A Three-Stemmed mRNA Pseudoknot in the SARS Coronavirus Frameshift Signal

Abstract

A wide range of RNA viruses use programmed −1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed −1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.

A Functional –1 Ribosomal Frameshift Signal in the Human Paraneoplastic Ma3 Gene

“…(A related finding was reported for a stem-loop sequence within loop 2 of the stimulatory pseudoknot for Ϫ1 frameshifting in SARS-CoV. The deletion of the stem-loop sequence had no appreciable effect, whereas mutations within the stem-loop sequence reduced frameshifting significantly (9,48,49).) Alternatively, the activity of the stem-loop may be artificially high because two sequences, GGUUC and GGCUC, further 3Ј in the vector sequence can potentially base pair with the stem-loop to form pseudoknots with large loops 2 (131 or 195 nt, respectively).…”

The role of programmed-1 ribosomal frameshifting in coronavirus propagation

“…It is often at least 2-orders of magnitude more efficient than baseline rates ribosomal error. The efficiency of a -1 PRF may range from 15-60% depending on the assay system and the amount of RNA flanking the core sequence (14)(15)(16). This suggests that the flanking sequences are of some importance; however, codon and reading frame constraints pose some limitations on analyses of these flanking sequences using current in vitro assay systems.…”

Ribosomal Frameshift Signals in Viral Genomes

“…Shown are diagrams of the sequence and base-pairing of three different coronavirus stimulatory elements. A) the avian infectious bronchitis virus two-stemmed pseudoknot (Brierley et al, 1989), B) the severe acute respiratory syndrome three-stemmed pseudoknot (Plant et al, 2005), and C) the kissing loops of the human coronavirus 229E stimulatory element (Herold & Siddell 1993). The heptameric slippery sites are shown in bold.…”

“…Additionally there is an example of a kissing stem-loop that promotes coronavirus frameshifting (Herold and Siddell, 1993). A lot of nuclease mapping and NMR data is available for some coronavirus signals but this does not reflect the diversity of nidovirus frameshift signals (Dos Ramos et al, 2004;Napthine et al, 1999;Plant et al, 2005Plant et al, , 2010Su et al, 2005).…”

“…Putative frameshift signals have been identified in several coronaviruses including bovine coronavirus, porcine epidemic diarrhia virus, transmissible gastroenteritis virus and human coronavirus OC43 (Bekaert & Rousset, 2005). The functionality of the IBV, human coronavirus 229E, Berne virus, murine hepatitis virus and SARS coronavirus frameshift signals have been demonstrated (Baranov et al, 2005;Brierley et al, 1987Brierley et al, , 1989Brierley et al, , 1991Brierley et al, , 1992Dos Ramos et al, 2004;Herold & Siddell, 1993;Plant et al, 2005Plant et al, , 2010Su et al, 2005).…”