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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 MDV1 infection on paip2 expression, PAPB1 level and localization.(A) Chicken embryo fibroblasts (CEF) were transfected with oncogenic BAC clone pRB1B5 of MDV1 or mock-transfected for 72 h. Total proteins were harvested and analysed by immunoblotting with the indicated antibodies. Quantification of the immunoblots from panel A using ImageQuant software is shown to the right. The results are from two independent experiments each in duplicate. (B) Total proteins were extracted from control samples or from samples taken from chicken infected with the oncogenic BAC clone pRB1B5 derived from archive samples. Proteins were analysed by immunoblotting as in panel A. Quantification of the immunoblots from panel B using ImageQuant software is shown to the right. The results are repeats from two different archive samples derived from the same chicken challenge experiment. (C) Indirect immunofluorescence of pRB1B5-infected CEF 72 h posttransfection. A series of optical sections were taken sequentially for each channel along the z-axis using a step size of 0.290 µm. The resulting 3D confocal image was reconstructed using IMARIS software. DAPI-staining shows the nucleus in blue, PABP1 in red and pp14 in green, the scale bar: 10 µm.
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pone-0114466-g004: Effect of MDV1 infection on paip2 expression, PAPB1 level and localization.(A) Chicken embryo fibroblasts (CEF) were transfected with oncogenic BAC clone pRB1B5 of MDV1 or mock-transfected for 72 h. Total proteins were harvested and analysed by immunoblotting with the indicated antibodies. Quantification of the immunoblots from panel A using ImageQuant software is shown to the right. The results are from two independent experiments each in duplicate. (B) Total proteins were extracted from control samples or from samples taken from chicken infected with the oncogenic BAC clone pRB1B5 derived from archive samples. Proteins were analysed by immunoblotting as in panel A. Quantification of the immunoblots from panel B using ImageQuant software is shown to the right. The results are repeats from two different archive samples derived from the same chicken challenge experiment. (C) Indirect immunofluorescence of pRB1B5-infected CEF 72 h posttransfection. A series of optical sections were taken sequentially for each channel along the z-axis using a step size of 0.290 µm. The resulting 3D confocal image was reconstructed using IMARIS software. DAPI-staining shows the nucleus in blue, PABP1 in red and pp14 in green, the scale bar: 10 µm.

Mentions: Infection with some herpesviruses such as HSV1 [13], [14] and KSHV [12] can trigger PABP1 to accumulate in the nucleus. By contrast, in cells infected with HCMV, another related herpesvirus, PABP1 does not redistribute to the nucleus but accumulates in the cytoplasm and its level increases in HCMV-infected cells [17], [19]. Given the functional importance of PABP1 for the IRES-driven expression of the IE pp14, we investigated the effect of MDV1 infection on PABP1 expression. Total proteins were isolated from control and MDV1-infected samples and the overall abundance of selected viral and host proteins were evaluated by immunoblotting (Fig. 4A and S2A and S2B Figure). There is no apparent effect on the level of PABP1 expression in primary CEF 72 hour post-transfection with the BAC clone of the oncogenic pRB1B5 as compared to control cells (Fig. 4A). MDV1 infection does not appear to affect the level of other translation initiation factors such eIF4E and eIF4A despite successful viral replication and viral antigens expression (Fig. 4A). Similar results are seen in tumour vs. control tissues from chicken inoculated with RB1B5 strain of MDV1 (Fig. 4B). Interestingly, the level of PABP-interacting protein 2 (paip2) in both pRB1B5 CEF-transfected and pRB1B5-derived tumours is about 50% less than that detected in control samples (Fig. 4A-4B). Significantly, paip2 is well known to preferentially inhibit translation of poly(A)-containing mRNA by interdicting PABP1 function [28].


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 MDV1 infection on paip2 expression, PAPB1 level and localization.(A) Chicken embryo fibroblasts (CEF) were transfected with oncogenic BAC clone pRB1B5 of MDV1 or mock-transfected for 72 h. Total proteins were harvested and analysed by immunoblotting with the indicated antibodies. Quantification of the immunoblots from panel A using ImageQuant software is shown to the right. The results are from two independent experiments each in duplicate. (B) Total proteins were extracted from control samples or from samples taken from chicken infected with the oncogenic BAC clone pRB1B5 derived from archive samples. Proteins were analysed by immunoblotting as in panel A. Quantification of the immunoblots from panel B using ImageQuant software is shown to the right. The results are repeats from two different archive samples derived from the same chicken challenge experiment. (C) Indirect immunofluorescence of pRB1B5-infected CEF 72 h posttransfection. A series of optical sections were taken sequentially for each channel along the z-axis using a step size of 0.290 µm. The resulting 3D confocal image was reconstructed using IMARIS software. DAPI-staining shows the nucleus in blue, PABP1 in red and pp14 in green, the scale bar: 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263670&req=5

pone-0114466-g004: Effect of MDV1 infection on paip2 expression, PAPB1 level and localization.(A) Chicken embryo fibroblasts (CEF) were transfected with oncogenic BAC clone pRB1B5 of MDV1 or mock-transfected for 72 h. Total proteins were harvested and analysed by immunoblotting with the indicated antibodies. Quantification of the immunoblots from panel A using ImageQuant software is shown to the right. The results are from two independent experiments each in duplicate. (B) Total proteins were extracted from control samples or from samples taken from chicken infected with the oncogenic BAC clone pRB1B5 derived from archive samples. Proteins were analysed by immunoblotting as in panel A. Quantification of the immunoblots from panel B using ImageQuant software is shown to the right. The results are repeats from two different archive samples derived from the same chicken challenge experiment. (C) Indirect immunofluorescence of pRB1B5-infected CEF 72 h posttransfection. A series of optical sections were taken sequentially for each channel along the z-axis using a step size of 0.290 µm. The resulting 3D confocal image was reconstructed using IMARIS software. DAPI-staining shows the nucleus in blue, PABP1 in red and pp14 in green, the scale bar: 10 µm.
Mentions: Infection with some herpesviruses such as HSV1 [13], [14] and KSHV [12] can trigger PABP1 to accumulate in the nucleus. By contrast, in cells infected with HCMV, another related herpesvirus, PABP1 does not redistribute to the nucleus but accumulates in the cytoplasm and its level increases in HCMV-infected cells [17], [19]. Given the functional importance of PABP1 for the IRES-driven expression of the IE pp14, we investigated the effect of MDV1 infection on PABP1 expression. Total proteins were isolated from control and MDV1-infected samples and the overall abundance of selected viral and host proteins were evaluated by immunoblotting (Fig. 4A and S2A and S2B Figure). There is no apparent effect on the level of PABP1 expression in primary CEF 72 hour post-transfection with the BAC clone of the oncogenic pRB1B5 as compared to control cells (Fig. 4A). MDV1 infection does not appear to affect the level of other translation initiation factors such eIF4E and eIF4A despite successful viral replication and viral antigens expression (Fig. 4A). Similar results are seen in tumour vs. control tissues from chicken inoculated with RB1B5 strain of MDV1 (Fig. 4B). Interestingly, the level of PABP-interacting protein 2 (paip2) in both pRB1B5 CEF-transfected and pRB1B5-derived tumours is about 50% less than that detected in control samples (Fig. 4A-4B). Significantly, paip2 is well known to preferentially inhibit translation of poly(A)-containing mRNA by interdicting PABP1 function [28].

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