The role of viral and cellular proteins in programmed -2 ribosomal frameshifting

Cellular proteins are encoded in DNA but synthesised by the ribosome through a messenger RNA (mRNA) 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, polypeptide synthesis starts, and as each subsequent triplet nucleotide "code" is decoded, one amino acid is added to a growing 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 frameshifting 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 a distinct C-terminus onto 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 a -1 frameshift (-1FS), 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 -1FS on the slippery sequence.

Recently, a novel example of a -2 frameshift signal (-2FS) has been unearthed in the porcine reproductive and respiratory syndrome virus (PRRSV). This relative of the SARS coronavirus is an economically important pathogen of pigs responsible for estimated losses of $600 million per annum in the U.S. alone. The PRRSV -2FS frameshifting signal has three unusual features that set it apart from the many examples of -1FS. First, the ribosome moves two nucleotides backwards on the mRNA rather then one. Secondly, and very surprisingly, there is no obvious stimulatory RNA secondary structure. Computational and manual inspection of the region does not reveal any stable base-pairing downstream of the slippery sequence. Thirdly, we have established in unpublished work that the viral protein nsp1 is required for efficient -2FS. This is the first example of a role for a virus protein in frameshifting. The PRRSV signal thus represents a highly novel translation system that warrants further investigation. In this application, we propose a detailed characterisation of the signals for -2FS in PRRSV and an investigation into how the viral protein mediates its stimulatory effect. We will test whether the nsp1 protein and/or cellular proteins can bind directly to the RNA downstream of the slippery sequence and affect ribosome function, or whether such proteins function by binding directly to the ribosome. We will also ask whether ribosomes pause upon encounter of a -2FS signal, as is commonly observed at -1FS signals. We will also investigate the role of frameshifting in the context of the PRRSV itself. New knowledge gained from our analysis will be used to search databases for other -2FS signals in viral and cellular genes.

Overall, the work will hopefully provide new information about the biology of gene expression and expand our knowledge of ribosome function and virus translation mechanisms. In the medium term, the work should help towards the development of vaccines and antiviral approaches to inhibit the replication of PRRSV.

This project will investigate the recently described programmed -2 ribosomal frameshifting (-2FS) signal of porcine reproductive and respiratory syndrome virus (PRRSV). What sets this signal apart from the more extensively studied -1 frameshift signal counterparts is the apparent absence of a stimulatory 3' mRNA secondary structure and the participation of trans-acting protein factors. We have recently obtained evidence that efficient -2FS at the PRRSV signal requires the participation of the viral non-structural protein, nsp1beta . A better understanding of the mechanism of -2FS will provide insights into this novel translation mechanism and will improve our understanding of how protein synthesis can be regulated and how ribosome function can be subverted. We plan first to confirm the key sequences required for -2FS and to optimise frameshifting assays. Subsequently we will define the role of viral and cellular proteins in -2FS and the mechanism(s) by which these factors act, through studies of RNA-protein and ribosome-protein interactions, and by investigating the induction of ribosomal pausing. Throughout the course of the work we will collaborate with the laboratories of Professors Eric Snijder and Ying Fang, established experts in the PRRSV field. These collaborations will give us the opportunity to test experimental observations in the context of the virus itself and may be informative in the design of new vaccines, a desirable goal given the huge economic impact of PRRSV outbreaks on pig farms. We also aim, through collaboration with our bioinformaticist Dr Andrew Firth, to identify new examples of -2FS in other viral and cellular genes, which would have broad-ranging implications.

We believe that this project will have impact in the following areas:

1. New tools for vaccines and antivirals.
The impact of PRRSV is approximately $600 million in losses each year to the U.S. swine industry alone, and several times this amount worldwide. The basic knowledge and tools that we will generate will inform on the development of live virus vaccines and in the long term, may highlight potential antiviral drug targets. Although the -1FS sites in HIV and other medically important viruses have been much lauded as potential drug targets for viral control (inhibiting frameshifting inhibits virus replication), results to date have been mixed. Nonetheless, the -2FS mechanism in PRRSV is a potential target for antiviral drugs (e.g. small molecule inhibitors or nucleic acid analogues). Given that the arterivirus frameshift-stimulatory mechanism appears to be strikingly different from the standard 3' RNA-structure-stimulated -1FS mechanism employed by HIV amongst others, the arterivirus mechanism is likely to provide very different opportunities for frameshift inhibition. Further, the trans-stimulatory role of the nsp1beta protein provides an additional target for frameshift knockdown.

2. Biotechnological use of inducible trans-stimulated -2FS gene expression constructs.
In addition to its application in disease control, the frameshift mechanism itself has significant applications in biotechnology development, in particular since few efficient and inducible frameshift signals are known. Such signals allow the efficient controlled expression of the second gene in a dicistronic construct. Data to date shows that the -2FS can be stimulated by nsp1beta expressed in trans, and therefore nsp1beta can be used as a molecular switch to activate and inactivate the expression of desired genes.

3. Development of search pattern(s) for querying other virus and cellular genomes (including human) for other examples of -2FS gene expression.
Characterization of the frameshift mechanism and other potential shift site motifs for -2FS will provide search patterns that may be applied to search for -2FS candidates in the genomes of other viruses and cellular organisms.

4. Increased understanding of a virus of significant agricultural/veterinary importance, and related viruses that may have zoonotic potential (cf. SARS-CoV).
The Arteriviridae family also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). The nsp2-encoding region of EAV is much smaller than in other arteriviruses and completely lacks the frameshift site and the TF ORF. However, both LDV and SHFV have the TF ORF and the frameshift site. LDV infects mice while SHFV infects primates. SHFV has been isolated from macaques (where it is lethal), red-tailed guenons and red colobus monkeys. This virus shows extreme genetic diversity; indeed the divergence between different SHFV isolates is similar to the divergence between LDV and PRRSV. It is likely that only a fraction of the diversity of arteriviruses in primates (and other mammals) has been sampled and the human zoonotic potential of these new viruses is not known. The bush meat trade and other close interactions between monkeys and humans increases the potential for zoonotic transmission with potentially global consequences (cf. SARS-CoV). A more detailed understanding of the replication strategies of arteriviruses is thus highly desirable.