Translation of Encephalomyocarditis Virus RNA by Internal Ribosomal Entry

Translation Initiation of the Insulin-like Growth Factor I Receptor mRNA Is Mediated by an Internal Ribosome Entry Site

“…Internal ribosome entry was first described for picornavirus (38),and different elements involved in the internal ribosome entry process are now well known. The rat IGR-IR 5Ј UTR mRNA does not present the conventional motifs of picornavirus IRES, such as a GNRA or CAAA loop (39). Nonetheless, our prediction of the secondary structure revealed that this 5Ј UTR mRNA presents a conserved polypyrimidine-rich stem-loop structure composed of two different loops.…”

The properties of chimeric picornavirus IRESes show that discrimination between internal translation initiation sites is influenced by the identity of the IRES and not just the context of the AUG codon

“…All picornavirus IRESes end with a 39-terminal AUG triplet located ;25 nt downstream of the start of the oligopyrimidine tract, and this AUG is considered to be the actual ribosome entry site (reviewed by Ehrenfeld & Semler, 1995;Hellen & Wimmer, 1995;Jackson & Kaminski, 1995)+ Where the various picornavirus IRESes differ is in what happens following internal ribosome entry at this site: whether it will be used as the primary translation initiation site, or whether the majority of ribosomes will initiate translation elsewhere+ They also differ in other respects: (1) their efficiency in various systems, (2) whether their activity is increased as a result of cleavage of eIF4G, and (3) whether their activity in rabbit reticulocyte lysates is stimulated by supplementation with HeLa cell cytoplasmic extracts+ Although the primary aim of this work was to use chimeric IRESes to determine whether the behavior of the ribosome on internal entry is dictated by the nature of the upstream IRES body or by the sequences at the actual entry site, we were also interested in which of these two parts influenced the other properties in which picornavirus IRESes differ+ With regard to efficiency, both in unsupplemented rabbit reticulocyte lysate and also in transfection assays of cultured cells, the hierarchy is generally agreed to be: rhinovirus , , enterovirus (e+g+ poliovirus) , , cardiovirus , aphthovirus IRESes (Borman et al+, 1995(Borman et al+, , 1997bRoberts et al+, 1998)+ The substitution of heterologous downstream sequences in the chimeric IRESes described here did not, in general, change the efficiency of internal initiation, which must therefore be largely governed by the nature of the upstream sequences, the body of the IRES+ What is perhaps most significant is that, apart from one exception, none of the chimeric IRESes were unexpectedly inefficient+ This argues that the two parts of the IRES are functionally independent: the upstream IRES segment and the downstream entry site region are not matched to each other in a functionally dedicated manner+ The exception was FMDV-PV mRNA, which was distinctly less efficient than FMDV-EMCV or XLFMDV mRNAs (Fig+ 3A)+ Stimulation of IRES activity by supplementation of in vitro assays with FMDV L protease is seen only with rhino-and enterovirus IRESes (with a very small effect on cardiovirus IRESes), and there is good evidence that stimulation is due to cleavage of eIF4G (Borman et al+, 1995(Borman et al+, , 1997a)+ Our results unambiguously show that the stimulation of poliovirus IRES activity by eIF4G cleavage is mediated via the body of the IRES, and not via the sequences at the putative ribosome entry site, as neither FMDV-PV mRNA (Fig+ 3A) nor EMCV-PV (Fig+ 5) showed a significant response to addition of L protease+ We did not obtain sufficiently consistent results in assays supplemented with HeLa cell extracts to be able to draw any conclusions as to whether the stimulation of poliovirus IRES activity by such extracts is mediated via the body of the IRES or via the sequences at the actual ribosome entry site+…”

“…Picornavirus RNAs are translated by a mechanism of internal ribosome entry dependent on an ;450 nt cisacting RNA element located within the 59-untranslated region (UTR), originally designated as the "internal ribosome entry site" (IRES)+ However, it is generally agreed that the actual ribosome entry site is not the total length of this large segment, but, if operationally defined as the most 59-proximal point at which initiation can occur, the entry or landing site is an AUG codon located 25 nt downstream of the start of a pyrimidine-rich tract at the 39 end of the IRES (for recent reviews, see Ehrenfeld & Semler, 1995;Hellen & Wimmer, 1995;Jackson & Kaminski, 1995)+ As has been described previously, the 59-proximal part of the oligopyrimidine tract is essential for IRES function in several different picornavirus species (Iizuka et al+, 1989;Jang & Wimmer, 1990;Kühn et al+, 1990;Meerovitch et al+, 1991;Nicholson et al+, 1991;Pestova et al+, 1991;Pilipenko et al+, 1992), but, on the other hand, the sequences between the oligopyrimidine tract and the AUG codon are not highly conserved between closely related species or even between different strains of the same species, although the length of this segment is conserved (Sangar et al+, 1987;Pritchard et al+, 1992)+ It has been suggested that this ;25-nt stretch serves as an unstructured spacer element (Kaminski et al+, 1994)+ On the basis of their IRES sequences, picornaviruses can be divided into three different groups: (1) hepatitis A virus, (2) the cardiovirus [e+g+, encephalomyocarditis virus (EMCV)] and aphthovirus family [e+g+, foot-and-mouth-disease virus (FMDV)], and (3) the enterovirus [e+g+, poliovirus (PV)] and rhinovirus family+ Several differences have been observed in the in vitro translation characteristics between IRESes from these different groups (Borman et al+, 1995)+ While IRESdriven translation of cardio-and aphthovirus RNAs is accurate and extremely efficient in the reticulocyte lysate, translation driven by the poliovirus or rhinovirus IRES is inaccurate and inefficient unless the system is supplemented with HeLa cell or Krebs II ascites cell extracts (Brown & Ehrenfeld, 1979;Dorner et al+, 1984;Svitkin et al+, 1988;Borman et al+, 1993Borman et al+, , 1995+ Another difference is that cleavage of eIF4G by picornavirus encoded proteases strongly stimulates translation driven by the polio-and the rhinovirus IRESes (Borman et al+, 1995;…”

Combination Therapy for Hepatitis C Virus with Heat-Shock Protein 90 Inhibitor 17-AAG and Proteasome Inhibitor MG132

“…The resulting 17-AAG treatment reduced NS3 levels, but MG132 treatment of NNC#2 cells did not block the degradation of HCV NS3 ( Figure 1D). This blocking efficiency significantly influenced the different activities of the virus IRES (that is, HCV IRES and EMCV IRES) [25][26][27][28]. In our assays, when Huh-7 cells exposed to MG132 were transfected with pHCV IRES luc or pEMCV IRES luc, HCV IRES activity was inhibited, but EMCV IRES was not ( Figure 2B).…”