Probing the mechanism of action of Shiftless, a host restriction factor targeting programmed ribosomal frameshifting.

Proteins are encoded in DNA but synthesised by the ribosome through a messenger RNA intermediate, that is copied from DNA. The process of protein synthesis is called translation. The mRNA is fed into the ribosome which moves along until a triplet start signal in the mRNA is recognised. At this point, amino acid biosynthesis starts and as each subsequent triplet nucleotide "code" is decoded, one amino acid is added to a growing polypeptide chain. The ribosome sticks to the triplet code (the reading frame) until it reaches a stop signal, at which point the completed protein is released. Some mRNAs, however, have embedded signals that instruct a proportion of the translating ribosomes to change reading frame, that is, to frameshift, at a defined position and to continue translation in an overlapping coding frame. Most examples of this programmed ribosomal frameshifting (PRF) come from viruses, although several have been found in cellular genes. Frameshift signals allow the synthesis of two proteins from a single mRNA and are most often used to attach additional amino acids onto the C-terminus of a protein. Many pathogenic viruses of animals and plants use frameshifting in the expression of virus proteins, including the retrovirus HIV and the SARS coronavirus. In almost all examples studied, the frameshifting event is -1 (-1 PRF), that is, the ribosome moves backwards by one nucleotide on the mRNA. The mRNA signals that induce frameshifting are composed of two elements, a "slippery sequence", where the ribosome changes frame and, immediately downstream, a stable region of double-stranded RNA (originating through base-pairing of self-complementary regions) referred to as the stimulatory RNA. The elements are spaced such that as the ribosome is decoding the slippery sequence it encounters the stimulatory RNA, and it is thought that a failure to properly unwind the stimulatory RNA leads to a -1 PRF on the slippery sequence. In addition to stimulatory RNAs, our laboratory has identified virus examples where efficient PRF requires the participation of proteins. In certain cardioviruses, viral protein 2A binds to the stimulatory RNA to promote PRF, and in the arterivirus porcine reproductive and respiratory syndrome virus, an important swine pathogen, PRF is enhanced by binding of viral nsp1b in complex with cellular poly(C) binding protein.

Recently, it was discovered that a cellular protein, Shiftless (SFL), can bind to PRF signals and block their function. SFL had previously been described as an inhibitor of Dengue virus replication and acts as a "restriction factor", that is, a protein induced by interferon upon virus infection and capable of reducing virus growth. SFL is the first example of a restriction factor that targets frameshifting and the first example of a protein that represses PRF. Excitingly, it shows repressive activity against all PRF signals tested to date. Given that this process is a key step in the replication of many pathogenic viruses of medical, veterinary and agricultural importance, any knowledge we can gain about the mechanism of action of SFL might be beneficial in designing strategies to target this process for antiviral intervention.

In this application, we propose a detailed characterisation of the SFL protein and how it functions using biochemical and structural biology methods. We aim to discover how SFL interacts with -1 PRF signals through RNA and ribosome binding assays. We plan to solve the structure of the protein alone, when bound to free ribosomes and when bound to ribosomes present on an mRNA at the frameshifting site. As part of our proposed studies on how SFL affects ribosome function, we will also determine whether SFL modulates the levels of cellular proteins and mRNAs when it is expressed. An understanding of how SFL functions will broaden our knowledge of translational control and provide new insights into how this restriction factor functions in blocking virus replication.

This project will investigate the recently described cellular restriction factor Shiftless (SFL) and how it targets sites of viral programmed ribosomal frameshifting (PRF), inhibiting the process and negatively modulating virus replication. PRF is a translational control mechanism widely used in the regulated expression of many viral, and some cellular proteins. The mRNA signals that induce PRF comprise a slippery sequence, where the ribosome changes into an overlapping frame, and a stimulatory RNA structure which promotes frameshifting by modulating the ribosomal elongation cycle. Stimulatory signals that function with the additional involvement of viral (cardiovirus 2A, arterivirus nsp1b) and cellular proteins (poly(C) binding protein) have also been described, but SFL is the first example of a repressive factor. The molecular basis of SFL action will be studied through a combination of functional assays, RNA-protein and ribosome-protein interaction studies and structural biology approaches. An understanding of how this trans-acting repressor functions will broaden our knowledge of translational control and provide new insights into ribosome structure and function, gene regulation, protein-protein and protein-nucleic acid interactions, virus replication strategies and virus-host interactions. As part of this project, we will examine the effect of SFL expression on host gene expression through ribosome profiling. Together with knowledge gleaned from structural and functional studies, these experiments will potentially be of additional benefit in understanding the reported restrictive activity of SFL in the replication of other viruses that do not utilise PRF.

1. New possibilities for antivirals.
The impact of virus diseases on health, wealth and welfare is enormous. Programmed ribosomal frameshifting (PRF) is a widespread phenomenon in the context of viral infection, and where it occurs, it is necessary for successful completion of the virus life cycle. Given that frameshifting is not widely used as a translational control strategy in healthy cells, global inhibition of PRF represents an attractive strategy for therapeutic intervention. Although the -1 PRF sites in HIV and other medically important viruses have been lauded as potential drug targets for viral control, results to date have been mixed. Shiftless represents the first known example of a biologically natural, non-toxic entity that appears to specifically target PRF without causing global, deleterious effects on ribosome function or the translation of host mRNAs. The basic knowledge and tools that we will generate from studies of Shiftless will allow us to elucidate the molecular basis of its action. In the medium term this could lay the foundations for the development of small molecule inhibitors and peptides that target the ribosome to block ribosomal frameshifting.

2. Insights into a an antiviral restriction factor.
A better understanding of the structure, biochemistry and cell biology of Shiftless will give insights into how it acts more generally in virus inhibition. This will highlight evolutionarily conserved mechanisms to subvert virus replication that might be broadly targetable.

3. A novel ribosome rescue pathway?
One interesting feature of SFL is that appears to recruit the termination complex (eRF3/eRF1) with resultant release of the nascent peptide, but in the absence of a stop codon. It will be informative to compare such non-canonical termination with those documented ribosome rescue mechanisms that serve to remove terminally-stalled ribosomes.

4. Insights into protein-RNA recognition/specificity
SFL appears to bind tightly to multiple different RNAs despite having low sequence homology to known RNA binding proteins. Determining the RNA binding mode of this protein will likely be generally informative about RNA protein interactions.