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. 2005 Feb 20;332(2):498-510.
doi: 10.1016/j.virol.2004.11.038.

Programmed ribosomal frameshifting in decoding the SARS-CoV genome

Pavel V Baranov  1 , Clark M Henderson, Christine B Anderson, Raymond F Gesteland, John F Atkins, Michael T Howard
Affiliations

Programmed ribosomal frameshifting in decoding the SARS-CoV genome

Pavel V Baranov et al. Virology. .

Abstract

Programmed ribosomal frameshifting is an essential mechanism used for the expression of orf1b in coronaviruses. Comparative analysis of the frameshift region reveals a universal shift site U_UUA_AAC, followed by a predicted downstream RNA structure in the form of either a pseudoknot or kissing stem loops. Frameshifting in SARS-CoV has been characterized in cultured mammalian cells using a dual luciferase reporter system and mass spectrometry. Mutagenic analysis of the SARS-CoV shift site and mass spectrometry of an affinity tagged frameshift product confirmed tandem tRNA slippage on the sequence U_UUA_AAC. Analysis of the downstream pseudoknot stimulator of frameshifting in SARS-CoV shows that a proposed RNA secondary structure in loop II and two unpaired nucleotides at the stem I-stem II junction in SARS-CoV are important for frameshift stimulation. These results demonstrate key sequences required for efficient frameshifting, and the utility of mass spectrometry to study ribosomal frameshifting.

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Figures

Fig. 1
Fig. 1
Sequence comparisons and predicted structures for coronoviral frameshift sites and stimulators. A. Alignment of the regions containing frameshift site and IBV-type pseudoknot. B. Alignment of the regions containing RNA stem loop structure that participates in formation of an elaborated pseudoknot or kissing stem loops. C. Secondary structures of predicted IBV-type pseudoknots. D. Secondary structures of predicted kissing stem loops. In panels A and B shading is used to show conserved nucleotides, frameshift sites are indicated with brown color, blue is used for the stem I and also for the stem III of ‘elaborated pseudoknots’ and red is used for each potential stem II. The predicted structures are shown, allowing for G:U and A:U pairing to indicate the longest possible stems The length of loop II and the positions of each loop and stem are indicated. Viral names are abbreviated; Avian Infectious Bronchitis Virus (IBV); Mouse Hepatitis Virus (MHV); Bovine Coronavirus (BCV); Human coronavirus OC43, HCVOC43; Transmissible Gastroenteritis Virus (TGV); Human Coronavirus 229E (HCV229E); Porcine Epidemic Diarrhea Virus (PEDV); Human Coronavirus NL63 (HCVNL63). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Mutagenic analysis of the frameshift site. A. The sequence of the frameshift region where changes were made is shown. The entire frameshift cassette including the pseudoknot was cloned into p2luc. Frameshift site is in bold, changes are high lighted in grey. B. A histogram of the results obtained by Dual luciferase assay indicating percent frameshifting in HEK293 cells relative to wildtype frameshifting levels.
Fig. 3
Fig. 3
Mutagenic analysis of the pseudoknot. A. The sequence of the pseudoknot region where changes were made is shown. The entire frameshift cassette was cloned into p2luc. Changed nucleotides are shown in bold. Nucleotides participating in formation of base pairs are highlighted in gray. B. A histogram of the results obtained by Dual luciferase assay indicating percent frameshifting in HEK293 cells relative to wildtype frameshifting levels.
Fig. 4
Fig. 4
Mutagenic analysis of loop II. A. The sequence of the loop II region where changes were made is shown. The entire frameshift cassette was cloned into p2luc. Changes are highlighted in black, dashes indicate deleted nucleotides. For SARSWT stem I of the pseudoknot is in gray, stem II of the pseudoknot in light gray. B. Potential RNA secondary structures in the loop II. C. A histogram of the results obtained by Dual luciferase assay indicating percent frameshifting in HEK293 cells (black bars) relative to wildtype frameshifting levels. Free energies of potential RNA secondary structures in loop II (light gray bars).
Fig. 5
Fig. 5
Mass spectrometry analysis of dual tagged frameshift product expressed in HEK293 cells. The mass spectrum between 40 and 50 kDa is shown. The expected mass for tandem frameshifting on U_UUA_AAC is 45702.9.
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