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. 2003 Mar;77(6):3353-9.
doi: 10.1128/jvi.77.6.3353-3359.2003.

Unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus

Oréda Boussadia  1 , Michael Niepmann, Laurent Créancier, Anne-Catherine Prats, François Dautry, Hélène Jacquemin-Sablon
Affiliations

Unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus

Oréda Boussadia et al. J Virol. 2003 Mar.

Abstract

Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). Unr, a cytoplasmic RNA-binding protein that contains five cold-shock domains and is encoded by the gene upstream of N-ras, stimulates translation directed by the human rhinovirus (HRV) IRES in vitro. To examine the role of Unr in translation of picornavirus RNAs in vivo, we derived murine embryonic stem (ES) cells in which either one (-/+) or both (-/-) copies of the unr gene were disrupted by homologous recombination. The activity of picornaviral IRES elements was analyzed in unr(+/+), unr(+/-), and unr(-/-) cell lines. Translation directed by the HRV IRES was severely impaired in unr(-/-) cells, as was that directed by the poliovirus IRES, revealing a requirement for Unr not previously observed in vitro. Transient expression of Unr in unr(-/-) cells efficiently restored the HRV and poliovirus IRES activities. In contrast, the IRES elements of encephalomyocarditis virus and foot-and-mouth-disease virus are not Unr dependent. Thus, Unr is a specific regulator of HRV and poliovirus translation in vivo and may represent a cell-specific determinant limiting replication of these viruses.

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Figures

FIG. 1.
FIG. 1.
Genotype of ES cell clones mutated at the unr locus. (A) Schematic representation of the 5′ end of the wild-type unr allele and of the mutated allele generated by homologous recombination; BamHI sites are indicated by the letter B. (B) Southern blot analysis of ES cell clones. Genomic DNA from eight independent ES cell clones (A, B, C, D, E, G, L, and K [lanes 1 to 8, respectively]) was digested with BamHI and hybridized with the unr probe P5 indicated in panel A. This probe detects a 4.3-kb fragment and a 2.2-kb fragment from the wild-type (wt) and mutated (mt) alleles, respectively.
FIG. 2.
FIG. 2.
Expression of unr in ES cell clones. (A) RNase protection analysis of RNA isolated from wild-type, unr+/−, and unr−/− ES cells. Probe UNK comprises unr, N-ras and Ki-ras sequences. Ten micrograms of total RNA from five ES clones of each genotype as shown in Fig. 1 was analyzed with probe UNK. The molecular size marker on the left was generated with end-labeled MspI fragments of pBR322. The protected fragments are indicated on the right. (B) Immunoblot of wild-type, heterozygous unr+/−, and homozygous unr−/− ES cells. Lysates (25 μg) prepared from genotyped ES cell clones were analyzed by Western blotting with anti-unr and anti-α-tubulin antibodies. Arrows indicate unr (85 kDa) and α-tubulin (55 kDa).
FIG. 3.
FIG. 3.
Effect of unr expression on the activity of picornaviral IRES elements. (A) The dicistronic constructs pCRpolL, pHRVL, pCREL, and pD128 containing the poliovirus, HRV, EMCV, and FMDV IRESs, respectively, and the control construct pCRHL were transfected in unr+/+, unr+/−, and unr−/− ES cells. Twenty-four hours after transfection, cells were analyzed for Fluc, Rluc, and CAT activities. The ratio of the enzymatic activities of the second cistron to the first cistron are presented by histograms for unr+/+ (clone L, black bars), unr+/− (clone K, gray bars), and unr−/− (clone A, light gray bars) cells. Fluc/Rluc ratios (left) or Fluc/CAT ratios (right) were normalized to the values obtained with control plasmids (pCRHL or pDmt, respectively). Three independent experiments were performed, each time in triplicate. Rluc and Fluc activities (relative light units) and CAT (cpm) activities are presented in the lower panel of the figure. (B) Northern blot analysis of total RNA extracted from transfected ES cells. Total cellular RNA was isolated from unr+/+ and unr−/− ES cells transfected with pHRVL (lanes 1, 2, 4, and 5) or pSGluc (lane 7), or from untransfected ES cells (lanes 3 and 6). Fifteen micrograms of total RNA was analyzed by Northern blotting with the Rluc and Fluc probes (top) and with a β-actin probe as a loading control (bottom). The migration of 28S and 18S rRNA is indicated on the right.
FIG. 4.
FIG. 4.
Rescue of HRV and poliovirus IRES activity by unr. unr+/+ and unr−/− ES cells were transfected with 1.5 μg of dicistronic plasmids containing the poliovirus, HRV, and EMCV IRESs, respectively. Each of the above plasmids was transfected together with 1 μg of either pSG5 (control) or pSG Flag-unr plasmids, respectively. Twenty-four hours after transfection, cells were analyzed for Fluc and Rluc activity (A) or Flag-unr protein expression (B). (A) The IRES activities in unr+/+ and unr−/− cells were determined as described in the legend to Fig. 3; the values were taken as 100 for each dicistronic plasmid cotransfected with pSG5 in unr+/+ cells. (B) Immunoblot analysis with anti-Flag antibody to detect the expression of Flag-unr protein. Lysates were prepared from unr+/+ (lanes 1 and 2) or unr−/− (lanes 3 and 4) ES cells transfected with 1 μg of pSG5 (lanes 1 and 3) or 1 μg of pSG Flag-unr (lanes 2 and 4) plasmids.
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