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Poly(A) binding protein 1 enhances cap-independent translation initiation of neurovirulence factor from avian herpesvirus.

Tahiri-Alaoui A, Zhao Y, Sadigh Y, Popplestone J, Kgosana L, Smith LP, Nair V - PLoS ONE (2014)

Bottom Line: We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek's disease virus serotype 1 (MDV1).We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail.RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1.

View Article: PubMed Central - PubMed

Affiliation: The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey, United Kingdom.

ABSTRACT
Poly(A) binding protein 1 (PABP1) plays a central role in mRNA translation and stability and is a target by many viruses in diverse manners. We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek's disease virus serotype 1 (MDV1). We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail. RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1. We propose a model that may offer a mechanistic explanation for the cap-independent enhancement of the activity of the 5L IRES by recruitment of a bona fide initiation protein to the 5' end of the message and that is, from the affinity binding data, still compatible with the formation of 'closed loop' structure of mRNA.

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Effect of deletions of the internal homo-polymeric sequences on the activity of the 5L IRES and mutation analysis of affinity interaction between the 5L IRES and PABP1.(A) Partial sequence from the full length 5L IRES from the immediate-early 1.8-kb mRNA that encodes pp14b isoform from Marek’s disease virus serotype 1. The 5L IRES spans nucleotides 129339-129798 (acc: AF243348). The internal poly-pyrimidine sequences C13 and U11 are boxed as well as the internal poly(A) sequences A11 and A9. (B) Schematic of the DNA constructs used for the luciferase reporter assay. In this vector the translation of R-Luc is controlled by the 5L IRES whereas the F-Luc is under the control of the intercistronic IRES (ICR). This configuration mimics the dual IRES bicistronic 1.8-kb mRNA from MDV1. DF-1 cells were transiently transfected with the indicated DNA vectors and after 24 h the cells were lysed and the luciferase activities were measured. The results are expressed as per cent change in luciferase activity relative to the control wild type sequence (5Lwt). (C) Northern blotting was performed on total RNA extracted from cells transfected with DNA constructs depicted in B. Hybridization was done with a random-primed 32P-labelled DNA fragment corresponding to the 5' end of the F-Luc open reading frame. Ethidium bromide-staining of the gel used for Northern blot is shown below the blot with 18S/28S rRNAs as size markers and loading control. (D) The mutated nucleotides within the internal poly(A) from the 5L IRES are underlined. The corresponding DNA vectors were used to transfect DF-1 cells as described in B. For simplicity, only the R-Luc values are shown as the F-Luc follows the same trend due to the coevolved synergistic functional relationship between the 5L IRES and the ICR IRES. The results are expressed as per cent change relative to the control wild type sequence (5Lwt). The experiment was repeated three times and the SEM is shown. (E) Purified recombinant human PABP1 (0.5 µM) was incubated with 32P-end labelled 5L IRES RNAs from wild type or from the indicated mutants and separated on a native 6% polyacrylamide gel by Electrophoretic Mobility Shift Assay (EMSA). It should be noted that the RNA was obtained by in vitro transcription and that it has no 3' poly (A) tail. The complex between PABP1 and the 5L IRES RNA was visualized by autoradiography using phosphor screen. The complex 5L IRES/PABP1 is observed in all combinations except with the mutants 5Lmt2 and 5Lmt2&3.
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pone-0114466-g001: Effect of deletions of the internal homo-polymeric sequences on the activity of the 5L IRES and mutation analysis of affinity interaction between the 5L IRES and PABP1.(A) Partial sequence from the full length 5L IRES from the immediate-early 1.8-kb mRNA that encodes pp14b isoform from Marek’s disease virus serotype 1. The 5L IRES spans nucleotides 129339-129798 (acc: AF243348). The internal poly-pyrimidine sequences C13 and U11 are boxed as well as the internal poly(A) sequences A11 and A9. (B) Schematic of the DNA constructs used for the luciferase reporter assay. In this vector the translation of R-Luc is controlled by the 5L IRES whereas the F-Luc is under the control of the intercistronic IRES (ICR). This configuration mimics the dual IRES bicistronic 1.8-kb mRNA from MDV1. DF-1 cells were transiently transfected with the indicated DNA vectors and after 24 h the cells were lysed and the luciferase activities were measured. The results are expressed as per cent change in luciferase activity relative to the control wild type sequence (5Lwt). (C) Northern blotting was performed on total RNA extracted from cells transfected with DNA constructs depicted in B. Hybridization was done with a random-primed 32P-labelled DNA fragment corresponding to the 5' end of the F-Luc open reading frame. Ethidium bromide-staining of the gel used for Northern blot is shown below the blot with 18S/28S rRNAs as size markers and loading control. (D) The mutated nucleotides within the internal poly(A) from the 5L IRES are underlined. The corresponding DNA vectors were used to transfect DF-1 cells as described in B. For simplicity, only the R-Luc values are shown as the F-Luc follows the same trend due to the coevolved synergistic functional relationship between the 5L IRES and the ICR IRES. The results are expressed as per cent change relative to the control wild type sequence (5Lwt). The experiment was repeated three times and the SEM is shown. (E) Purified recombinant human PABP1 (0.5 µM) was incubated with 32P-end labelled 5L IRES RNAs from wild type or from the indicated mutants and separated on a native 6% polyacrylamide gel by Electrophoretic Mobility Shift Assay (EMSA). It should be noted that the RNA was obtained by in vitro transcription and that it has no 3' poly (A) tail. The complex between PABP1 and the 5L IRES RNA was visualized by autoradiography using phosphor screen. The complex 5L IRES/PABP1 is observed in all combinations except with the mutants 5Lmt2 and 5Lmt2&3.

Mentions: We have previously reported the presence of a functional IRES within the 5' leader of an IE mRNA from MDV1 [22]. The 5L IRES is part of a naturally occurring bicistronic mRNA that contains another functional IRES within the inter-cistronic region [23]. We showed that both IRES elements function synergistically and proposed an allosteric model for their activity [22]. The 5L IRES controls the expression of viral pp14 that we have recently shown to mediate the neurovirulence phenotype of MDV1 [20]. An important feature of the sequence of the 5L IRES is that it contains two sets of internal poly-pyrimidine sequences; one is C13 and the second is U11 (Fig. 1A). In addition there are two adjacent poly(A) sequences, A11 and A9 that are separated by one cytosine (Fig. 1A). Deletion of the C13 and of the U11 does not affect the activity of the 5L reporter, however, deletion of the A11 and A9 poly(A) reduces the activity of the reporter by more than 75% (Fig. 1B), and these effects are unlikely to be due to altered RNA stability or abundance as indicated from Northern blot analysis (Fig. 1C). The concomitant reduction of the ICR IRES activity does not indicate nonspecific effect but is a manifestation of the coevolved functional relationship between the two IRESes that we have previously reported [22]. To further investigate the role of each of the internal poly(A) sequences in the activity of the 5L IRES within the reporter mRNA, we made single mutations as depicted in (Fig. 1D). The constructs were designed in a configuration that mimics the naturally occurring viral bicistronic dual IRES [22] and were used to transfect DF-1 cells. After 24 hours incubation the luciferase activities were measured (Fig. 1D). For simplicity only the activity of the R-Luc that is under the control of the 5L IRES is shown; as the activity of the F-Luc (controlled by the ICR IRES) followed the same trend. Mutating the internal C to A in the 5Lmt1 did not affect the activity of the 5L reporter that remained similar to the control 5Lwt. In the 5Lmt2, where the A11 tracts were disturbed by mutating the middle A to G, the activity of the 5L reporter decreased by about 80%. Combining mutations mt1 and mt2 within the construct 5Lmt1&2 restored the activity of the 5L reporter to its wild type level; suggesting that A11 is the optimal requirement for maintaining full 5L IRES activity within reporter mRNA. This is supported by the results from the constructs 5Lmt3, 5Lmt1&3, 5Lmt2&3 and 5Lmt1&2&3 (Fig. 1D).


Poly(A) binding protein 1 enhances cap-independent translation initiation of neurovirulence factor from avian herpesvirus.

Tahiri-Alaoui A, Zhao Y, Sadigh Y, Popplestone J, Kgosana L, Smith LP, Nair V - PLoS ONE (2014)

Effect of deletions of the internal homo-polymeric sequences on the activity of the 5L IRES and mutation analysis of affinity interaction between the 5L IRES and PABP1.(A) Partial sequence from the full length 5L IRES from the immediate-early 1.8-kb mRNA that encodes pp14b isoform from Marek’s disease virus serotype 1. The 5L IRES spans nucleotides 129339-129798 (acc: AF243348). The internal poly-pyrimidine sequences C13 and U11 are boxed as well as the internal poly(A) sequences A11 and A9. (B) Schematic of the DNA constructs used for the luciferase reporter assay. In this vector the translation of R-Luc is controlled by the 5L IRES whereas the F-Luc is under the control of the intercistronic IRES (ICR). This configuration mimics the dual IRES bicistronic 1.8-kb mRNA from MDV1. DF-1 cells were transiently transfected with the indicated DNA vectors and after 24 h the cells were lysed and the luciferase activities were measured. The results are expressed as per cent change in luciferase activity relative to the control wild type sequence (5Lwt). (C) Northern blotting was performed on total RNA extracted from cells transfected with DNA constructs depicted in B. Hybridization was done with a random-primed 32P-labelled DNA fragment corresponding to the 5' end of the F-Luc open reading frame. Ethidium bromide-staining of the gel used for Northern blot is shown below the blot with 18S/28S rRNAs as size markers and loading control. (D) The mutated nucleotides within the internal poly(A) from the 5L IRES are underlined. The corresponding DNA vectors were used to transfect DF-1 cells as described in B. For simplicity, only the R-Luc values are shown as the F-Luc follows the same trend due to the coevolved synergistic functional relationship between the 5L IRES and the ICR IRES. The results are expressed as per cent change relative to the control wild type sequence (5Lwt). The experiment was repeated three times and the SEM is shown. (E) Purified recombinant human PABP1 (0.5 µM) was incubated with 32P-end labelled 5L IRES RNAs from wild type or from the indicated mutants and separated on a native 6% polyacrylamide gel by Electrophoretic Mobility Shift Assay (EMSA). It should be noted that the RNA was obtained by in vitro transcription and that it has no 3' poly (A) tail. The complex between PABP1 and the 5L IRES RNA was visualized by autoradiography using phosphor screen. The complex 5L IRES/PABP1 is observed in all combinations except with the mutants 5Lmt2 and 5Lmt2&3.
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Related In: Results  -  Collection

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Show All Figures
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pone-0114466-g001: Effect of deletions of the internal homo-polymeric sequences on the activity of the 5L IRES and mutation analysis of affinity interaction between the 5L IRES and PABP1.(A) Partial sequence from the full length 5L IRES from the immediate-early 1.8-kb mRNA that encodes pp14b isoform from Marek’s disease virus serotype 1. The 5L IRES spans nucleotides 129339-129798 (acc: AF243348). The internal poly-pyrimidine sequences C13 and U11 are boxed as well as the internal poly(A) sequences A11 and A9. (B) Schematic of the DNA constructs used for the luciferase reporter assay. In this vector the translation of R-Luc is controlled by the 5L IRES whereas the F-Luc is under the control of the intercistronic IRES (ICR). This configuration mimics the dual IRES bicistronic 1.8-kb mRNA from MDV1. DF-1 cells were transiently transfected with the indicated DNA vectors and after 24 h the cells were lysed and the luciferase activities were measured. The results are expressed as per cent change in luciferase activity relative to the control wild type sequence (5Lwt). (C) Northern blotting was performed on total RNA extracted from cells transfected with DNA constructs depicted in B. Hybridization was done with a random-primed 32P-labelled DNA fragment corresponding to the 5' end of the F-Luc open reading frame. Ethidium bromide-staining of the gel used for Northern blot is shown below the blot with 18S/28S rRNAs as size markers and loading control. (D) The mutated nucleotides within the internal poly(A) from the 5L IRES are underlined. The corresponding DNA vectors were used to transfect DF-1 cells as described in B. For simplicity, only the R-Luc values are shown as the F-Luc follows the same trend due to the coevolved synergistic functional relationship between the 5L IRES and the ICR IRES. The results are expressed as per cent change relative to the control wild type sequence (5Lwt). The experiment was repeated three times and the SEM is shown. (E) Purified recombinant human PABP1 (0.5 µM) was incubated with 32P-end labelled 5L IRES RNAs from wild type or from the indicated mutants and separated on a native 6% polyacrylamide gel by Electrophoretic Mobility Shift Assay (EMSA). It should be noted that the RNA was obtained by in vitro transcription and that it has no 3' poly (A) tail. The complex between PABP1 and the 5L IRES RNA was visualized by autoradiography using phosphor screen. The complex 5L IRES/PABP1 is observed in all combinations except with the mutants 5Lmt2 and 5Lmt2&3.
Mentions: We have previously reported the presence of a functional IRES within the 5' leader of an IE mRNA from MDV1 [22]. The 5L IRES is part of a naturally occurring bicistronic mRNA that contains another functional IRES within the inter-cistronic region [23]. We showed that both IRES elements function synergistically and proposed an allosteric model for their activity [22]. The 5L IRES controls the expression of viral pp14 that we have recently shown to mediate the neurovirulence phenotype of MDV1 [20]. An important feature of the sequence of the 5L IRES is that it contains two sets of internal poly-pyrimidine sequences; one is C13 and the second is U11 (Fig. 1A). In addition there are two adjacent poly(A) sequences, A11 and A9 that are separated by one cytosine (Fig. 1A). Deletion of the C13 and of the U11 does not affect the activity of the 5L reporter, however, deletion of the A11 and A9 poly(A) reduces the activity of the reporter by more than 75% (Fig. 1B), and these effects are unlikely to be due to altered RNA stability or abundance as indicated from Northern blot analysis (Fig. 1C). The concomitant reduction of the ICR IRES activity does not indicate nonspecific effect but is a manifestation of the coevolved functional relationship between the two IRESes that we have previously reported [22]. To further investigate the role of each of the internal poly(A) sequences in the activity of the 5L IRES within the reporter mRNA, we made single mutations as depicted in (Fig. 1D). The constructs were designed in a configuration that mimics the naturally occurring viral bicistronic dual IRES [22] and were used to transfect DF-1 cells. After 24 hours incubation the luciferase activities were measured (Fig. 1D). For simplicity only the activity of the R-Luc that is under the control of the 5L IRES is shown; as the activity of the F-Luc (controlled by the ICR IRES) followed the same trend. Mutating the internal C to A in the 5Lmt1 did not affect the activity of the 5L reporter that remained similar to the control 5Lwt. In the 5Lmt2, where the A11 tracts were disturbed by mutating the middle A to G, the activity of the 5L reporter decreased by about 80%. Combining mutations mt1 and mt2 within the construct 5Lmt1&2 restored the activity of the 5L reporter to its wild type level; suggesting that A11 is the optimal requirement for maintaining full 5L IRES activity within reporter mRNA. This is supported by the results from the constructs 5Lmt3, 5Lmt1&3, 5Lmt2&3 and 5Lmt1&2&3 (Fig. 1D).

Bottom Line: We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek's disease virus serotype 1 (MDV1).We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail.RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1.

View Article: PubMed Central - PubMed

Affiliation: The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey, United Kingdom.

ABSTRACT
Poly(A) binding protein 1 (PABP1) plays a central role in mRNA translation and stability and is a target by many viruses in diverse manners. We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek's disease virus serotype 1 (MDV1). We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail. RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1. We propose a model that may offer a mechanistic explanation for the cap-independent enhancement of the activity of the 5L IRES by recruitment of a bona fide initiation protein to the 5' end of the message and that is, from the affinity binding data, still compatible with the formation of 'closed loop' structure of mRNA.

Show MeSH
Related in: MedlinePlus