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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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Reverse genetic mutation analysis shows that MDV1 miRNAs from Lat-cluster are responsible for paip2 repression.(A) Schematic representation of the bicistronic transcripts that we and others have cloned as cDNA and that encode for pp14a and pp14b isoforms, modified from Tahiri-Alaoui et al, J. Virol. Dec. 2009, Vol.83, No. 24, p12769-12778. (B) & (C) Chicken embryo fibroblasts (CEF) were transfected with BAC clone pRB1B5 Lat-miR-Revertant or pRB1B5 Lat-miR- deletion, respectively. RNA and proteins were simultaneously extracted using Trizol at the indicated time points. Viral and host proteins were detected by immunoblotting with the indicated antibodies. (D) Quantitative RT-PCR of host (paip2) and of viral transcripts (pp14a and pp14b isoforms) at the indicated time points. GAPDH is used as the endogenous control and time zero is used as the calibrator. All experiments were repeated three times and the error bars indicate the SEM.
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pone-0114466-g006: Reverse genetic mutation analysis shows that MDV1 miRNAs from Lat-cluster are responsible for paip2 repression.(A) Schematic representation of the bicistronic transcripts that we and others have cloned as cDNA and that encode for pp14a and pp14b isoforms, modified from Tahiri-Alaoui et al, J. Virol. Dec. 2009, Vol.83, No. 24, p12769-12778. (B) & (C) Chicken embryo fibroblasts (CEF) were transfected with BAC clone pRB1B5 Lat-miR-Revertant or pRB1B5 Lat-miR- deletion, respectively. RNA and proteins were simultaneously extracted using Trizol at the indicated time points. Viral and host proteins were detected by immunoblotting with the indicated antibodies. (D) Quantitative RT-PCR of host (paip2) and of viral transcripts (pp14a and pp14b isoforms) at the indicated time points. GAPDH is used as the endogenous control and time zero is used as the calibrator. All experiments were repeated three times and the error bars indicate the SEM.

Mentions: To further investigate the biological relevance of the viral miRNA-mediated paip2 repression during viral infection and its effect on pp14 expression, we used reverse genetics mutagenesis [35] and deleted both copies of cluster 3 miRNAs from the latency-associated region of the pRB1B5 BAC clone [36]. Cluster 3 contains miR-M6, miR-M7, miR-M8, miR-M10 and miR-M13 [35], all the 4 miRNAs that seem to mediate paip2 translation repression. Reconstruction of the mutant viruses in primary CEF transfected with the BAC DNA and analysis of in vitro growth kinetics show that the mutant viruses replicate with comparable kinetics, but slightly slower than the parent pRB1B5 (S6 Figure); therefore only the mutant viruses are used for this studies. Primary CEF were transfected with BAC DNA from the mutant viruses and the cells were lysed at the indicated times points with TRIzol then RNA and proteins were simultaneously extracted and analysed by immunoblotting and quantitative RT-PCR (Fig. 6). Immunoblotting shows that there is significantly higher level of pp14b isoform compared to that of pp14a isoform in CEF transfected with pRB1B5-Lat-miR-Δ and with pRB1B5-Lat-miR-Rev 48 hours post transfection (Fig. 6). The two pp14 isoforms differ by the composition of their N-termini as a result of differential splicing which gives two splice isoforms the translation of which is either cap-dependent for the pp14a or 5L IRES-driven for the pp14b [22], and as depicted in Fig. 6A. The expression level of both pp14 isoforms increases over time in CEF transfected with pRB1B5-Lat-miR-Rev (Fig. 6B); however and in contrast to pp14a, the level of pp14b decreases over time in CEF transfected with pRB1B5-Lat-miR-Δ (Fig. 6C). As judged from the quantitative real-time RT-PCR results (Fig. 6D), the differences in expression level between both pp14 isoforms cannot be solely explained by the differential accumulation of their respective transcripts that follows the same trend. We can clearly see that the levels of pp14b transcripts are consistently higher than those of the pp14a transcripts which also continue to increase over time, confirming our previous findings [22]. Interestingly, the continued increase of pp14b isoform in CEF transfected with pRB1B5-Lat-miR-Rev correlates with significant decrease in the level of paip2 protein (Fig. 6B) that itself is concomitant with increased level of MDV1 miRNAs, M7, M8, M10 and M13 (S7 Figure). There is no detectable difference over the time points examined in the abundance of paip2 mRNAs between CEF transfected with both mutant BAC DNAs (Fig. 6D). Additionally, we show that BAC DNA mutagenesis does not affect the expression of other viral miRNAs such as miR-M4 from cluster 1 (S8 Figure). These results indicate a direct link between MDV1-miRNAs expressed from the Lat-cluster and paip2-mediated translation repression. At this stage we do not know the relative contribution of each of the viral miRNAs to the overall paip2 translation repression. Additional evidence supporting the link between viral miRNAs-mediated paip2 repression and the enhanced activity of 5L IRES came from siRNA paip2-mediated repression experiments that allowed rescuing pp14b (under 5L IRES control) expression in CEF infected by pRB1B5 Lat-miR-Δ to levels comparable to those observed with pRB1B5 Lat-miR-Rev (Fig. 7). These RNAi rescue experiments clearly show that the level of paip2 is reduced by siRNA against paip2 as compared to control siRNA in CEF-infected with mutant viruses and in CEF-control, whereas the level of PABP1 expression remains unchanged under all conditions (Fig. 7). Significantly, none of these changes appear to affect the expression level of another IE protein, pp38 isoforms or the expression level of PABP1 from the host. Overall, viral miRNA-mediated paip2 repression illustrates a finely tuned transcript-specific translation control strategy that appears to specifically affect the accumulation of pp14b isoform which is under 5L IRES control, whereas the cap-dependent pp14a isoform expression remains unaffected. Our results also show that although the optimal 5L IRES activity requires the presence of PABP1, MDV1 infection does not appear to cause increased accumulation of PABP1, but the virus is instead using a strategy that ensure the availability of an active pool of PABP1.


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)

Reverse genetic mutation analysis shows that MDV1 miRNAs from Lat-cluster are responsible for paip2 repression.(A) Schematic representation of the bicistronic transcripts that we and others have cloned as cDNA and that encode for pp14a and pp14b isoforms, modified from Tahiri-Alaoui et al, J. Virol. Dec. 2009, Vol.83, No. 24, p12769-12778. (B) & (C) Chicken embryo fibroblasts (CEF) were transfected with BAC clone pRB1B5 Lat-miR-Revertant or pRB1B5 Lat-miR- deletion, respectively. RNA and proteins were simultaneously extracted using Trizol at the indicated time points. Viral and host proteins were detected by immunoblotting with the indicated antibodies. (D) Quantitative RT-PCR of host (paip2) and of viral transcripts (pp14a and pp14b isoforms) at the indicated time points. GAPDH is used as the endogenous control and time zero is used as the calibrator. All experiments were repeated three times and the error bars indicate the SEM.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263670&req=5

pone-0114466-g006: Reverse genetic mutation analysis shows that MDV1 miRNAs from Lat-cluster are responsible for paip2 repression.(A) Schematic representation of the bicistronic transcripts that we and others have cloned as cDNA and that encode for pp14a and pp14b isoforms, modified from Tahiri-Alaoui et al, J. Virol. Dec. 2009, Vol.83, No. 24, p12769-12778. (B) & (C) Chicken embryo fibroblasts (CEF) were transfected with BAC clone pRB1B5 Lat-miR-Revertant or pRB1B5 Lat-miR- deletion, respectively. RNA and proteins were simultaneously extracted using Trizol at the indicated time points. Viral and host proteins were detected by immunoblotting with the indicated antibodies. (D) Quantitative RT-PCR of host (paip2) and of viral transcripts (pp14a and pp14b isoforms) at the indicated time points. GAPDH is used as the endogenous control and time zero is used as the calibrator. All experiments were repeated three times and the error bars indicate the SEM.
Mentions: To further investigate the biological relevance of the viral miRNA-mediated paip2 repression during viral infection and its effect on pp14 expression, we used reverse genetics mutagenesis [35] and deleted both copies of cluster 3 miRNAs from the latency-associated region of the pRB1B5 BAC clone [36]. Cluster 3 contains miR-M6, miR-M7, miR-M8, miR-M10 and miR-M13 [35], all the 4 miRNAs that seem to mediate paip2 translation repression. Reconstruction of the mutant viruses in primary CEF transfected with the BAC DNA and analysis of in vitro growth kinetics show that the mutant viruses replicate with comparable kinetics, but slightly slower than the parent pRB1B5 (S6 Figure); therefore only the mutant viruses are used for this studies. Primary CEF were transfected with BAC DNA from the mutant viruses and the cells were lysed at the indicated times points with TRIzol then RNA and proteins were simultaneously extracted and analysed by immunoblotting and quantitative RT-PCR (Fig. 6). Immunoblotting shows that there is significantly higher level of pp14b isoform compared to that of pp14a isoform in CEF transfected with pRB1B5-Lat-miR-Δ and with pRB1B5-Lat-miR-Rev 48 hours post transfection (Fig. 6). The two pp14 isoforms differ by the composition of their N-termini as a result of differential splicing which gives two splice isoforms the translation of which is either cap-dependent for the pp14a or 5L IRES-driven for the pp14b [22], and as depicted in Fig. 6A. The expression level of both pp14 isoforms increases over time in CEF transfected with pRB1B5-Lat-miR-Rev (Fig. 6B); however and in contrast to pp14a, the level of pp14b decreases over time in CEF transfected with pRB1B5-Lat-miR-Δ (Fig. 6C). As judged from the quantitative real-time RT-PCR results (Fig. 6D), the differences in expression level between both pp14 isoforms cannot be solely explained by the differential accumulation of their respective transcripts that follows the same trend. We can clearly see that the levels of pp14b transcripts are consistently higher than those of the pp14a transcripts which also continue to increase over time, confirming our previous findings [22]. Interestingly, the continued increase of pp14b isoform in CEF transfected with pRB1B5-Lat-miR-Rev correlates with significant decrease in the level of paip2 protein (Fig. 6B) that itself is concomitant with increased level of MDV1 miRNAs, M7, M8, M10 and M13 (S7 Figure). There is no detectable difference over the time points examined in the abundance of paip2 mRNAs between CEF transfected with both mutant BAC DNAs (Fig. 6D). Additionally, we show that BAC DNA mutagenesis does not affect the expression of other viral miRNAs such as miR-M4 from cluster 1 (S8 Figure). These results indicate a direct link between MDV1-miRNAs expressed from the Lat-cluster and paip2-mediated translation repression. At this stage we do not know the relative contribution of each of the viral miRNAs to the overall paip2 translation repression. Additional evidence supporting the link between viral miRNAs-mediated paip2 repression and the enhanced activity of 5L IRES came from siRNA paip2-mediated repression experiments that allowed rescuing pp14b (under 5L IRES control) expression in CEF infected by pRB1B5 Lat-miR-Δ to levels comparable to those observed with pRB1B5 Lat-miR-Rev (Fig. 7). These RNAi rescue experiments clearly show that the level of paip2 is reduced by siRNA against paip2 as compared to control siRNA in CEF-infected with mutant viruses and in CEF-control, whereas the level of PABP1 expression remains unchanged under all conditions (Fig. 7). Significantly, none of these changes appear to affect the expression level of another IE protein, pp38 isoforms or the expression level of PABP1 from the host. Overall, viral miRNA-mediated paip2 repression illustrates a finely tuned transcript-specific translation control strategy that appears to specifically affect the accumulation of pp14b isoform which is under 5L IRES control, whereas the cap-dependent pp14a isoform expression remains unaffected. Our results also show that although the optimal 5L IRES activity requires the presence of PABP1, MDV1 infection does not appear to cause increased accumulation of PABP1, but the virus is instead using a strategy that ensure the availability of an active pool of PABP1.

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