus. Tagged knock-in of host proteins can facilitate interaction studies.
Overexpression
Overexpression of host translation factors or viral proteins can enhance or inhibit termination-reinitiation. For example, overexpressing eIF3 or eIF2 may increase reinitiation efficiency, while overexpressing dominant-negative mutants can block it. This approach helps identify rate-limiting factors.
How EDITGENE Supports viral translational termination-reinitiation Research
Researchers studying viral translational termination-reinitiation-related genes often need to determine whether a candidate gene is causally involved in the process or merely correlated with it. EDITGENE provides a comprehensive suite of CRISPR-based services to enable precise genetic manipulation and functional validation.
Contact EDITGENE today to design your custom CRISPR model for viral translational termination-reinitiation research.
Frequently Asked Questions About viral translational termination-reinitiation
What is viral translational termination-reinitiation (GO:0075525)?
It is a biological process in which ribosomes translate an upstream open reading frame (ORF) on a viral mRNA, terminate, and then a proportion of 40S subunits remain tethered to the mRNA and reinitiate translation at a downstream ORF, allowing expression of multiple proteins from a single dicistronic mRNA.
Which viruses use termination-reinitiation?
Influenza B virus, hepatitis B virus, and bovine norovirus are well-characterized examples that use termination-reinitiation to express essential proteins.
What genes are involved in viral translational termination-reinitiation?
Key genes include viral genes such as influenza B virus BM2 and M1, hepatitis B virus polymerase, and bovine norovirus ORF1 and ORF2, as well as host genes encoding ribosomal proteins (e.g., RPS3) and translation initiation factors (e.g., eIF3, eIF2).
How is termination-reinitiation regulated?
It is regulated by cis-acting RNA structures in the viral mRNA, the length of the upstream ORF, the spacing between ORFs, and host translation factors such as eIF3 and eIF2.
What is the difference between termination-reinitiation and canonical translation?
In canonical translation, ribosomes dissociate from the mRNA after termination. In termination-reinitiation, a proportion of 40S subunits remain on the mRNA and reinitiate at a downstream ORF, enabling dicistronic expression.
Why is termination-reinitiation important for viral replication?
It allows viruses with compact genomes to express multiple proteins from a single mRNA, conserving coding capacity and regulating protein stoichiometry, which is essential for viral replication and assembly.
Can termination-reinitiation be targeted for antiviral therapy?
Yes, disrupting the RNA structures or ribosome interactions required for reinitiation could inhibit viral replication. This is an active area of research.
What methods are used to study termination-reinitiation?
Common methods include dual-luciferase reporter assays, Ribo-seq, RNA structure probing (e.g., SHAPE-MaP), and CRISPR-based genetic screens.
What is the role of RNA structures in termination-reinitiation?
Conserved RNA structures in viral mRNAs interact with the ribosome to promote 40S subunit retention and efficient reinitiation. Mutations that disrupt these structures reduce reinitiation.
How can CRISPR help study termination-reinitiation?
CRISPR can be used to knockout, knock-in, or overexpress candidate host genes to determine their role in termination-reinitiation. Genome-wide screens can identify novel regulators.
Conclusion
Viral translational termination-reinitiation (GO:0075525) is a fascinating and medically relevant translation strategy used by diverse RNA viruses to express multiple proteins from a single dicistronic mRNA. It relies on the retention of 40S ribosomal subunits after termination and their reinitiation at a downstream ORF, a process regulated by viral RNA structures and host translation factors. Understanding this mechanism not only sheds light on fundamental translation control but also offers potential avenues for antiviral development. Continued research using advanced techniques such as Ribo-seq and CRISPR screening will further elucidate the molecular details and therapeutic potential of this process.
References
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- 2. Sherlock ME et al.. 2023. A conserved class of viral RNA structures regulate translation reinitiation through dynamic ribosome interactions.. bioRxiv PMID: 37808774
- 3. Powell ML et al.. 2011. Further characterisation of the translational termination-reinitiation signal of the influenza B virus segment 7 RNA.. PLoS One 6(2):e16822 PMID: 21347434
- 4. Sherlock ME et al.. 2025. A conserved class of viral RNA structures regulates translation reinitiation through dynamic ribosome interactions.. Cell Rep 44(2):115236 PMID: 39893634
- 5. Hwang WL et al.. 1998. Translational regulation of hepatitis B virus polymerase gene by termination-reinitiation of an upstream minicistron in a length-dependent manner.. J Gen Virol 79 ( Pt 9):2181-9 PMID: 9747727
- 6. Sorokin II et al.. 2021. Non-Canonical Translation Initiation Mechanisms Employed by Eukaryotic Viral mRNAs.. Biochemistry (Mosc) 86(9):1060-1094 PMID: 34565312
- 7. Powell ML et al.. 2008. Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein.. RNA 14(11):2394-406 PMID: 18824510
- 8. McCormick CJ et al.. 2008. Translation termination reinitiation between open reading frame 1 (ORF1) and ORF2 enables capsid expression in a bovine norovirus without the need for production of viral subgenomic RNA.. J Virol 82(17):8917-21 PMID: 18579601
