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. 2010 Feb 5;397(1):14-22.
doi: 10.1016/j.virol.2009.11.006. Epub 2009 Nov 27.

The 5'CL-PCBP RNP complex, 3' poly(A) tail and 2A(pro) are required for optimal translation of poliovirus RNA

Sushma A Ogram  1 , Allyn Spear, Nidhi Sharma, James B Flanegan
Affiliations

The 5'CL-PCBP RNP complex, 3' poly(A) tail and 2A(pro) are required for optimal translation of poliovirus RNA

Sushma A Ogram et al. Virology. .

Abstract

In this study, we showed that the 5'CL-PCBP complex, 3' poly(A) tail and viral protein 2A(pro) are all required for optimal translation of PV RNA. The 2A(pro)-mediated stimulation of translation was observed in the presence or absence of both the 5'CL and the 3' poly(A) tail. Using protein-RNA tethering, we established that the 5'CL-PCBP complex is required for optimal viral RNA translation and identified the KH3 domain of PCBP2 as the functional region. We also showed that the 5'CL-PCBP complex and the 3' poly(A) tail stimulate translation independent of each other. In addition to the independent function of each element, the 5'CL and the 3' poly(A) tail function synergistically to stimulate and prolong translation. These results are consistent with a model in which the 5'CL-PCBP complex interacts with the 3' poly(A)-PABP complex to form a 5'-3' circular complex that facilitates ribosome reloading and stimulates PV RNA translation.

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Figures

Fig. 1
Fig. 1
Schematic of poliovirus RNAs utilized in this study. (A) Diagram of the full-length PV1 RNA which encodes all of the viral proteins. (B) Diagram of PV1p50 RNA in which nucleotides 867–6011 from PV1 RNA are deleted. This RNA contains the authentic 5’ NTR, IRES, 3’ NTR and poly(A) tail of PV1 RNA. It also contains the authentic initiation and stop codons of the viral polyprotein.
Fig. 2
Fig. 2
Requirement of the 5’CL-PCBP complex and 3’NTR(A)80 for efficient PV RNA translation. (A) Translation of either PV1p50, PV1p50(C24A), PV1p50(Δ3’NTR(A)80) or PV1p50(C24A/Δ3’NTR(A)80) RNA at a concentration of 34 µg/ml was measured by pulse-labeling for 1 h over a period of 4 h at 34 °C. The reactions were pulse-labeled with 15 µCi [35S]-methionine for 1 h at the indicated time points. At the end of the pulse, 4µl of the translation reaction was solubilized in 40 µl 1X SDS sample buffer. Labeled p50 protein synthesized was analyzed by 9–18% SDS-PAGE and visualized by autoradiograpy. (B) The amount of labeled p50 synthesized during each hour of the pulse was quantitated using a PhosphorImager.
Fig. 3
Fig. 3
RNA stability of PV1p50 and PV1p50(Δ3’NTR(A)80) RNA in HeLa S10 reactions. 32P-labeled PV1p50 or PV1p50(Δ3’NTR(A)80) RNAs were added to reactions at a final concentration of 34 µg/ml and incubated for 4 h at 34 °C. Samples were removed at the indicated time points and the amount of labeled RNA that remained intact was determined by precipitation in tricholoroacetic acid. The amount of labeled RNA recovered at each time point was calculated as a percentage of the amount of input RNA.
Fig. 4
Fig. 4
Requirement of the 3’ poly (A) tail, independent of the 3’NTR, for efficient PV RNA translation. Translation of either PV1p50, PV1p50(Δ3’NTR(A)80) or PV1p50(Δ(A)80) RNA at a concentration of 34 µg/ml was measured by pulse-labeling for 1 h over a period of 3 h as described in Fig. 2 The amount of labeled protein synthesized during each hour of the pulse was quantitated using a PhosphorImager. To quantify the cumulative level of protein synthesized, the amount of labeled protein synthesized during each hour of the pulse was added to the previous total during the 4 h time period and expressed as a function of time.
Fig. 5
Fig. 5
Tethering PCBP2 to the 5’CL enhances PV RNA translation. Translation of (A) PV1p50 RNA and (B) PV1p50(5’CLMS2) RNA was measured in the presence of either (MS2)2 or (MS2)2PCBP2 fusion protein expression RNA. Stem-loop ‘b’ in PV1p50(5’CLMS2) RNA was replaced with the cognate binding site of the MS2 protein and is bound by (MS2)2 or (MS2)2PCBP2 fusion protein. The amount of labeled protein synthesized during the 4 h time period was calculated and depicted as described for Fig. 4 An equimolar RNA ratio was maintained in these reactions. The reactions shown in panels (A) and (B) were performed at the same time and analyzed on the same gel.
Fig. 6
Fig. 6
Tethering the KH3 region of PCBP2 to the 5’CL enhances PV RNA translation (A) Schematic of the structure of full length PCBP2 containing the three KH domains and the individual protein fragments containing each of the KH domains. (B) Translation of PV1p50(5’CLMS2) RNA was measured in the presence of either (MS2)2PCBP2, (MS2)2KH1, (MS2)2KH2 or (MS2)2KH3 fusion protein expression RNA as before. The amount of labeled protein synthesized during the 4 h time period was calculated and depicted as described for Fig. 4 An equimolar RNA ratio was maintained in these reactions.
Fig. 7
Fig. 7
Requirement of the 5’CL-PCBP complex for efficient PV RNA translation in the presence or absence of active 2Apro. (A) Translation of PV1p50 and PV1p50(C24A) RNA was measured in the presence of either 2Apro or 2A(C109A) RNA. The molar ratio of reporter RNA to the 2A expression RNAs was 1:2. (B) The amount of labeled p50 synthesized during each hour of the pulse was quantitated using a PhosphorImager.
Fig. 8
Fig. 8
Requirement of the 3’ poly(A) tail for efficient PV RNA translation in the presence or absence of active 2Apro. (A) Translation of PV1p50 and PV1p50(Δ3’NTR(A)80) RNA was measured in the presence of either 2Apro or 2A(C109A) RNA. The molar ratio of reporter RNA to the 2A expression RNAs was 1:2. (B) The amount of labeled p50 synthesized during each hour of the pulse was quantitated using a PhosphorImager.
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