Characterization of the stimulators of protein-directed ribosomal frameshifting in Theiler's murine encephalomyelitis virus

AbstractMany viruses utilize programmed –1 ribosomal frameshifting (–1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a ‘slippery’ sequence and a 3′-adjacent RNA structure. Recently, we showed that −1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.

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Structural and molecular basis for protein-stimulated ribosomal frameshifting in Theiler’s murine encephalomyelitis virus

10.1101/2020.08.11.245068 ◽

2020 ◽

Cited By ~ 1

Author(s):

Chris H. Hill ◽

Georgia Cook ◽

Sawsan Napthine ◽

Anuja Kibe ◽

Katherine Brown ◽

...

Keyword(s):

Translational Control ◽

Rna Binding ◽

Critical Role ◽

Ribosome Profiling ◽

Encephalomyocarditis Virus ◽

Theiler's Murine Encephalomyelitis Virus ◽

Stem Loop ◽

Ribosomal Frameshifting ◽

Theiler’S Murine Encephalomyelitis Virus ◽

Encephalomyelitis Virus

AbstractThe 2A protein of Theiler’s murine encephalomyelitis virus (TMEV) is required for stimulating programmed −1 ribosomal frameshifting (PRF) during infection. However, the amino acid sequence of TMEV 2A shares only 27% identity with the 2A orthologue from the related cardiovirus encephalomyocarditis virus (EMCV) for which a structure has been recently determined. Here we present the X-ray crystal structure of TMEV 2A, revealing that, despite the low sequence identity, the overall beta-shell architecture is retained, and the location of previously described mutations on this structure suggests a common RNA binding mode. We determine the minimal stimulatory element in the viral RNA required for 2A binding and show that 2A binds to this element with 1:1 stoichiometry and nanomolar affinity. We also demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence that an alternative pseudoknot-like conformation recently demonstrated for EMCV is a conserved feature of cardiovirus stimulatory elements. We go on to examine frameshifting in infected cells by ribosome profiling and metabolic labelling. We observe PRF efficiencies of up to 85%, highlighting this as the most efficient example of −1 PRF in any natural system thus far characterised. Furthermore, we document a series of ribosomal pauses in and around the site of PRF with potential implications for our understanding of translational control in cardioviruses.

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Theiler’s murine encephalomyelitis virus contrasts with encephalomyocarditis and foot-and-mouth disease viruses in its functional utilization of the StopGo non-standard translation mechanism

Journal of General Virology ◽

10.1099/vir.0.047571-0 ◽

2013 ◽

Vol 94 (2) ◽

pp. 348-353 ◽

Cited By ~ 7

Author(s):

G. Loughran ◽

J. E. Libbey ◽

S. Uddowla ◽

M. F. Scallan ◽

M. D. Ryan ◽

...

Keyword(s):

Amino Acid ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Encephalomyocarditis Virus ◽

Theiler's Murine Encephalomyelitis Virus ◽

Theiler’S Murine Encephalomyelitis Virus ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Encephalomyelitis Virus

The picornaviruses’ genome consists of a positive-sense ssRNA. Like many picornaviruses, cardioviruses synthesize two distinct polyprotein precursors from adjacent but non-overlapping genome segments. Both the [L-1ABCD-2A] and the [2BC-3ABCD] polyproteins are proteolytically processed to yield mature capsid and non-structural proteins, respectively. An unusual translational event, known as ‘StopGo’ or ‘Stop-Carry on’, is responsible for the release of the [L-1ABCD-2A] polyprotein from the ribosome and synthesis of the N-terminal amino acid of the [2BC-3ABCD] polyprotein. A common feature of these viruses is the presence of a highly conserved signature sequence for StopGo: –D(V/I)ExNPG↓P–, where –D(V/I)ExNPG are the last 7 aa of 2A, and the last P- is the first amino acid of 2B. Here, we report that, in contrast to encephalomyocarditis virus and foot-and-mouth disease virus, a functional StopGo does not appear to be essential for Theiler’s murine encephalomyelitis virus viability when tested in vitro and in vivo.

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Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus

Journal of Virology ◽

10.1128/jvi.01043-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8580-8589 ◽

Cited By ~ 13

Author(s):

Leanne K. Finch ◽

Roger Ling ◽

Sawsan Napthine ◽

Allan Olspert ◽

Thomas Michiels ◽

...

Keyword(s):

Virus Infection ◽

Loop Structure ◽

Theiler's Murine Encephalomyelitis Virus ◽

Stem Loop ◽

Ribosomal Frameshifting ◽

Content Type ◽

Reading Frame ◽

Theiler’S Murine Encephalomyelitis Virus ◽

Stem Loop Structure ◽

Encephalomyelitis Virus

ABSTRACTTheiler's murine encephalomyelitis virus(TMEV) is a member of the genusCardiovirusin thePicornaviridae, a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus,Encephalomyocarditis virus, a programmed −1 ribosomal frameshift (−1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that −1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3′ RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient −1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses.IMPORTANCEMany viruses utilize programmed −1 ribosomal frameshifting (−1 PRF) to produce different protein products at a defined ratio, or to translate overlapping ORFs to increase coding capacity. With few exceptions, −1 PRF occurs on specific “slippery” heptanucleotide sequences and is stimulated by RNA structure beginning 5 to 9 nucleotides (nt) downstream of the slippery site. Here we describe an unusual case of −1 PRF in Theiler's murine encephalomyelitis virus (TMEV) that is extraordinarily efficient (74 to 82% of ribosomes shift into the alternative reading frame) and, in stark contrast to other examples of −1 PRF, is dependent upon a stem-loop structure beginning 14 nt downstream of the slippery site. Furthermore, in TMEV-based reporter constructs in transfected cells, efficient frameshifting is critically dependent upon virus infection. We suggest that TMEV evolved frameshifting as a novel mechanism for removing ribosomes from the message (a “ribosome sink”) to downregulate synthesis of the 3′-encoded replication proteins.

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