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Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.

Finch LK, Ling R, Napthine S, Olspert A, Michiels T, Lardinois C, Bell S, Loughran G, Brierley I, Firth AE - J. Virol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

ABSTRACT
Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, 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.

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Analysis of frameshifting in the viral context. (A) Radiolabeled TMEV translation products. BHK-21 cells were infected with either WT, SCM, SS, or LVWT viruses at an MOI of 8 or mock infected. Cells were labeled from 6 to 7 h p.i. and harvested at 7 h p.i., and proteins were separated by SDS-PAGE. Note that the more slowly migrating 2A band for LVWT may contain both 2A-2B* and a 2A-2B cleavage product. All samples were run on the same gel; an irrelevant lane has been excised. (B) Relative amounts of TMEV proteins. Individual band intensities for WT, LVWT, and SCM were normalized first by methionine content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized band for SS. Each bar represents the mean (± standard deviation) from all biological repeats in which the corresponding band could be resolved and quantified (see the text). (C) Frameshifting efficiency. The intensity in each of the VP0, VP3, VP1, and 2C bands for WT, LVWT, and SCM viruses was normalized first by methionine content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized values for SS. Then the value for 2C (downstream product) was divided by the average of the values for VP0, VP3, and VP1 (upstream products). Subtracting this value from 1 and multiplying the result by 100 gives the percent frameshifting efficiency. Each bar represents the mean value (± standard deviation) from five, five, and two biological repeats for WT, LVWT, and SCM viruses, respectively.
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Figure 3: Analysis of frameshifting in the viral context. (A) Radiolabeled TMEV translation products. BHK-21 cells were infected with either WT, SCM, SS, or LVWT viruses at an MOI of 8 or mock infected. Cells were labeled from 6 to 7 h p.i. and harvested at 7 h p.i., and proteins were separated by SDS-PAGE. Note that the more slowly migrating 2A band for LVWT may contain both 2A-2B* and a 2A-2B cleavage product. All samples were run on the same gel; an irrelevant lane has been excised. (B) Relative amounts of TMEV proteins. Individual band intensities for WT, LVWT, and SCM were normalized first by methionine content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized band for SS. Each bar represents the mean (± standard deviation) from all biological repeats in which the corresponding band could be resolved and quantified (see the text). (C) Frameshifting efficiency. The intensity in each of the VP0, VP3, VP1, and 2C bands for WT, LVWT, and SCM viruses was normalized first by methionine content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized values for SS. Then the value for 2C (downstream product) was divided by the average of the values for VP0, VP3, and VP1 (upstream products). Subtracting this value from 1 and multiplying the result by 100 gives the percent frameshifting efficiency. Each bar represents the mean value (± standard deviation) from five, five, and two biological repeats for WT, LVWT, and SCM viruses, respectively.

Mentions: [35S]methionine-labeled proteins from infected cell lysates were separated by electrophoresis and quantified by phosphorimaging. The expression levels of products encoded downstream of the predicted shift site were observed to be much lower, relative to the upstream products, for WT virus than for the SS mutant (Fig. 3A), indicating that a substantial proportion of ribosomes leave the polyprotein frame in the vicinity of the frameshift site and that this departure is mediated by the sequence of the predicted frameshift site. This is most obvious when the intensities of the 2C and VP1 proteins are compared. The intensity of each band depends on the number of methionines present and the abundance of the protein. There is clearly much more of the downstream product 2C relative to VP1 in the SS sample than in the WT, LVWT, and SCM samples.


Characterization of Ribosomal Frameshifting in Theiler's Murine Encephalomyelitis Virus.

Finch LK, Ling R, Napthine S, Olspert A, Michiels T, Lardinois C, Bell S, Loughran G, Brierley I, Firth AE - J. Virol. (2015)

Analysis of frameshifting in the viral context. (A) Radiolabeled TMEV translation products. BHK-21 cells were infected with either WT, SCM, SS, or LVWT viruses at an MOI of 8 or mock infected. Cells were labeled from 6 to 7 h p.i. and harvested at 7 h p.i., and proteins were separated by SDS-PAGE. Note that the more slowly migrating 2A band for LVWT may contain both 2A-2B* and a 2A-2B cleavage product. All samples were run on the same gel; an irrelevant lane has been excised. (B) Relative amounts of TMEV proteins. Individual band intensities for WT, LVWT, and SCM were normalized first by methionine content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized band for SS. Each bar represents the mean (± standard deviation) from all biological repeats in which the corresponding band could be resolved and quantified (see the text). (C) Frameshifting efficiency. The intensity in each of the VP0, VP3, VP1, and 2C bands for WT, LVWT, and SCM viruses was normalized first by methionine content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized values for SS. Then the value for 2C (downstream product) was divided by the average of the values for VP0, VP3, and VP1 (upstream products). Subtracting this value from 1 and multiplying the result by 100 gives the percent frameshifting efficiency. Each bar represents the mean value (± standard deviation) from five, five, and two biological repeats for WT, LVWT, and SCM viruses, respectively.
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Figure 3: Analysis of frameshifting in the viral context. (A) Radiolabeled TMEV translation products. BHK-21 cells were infected with either WT, SCM, SS, or LVWT viruses at an MOI of 8 or mock infected. Cells were labeled from 6 to 7 h p.i. and harvested at 7 h p.i., and proteins were separated by SDS-PAGE. Note that the more slowly migrating 2A band for LVWT may contain both 2A-2B* and a 2A-2B cleavage product. All samples were run on the same gel; an irrelevant lane has been excised. (B) Relative amounts of TMEV proteins. Individual band intensities for WT, LVWT, and SCM were normalized first by methionine content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized band for SS. Each bar represents the mean (± standard deviation) from all biological repeats in which the corresponding band could be resolved and quantified (see the text). (C) Frameshifting efficiency. The intensity in each of the VP0, VP3, VP1, and 2C bands for WT, LVWT, and SCM viruses was normalized first by methionine content, then by the means of these values for VP0, VP1, and VP3 (to control for lane loading), and then by the corresponding similarly normalized values for SS. Then the value for 2C (downstream product) was divided by the average of the values for VP0, VP3, and VP1 (upstream products). Subtracting this value from 1 and multiplying the result by 100 gives the percent frameshifting efficiency. Each bar represents the mean value (± standard deviation) from five, five, and two biological repeats for WT, LVWT, and SCM viruses, respectively.
Mentions: [35S]methionine-labeled proteins from infected cell lysates were separated by electrophoresis and quantified by phosphorimaging. The expression levels of products encoded downstream of the predicted shift site were observed to be much lower, relative to the upstream products, for WT virus than for the SS mutant (Fig. 3A), indicating that a substantial proportion of ribosomes leave the polyprotein frame in the vicinity of the frameshift site and that this departure is mediated by the sequence of the predicted frameshift site. This is most obvious when the intensities of the 2C and VP1 proteins are compared. The intensity of each band depends on the number of methionines present and the abundance of the protein. There is clearly much more of the downstream product 2C relative to VP1 in the SS sample than in the WT, LVWT, and SCM samples.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

ABSTRACT
Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae, 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.

Show MeSH
Related in: MedlinePlus