Programmed -1 ribosomal frameshifting (-1 PRF) is a mechanism used to regulate gene expression at the level of protein synthesis. As ribosomes translate one ORF they encounter a signal in the mRNA that directs a fraction of them to shift into an alternative downstream ORF which is in the -1 phase relative to the initiating upstream ORF (). In viruses -1 PRF usually results in a C-terminally extended polyprotein containing additional function not present in the upstream ORF. The use of a -1 PRF mechanism for expression of a viral gene was first published in 1985 for the Rous sarcoma virus (17
) and subsequently for other retroviruses (18
). The first complete coronavirus sequence was published in 1987 (IBV; 19) and later that same an in vitro
translation system was used to demonstrate that a -1 PRF mechanism was used to translate ORF1ab (10
). In subsequent years, the IBV frameshift signal has been extensively analyzed by the Brierley and co-workers to become one of the most well characterized -1 PRF signals.
Figure 3 Programmed -1 Ribosomal Frameshifting. During translation features intrinsic to the mRNA being decoded manipulate the ribosome such that the reading frame is altered. (A) The ribosome pauses over a heptameric slippery site (UUUAAAC in coronaviruses). (more ...)
-1 PRF signals are usually composed of a “slippery site” followed by a stimulatory structure. These two elements are typically separated by a short spacer region. The slippery site is composed of a heptameric sequence such that the A- and P-site tRNAs can un-pair from the mRNA and re-pair in the -1 reading frame (20
; ). The nucleotides surrounding the heptameric slippery site have been shown to have a limited effect on frameshifting efficiencies. Experiments altering the spacer region between the slippery site and stimulatory element reduced frameshifting efficiency suggesting that there might be some optimal spacer sequence (27
). The three nucleotides 5′ of the heptameric sequence also affect -1 PRF efficiency suggesting a role for the exiting tRNA in the ribosomal E-site (24
). The stimulatory element has been shown to contribute significantly to -1 PRF efficiencies.
While the stimulatory structure was initially postulated to be a simple mRNA stem-loop studies of the IBV -1 PRF signal provided the first evidence for the requirement of a more complex mRNA pseudoknot (27; ). Subsequently mRNA pseudoknots were identified in the frameshift signals of a wide variety of plant and animal viruses. As additional viral sequences became available more elaborate stimulatory structures were identified in coronaviruses. These include “kissing loops” (32
), and three stemmed mRNA pseudoknots, which were predicted for the coronavirus and the related torovirus Berne virus (33
), and subsequently demonstrated by nuclease mapping for the SARS coronavirus (15
). The variation in these stimulatory elements suggests that the additional features might be required for fine-tuning frameshifting efficiency or, alternatively, involved in additional viral functions. Interestingly, efficient frameshifting was observed when the third stem was deleted from the SARS-CoV pseudoknot, or when a similar region was deleted from the IBV stimulatory structure, suggesting that these regions are not required to modulate -1 PRF (15
). However, it is clear from mutational analyses that when the third stem is present that it has an effect on -1 PRF (14
). Furthermore, additional sequence upstream of the core frameshift signal has been shown to affect -1 PRF efficiency in SARS-CoV (16
). Thus, although core essential elements of the frameshift signal have been defined, the scope of factors, either cis- or trans-acting, has not yet been revealed.
Figure 4 Coronavirus Frameshift Signals. Two to three RNA stems following a heptameric slippery site comprise the coronavirus frameshift signal. The slippery site is underlined. The proposed IBV, SARS-CoV and HCoV-229E pseudoknot structures are shown (A to C respectively). (more ...)
A number of models have been proposed to describe the mechanism by which -1 PRF occurs (20
). All the models posit that the stimulatory element causes a pause in translation and that base-pairing is required at the non-wobble positions of at least two tRNA molecules to the mRNA after the frameshift (). Differences among the models are centered on the timing of the frameshift within the context of the elongation cycle. The detection of two different frameshift products by protein sequencing (18
) suggests that the different models may not be mutually exclusive. Analysis of frameshifting is complicated somewhat by the availability of malleable experimental systems imitating the appropriate host cell. It has been shown that prokaryotic ribosomes decipher coronavirus frameshift signals quite differently from yeast, plant or mammalian ribosomes (25
). Thus a suitable system must be used to draw purposeful conclusions from in vitro
analyses of -1 PRF. The prevalence of coronaviruses and their spread among a wide range of mammals including bats (1
) suggests that analyses in mammalian cells are appropriate in most instances.