A new pathway of translational regulation mediated by eukaryotic initiation factor 3

Abstract

We report a new pathway of translation regulation that may operate in interferon-treated or virusinfected mammalian cells. This pathway is activated by P56, a protein whose synthesis is strongly induced by interferons or double-stranded RNA. Using a yeast two-hybrid screen, we identi®ed the P48 subunit of the mammalian translation initiation factor eIF-3 as a protein that interacts with P56. The P56±P48 interaction was con®rmed in human cells by co-immunoprecipitation assays and confocal microscopy. Gel ®ltration assays revealed that P56 binds to the large eIF-3 complex that contains P48. Puri®ed recombinant P56 inhibited in vitro translation of reporter mRNAs in a dose-dependent fashion, and that inhibition was reversed by the addition of puri®ed eIF-3. In vivo, expression of transfected P56 or induction of the endogenous P56 by interferon caused an inhibition of overall cellular protein synthesis and the synthesis of a transfected reporter protein. As expected, a P56 mutant that does not interact with P48 and eIF-3 failed to inhibit protein synthesis in vitro and in vivo. Keywords: double-stranded RNA/eIF-3/interferon/P56/ translational regulation IntroductionAlthough the antiviral effect of interferon (IFN) is the most well known, IFNs have many other effects on cell physiology (Stark et al., 1998). The multiple effects of IFNs are mediated by the numerous cellular proteins whose synthesis is induced by IFNs (Sen and Ransohoff, 1993). However, the speci®c biochemical and cellular functions of most of these proteins are unknown. Among the notable exceptions are the enzymes PKR and 2±5(A) synthetases that are known to affect virus replication and cell growth (Stark et al., 1998). Here, we report the function of another IFN-inducible protein, P56.The cDNA for P56 was cloned in the process of cloning IFN-inducible mRNAs (Chebeth et al., 1983;Kusari and Sen, 1986). We have studied extensively the transcriptional regulation of 561 mRNA that encodes P56 (Bandyopadhyay et al., 1990). The mRNA is undetectable in untreated cells but it is induced rapidly upon IFN treatment (Kusari and Sen, 1986). The 561 mRNA level is quite high in IFN-treated cells and, in a recent gene array analysis, it scored as the most abundant IFN-induced mRNA among >100 such mRNAs (Der et al., 1998). Because the 561 mRNA and the encoded protein, P56, both turn over rapidly, cells are depleted of them quickly after IFN treatment ceases. The 561 gene is transcriptionally induced not only by IFN but also by double-stranded (ds) RNA or virus infection (Tiwari et al., 1987). Our studies using various mutant cells have clearly established that the Jak±STAT pathway used by IFN to induce it is dispensable for its induction by dsRNA or viruses (Bandyopadhyay et al., 1995). Thus, there are alternative ways to induce the 561 gene, and the encoded protein, P56, may have cellular functions beyond the IFN system.Because the cellular level of P56 is drastically enhanced upon treatment of cells with IFN or dsRNA, we suspected that it has important cellular fu...

Mouse p56 Blocks a Distinct Function of Eukaryotic Initiation Factor 3 in Translation Initiation

“…Previous studies from our laboratory have characterized the human viral stress-inducible protein p56, a 56-kDa protein (1). Human p56 (Hup56) has been shown to act as an inhibitor of protein synthesis through its association with the "e" subunit of eukaryotic initiation factor 3 (eIF3 1 ; the e subunit is also known as p48 or Int6) (2,3). We have shown recently (4) that the inhibitory activity of human p56 occurs at the step of ternary complex stabilization by eIF3, a key step in the initiation pathway for protein synthesis in eukaryotes.…”

Differential proteomic analysis of respiratory samples from patients suffering from influenza

“…The IFIT family proteins contain a TPR (tetratricopeptide repeat) domain, a 34 amino acid motif folding into a helix-turn-helix structure, which mediates protein interactions [9]. IFIT1 and IFIT2 are involved in a nonspecific antiviral program through their direct interactions with eukaryotic initiation factor 3, which subsequently suppresses more than 60 % of translation in cells and viruses during protein synthesis [13,15,38]. The IFIT family, especially IFIT1 and IFIT3, restrict DNA and RNA virus replication, such as hepatitis B virus (HBV), human papillomavirus (HPV), hepatitis C virus (HCV), West Nile virus (WNV) and others [26,30].…”

Identification and expression analysis of two IFN-inducible genes in crucian carp (Carassius auratus L.)

“…Previous reports demonstrated that the last three specific TPR motifs in the C-terminal region are required for interaction of IFI56 with the translation initiation factor eIF-3 (Guo and Sen, 2000;Guo et al, 2000a), which is critical for human IFI56 inhibiting cellular protein synthesis, possibly including viral protein synthesis. In the present study, all 10 TPR motifs were found in CaIFI58 as compared to human IFI58.…”

“…These genes encode structurally related proteins, which contain multiple tetratricopeptide repeat (TPR) domains that are a loosely conserved 34-amino acid residue repeat unit involved in specific protein -protein interactions (Lamb et al, 1995;Das et al, 1998). Recently, one cellular function of IFI56 has been revealed by the discovery that IFI56 binds to the large multimeric complex of eIF-3 via p48 and blocks its function in initiating protein synthesis, which is presumed to mediate inhibiting viral replication and cell growth (Guo and Sen, 2000;Guo et al, 2000a), and also possibly mediate cell apoptosis (Grandvaux et al, 2002).…”