Internal translation initiation on poliovirus RNA: further characterization of La function in poliovirus translation in vitro

Abstract

Initiation of poliovirus RNA translation by internal entry of ribosomes is believed to require the participation of transacting factors. The mechanism of action of these factors is poorly defined. The limiting amount of one of these factors, La protein, in rabbit reticulocyte lysates (RRL) has been postulated to partially explain the inefficient translation of poliovirus RNA in this system. To further characterize La activity in translation and to identify other potential limiting factors, we assayed the ability of La protein as well as purified initiation factors, eIF-2, guanine nucleotide exchange factor (GEF), eIF-4A, eIF-4B, eIF-4F, and eIF-3, to stimulate the synthesis of P1, the capsid precursor protein, in poliovirus type 1 (Mahoney) RNAprogrammed RRL. Of the proteins tested, only La, GEF, and to some extent eIF-2 stimulated the synthesis of P1. The enhanced translation of P1 in response to La occurred concomitantly with the inhibition of synthesis of most aberrant polypeptides, resulting from initiation in the middle of the genome. Deletion of the carboxy-terminal half (214 amino acids) of La did not decrease its binding to the poliovirus 5' untranslated region but abrogated the stimulatory and correcting activity in translation. In contrast to La, GEF and eIF-2 stimulated the overall translation and increased the synthesis of aberrant products as well as P1. Neither La, GEF, nor any other factor stimulated translation of encephalomyocarditis virus RNA in RRL. The implications of these findings for the mechanism of internal translation initiation on picornavirus RNAs are discussed.

General RNA binding proteins render translation cap dependent.

“…In the case of poliovirus, several aberrant proteins are synthesized from internal AUGs that are not used in vivo (Dorner et al, 1984). The synthesis of the aberrant proteins is suppressed by the addition of La (Svitkin et al, 1994a) and to some 7152 (5) 0-0 -B extent by hnRNP I/PTB, hnRNP Al and p50 (Y.V.Svitkin, unpublished observations).…”

“…Preparation of RNA binding proteins and translation factors Recombinant La protein was overexpressed in Ecoli and purified as described previously (Svitkin et al, 1994a), except that KCI was substituted for NaCl in the buffers used for the elution of La from the heparin-Sepharose, poly(U)-Sepharose and S-Sepharose columns. This modification significantly improved the yield of La.…”

“…Recombinant eIF-4A, eIF-4E, eIF-4E-BPI and eIF-4E-BP2 (Pause et al, 1994a,b) were obtained from A.Pause. All proteins were stored in buffer A (Svitkin et al, 1994a) at -70°C.…”

“…site, which is cap independent and mediated by an internal ribosome entry site (IRES), is stimulated by La. In addition, La inhibits initiation from aberrant translation start sites of poliovirus genomic RNA Svitkin et al, 1994a). Since reticulocyte lysate is also less dependent on the cap for translation, we wished to examine the potential importance of La protein for capdependent translation.…”

End-to-end communication in the modulation of translation by mammalian RNA viruses

“…Various cellular proteins may play a role in this process. For example, the cellular proteins eukaryotic elongation factor 1A (eEFIA), La autoantigen (La), murine proliferationassociated protein-1 (Mppl), poly-r(C)-binding protein (PCBP), and polypyrimidine tract binding protein (PTB) have been shown to bind the 5 UTR of picornaviruses and to enhance translation from picornavirus IRES elements (Blyn et al, 1996(Blyn et al, , 1997Borman et al, 1993;Florez et al, 2005;Hellen et al, 1993;Kolupaeva et al, 1996;Meerovitch et al, 1993;Pilipenko et al, 2000;Svitkin et al, 1994), and some of these cellular proteins have also been observed to bind to the 3 UTR of the Norwalk calicivirus (La, PTB, and PABP) (Gutierrez-Escolano et al, 2003) and the HCV genomic RNA (PTB) ( Fig. 1D) (Ito and Lai, 1999).…”