Molecular analysis of a novel translation 'termination-reinitiation' signal

Lead Research Organisation: University of Cambridge
Department Name: Pathology

Abstract

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Technical Summary

This project will investigate the mechanism of an unconventional translational event operating in feline calicivirus. The sole sub-genomic viral RNA detected in infected cells is dicistronic, with a long 5 prime-ORF (encoding the capsid protein precursor) that overlaps a short downstream ORF by 4 nucleotides (AUGA). In virus-infected cells and in vitro systems, the downstream ORF is expressed at approximately 10-20 per cent of the level of the upstream ORF. Downstream ORF expression appears to occur by an unusual termination-reinitiation event, superficially similar to the translational coupling seen with some prokaryotic polycistronic mRNAs (e.g. ribosomal protein mRNAs), and does not seem to involve leaky scanning, shunting, IRES¿s or ribosomal frameshifting. The global objective of this project is to characterise the mechanism of expression of the downstream ORF. This will involve pursuing the following experimental objectives: mapping the segments flanking the termination-reinitiation site that are required for reinitiation; determination of the initiation factor requirements of the process; identification of protein factors that interact with the critical segment; determination of the mRNA secondary structure of this region; molecular genetic analysis of the role of structures identified in this region and of the termination and initiation codons; analysis of the effects of modulation of reinitiation efficiency on virus replication.
 
Description 1. We characterised the termination-reinitiation signal of feline calicivirus (FCV) and demonstrated a role for initiation factor 3 in the process of reinitiation. This mechanism of reinitiation after translation of a long open reading frame (ORF) revealed why such events do not generally occur in mammalian mRNA translation.
2. We characterised the primary sequence and mRNA secondary structural requirements for efficient termination reinitiation in FCV, influenza B virus (BM2) and murine norovirus (MNV). We demonstrated that an interaction between a viral mRNA primary sequence and helix 26 of ribosomal 18S RNA is essential for termination-reinitiation in FCV, BM2 and MNV. We also showed that the 18S rRNA complementary region is presented as part of a metastable stem-loop structure.
3. We were able to demonstrate that reducing the frequency of reinitiation of the influenza BM2 ORF was detrimental to influenza B virus replication.
Exploitation Route These studies outlined the basic signals for translation termination-reinitiation and have aided the subsequent identification of novel examples of the phenomenon. Ultimately, the knowledge may prove useful in targeted antiviral therapy.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description Alternate Recodings II 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact This was a scientific workshop bringing together participants from diverse biological fields. The aim was to alert scientists to the huge amount of related work that was going on in smaller laboratories that few had appreciated.

Several collaborative proposals were made at the meeting and I personally have been contacted for advice on numerous occasions since the workshop.
Year(s) Of Engagement Activity 2014
URL http://events.embo.org/14-recoding/