Abstract: Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (L(pro)) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2A(pro)). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV L(pro) specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2A(pro) in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV L(pro) in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV L(pro) and rhinovirus 2A(pro) enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.
Abstract: Several picornaviruses shut down host cellular protein synthesis by proteolytic cleavage of the eukaryotic initiation factor (eIF) 4GI and eIF4GII isoforms. Viral RNA translation is maintained by a cap-independent mechanism. Here, we identify the human rhinovirus 2 2A(pro) cleavage site in eIF4GII in vitro as PLLNV(699)*GSR; this sequence lies seven amino acids C-terminal to the cleavage site previously identified in eIF4GI (LSTR681*GPP).
Abstract: Infection of mammalian cells with picornaviruses like entero-, rhino-, and aphthoviruses leads to an inhibition of cap-dependent cellular protein synthesis by the cleavage of both translation initiation factors, eIF4GI and eIF4GII. In entero- and rhinovirus infection this cleavage process is mediated by the viral 2A proteinase (2A(pro)). In order to discriminate between a direct mode of eIF4G cleavage and an indirect cleavage via activation of a cellular proteinase, a thermosensitive 2A(pro) mutant (ts-2A(pro)) of human rhinovirus 2 was employed. Temperature shift experiments of cytoplasmic HeLa cell extracts incubated with ts-2A(pro) strongly support a direct mode of cleavage of eIF4GI and eIF4GII by the viral 2A(pro).
Abstract: The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two-dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C-terminal one-third of the protein. Mass spectrometry and mutational analyses identified the serum-stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide-3-kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12-rapamycin-associated protein/mammalian target of rapamycin), prevent the serum-induced phosphorylation of these residues. Finally, the phosphorylation state of N-terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C-terminus to kinases is modulated by this pathway(s).
Abstract: A cell line was generated that expresses the poliovirus 2A protease in an inducible manner. Tightly controlled expression was achieved by utilizing the muristerone A-regulated expression system. Upon induction, cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI) and eIF4GII is observed, with the latter being cleaved in a somewhat slower kinetics. eIF4G cleavage was accompanied by a severe inhibition of protein synthesis activity. Upon induction of the poliovirus 2A protease, the cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-size DNA ladder, and positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining; hence, their death can be characterized as apoptosis. These results indicate that the expression of the 2A protease in mammalian cells is sufficient to induce apoptosis. We suggest that the poliovirus 2A protease induces apoptosis either by arresting cap-dependent translation of some cellular mRNAs that encode proteins required for cell viability, by preferential cap-independent translation of cellular mRNAs encoding apoptosis inducing proteins, or by cleaving other, yet unidentified cellular target proteins.
Abstract: Eukaryotic translation initiation factor 4G (eIF4G), which has two homologs known as eIF4GI and eIF4GII, functions in a complex (eIF4F) which binds to the 5' cap structure of cellular mRNAs and facilitates binding of capped mRNA to 40S ribosomal subunits. Disruption of this complex in enterovirus-infected cells through eIF4G cleavage is known to block this step of translation initiation, thus leading to a drastic inhibition of cap-dependent translation. Here, we show that like eIF4GI, the newly identified homolog eIF4GII is cleaved during apoptosis in HeLa cells and can serve as a substrate for caspase 3. Proteolysis of both eIF4GI and eIF4GII occurs with similar kinetics and coincides with the profound translation inhibition observed in cisplatin-treated HeLa cells. Both eIF4GI and eIF4GII can be cleaved by caspase 3 with similar efficiency in vitro, however, eIF4GII is processed into additional fragments which destroy its core central domain and likely contributes to the shutoff of translation observed in apoptosis. Cell Death and Differentiation (2000) 7, 1234 - 1243.
Abstract: For many members of the Picornaviridae family, infection of cells results in a shutoff of host protein synthesis. For rhinoviruses and enteroviruses, the shutoff has been explained in part by the cleavage of eukaryotic initiation factor 4GI (eIF4GI), a component of the cap-binding protein complex eIF4F. The cleavage of eIF4GI is mediated by the virus-specific proteinase 2Apro and results in inhibition of cap-dependent, but not cap-independent, translation. The inhibition of host protein synthesis after infection with human rhinovirus 14 (HRV-14) lags behind the cleavage of eIF4GI. Recently, we discovered a functional homolog of eIF4GI, termed eIF4GII, and showed that cleavage of eIF4GII coincides with the shutoff of host cell protein synthesis after poliovirus infection (Gradi et al., Proc. Natl. Acad. Sci. USA 95:11089-11094, 1998). We wished to determine whether eIF4GII cleavage kinetics could also explain the lack of correlation between the kinetics of eIF4GI cleavage and the shutoff of host protein synthesis after rhinovirus infection. In this study, we examined the correlation between human rhinovirus-induced shutoff of host protein synthesis and cleavage of eIF4GI and eIF4GII. In HRV-14-infected HeLa cells, almost no intact eIF4GI could be detected by 4 h postinfection, while only 4% of eIF4GII was cleaved at this time. By 6 h, however, 67% of eIF4GII was cleaved, and this cleavage coincided with a significant (60%) decline of host translation. These results suggest that cleavage of both eIF4GI and eIF4GII is required for HRV-mediated inhibition of host cell protein synthesis and that the cleavage of eIF4GII is the rate-limiting step in the shutoff of host cell protein synthesis after rhinovirus infection.
Abstract: Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.
Abstract: Eukaryotic initiation factor (eIF) 4GI is a component of the cap-binding protein complex eIF4F, which is required for cap-dependent translation. Infection of cells by poliovirus results in a precipitous decline of host cell protein synthesis, which is preceded by the cleavage of eIF4GI. Cleavage of eIF4GI results in the inhibition of cap-dependent translation. Poliovirus translation is not affected by eIF4GI cleavage, however, because poliovirus mRNA is translated by a cap-independent mechanism. Cleavage of eIF4GI alone cannot explain the shutoff of host protein synthesis, because after infection in the presence of inhibitors of virus replication, eIF4GI is cleaved, yet host protein synthesis is only partially inhibited. Here we show that eIF4GII, a recently discovered functional homolog of eIF4GI, is more resistant to poliovirus-mediated cleavage than eIF4GI, and that its proteolysis is concomitant with the shutoff of host cell protein synthesis. Moreover, infection with poliovirus in the presence of inhibitors of virus replication resulted in efficient cleavage of eIF4GI, but only partial proteolysis of eIF4GII. Thus, cleavage of both eIF4GI and eIF4GII appears to be required for the shutoff of host protein synthesis after poliovirus infection. These results explain several earlier reports documenting the lack of correlation between eIF4GI cleavage and inhibition of cellular mRNA translation after poliovirus infection.
Abstract: Most eukaryotic mRNAs possess a 5' cap and a 3' poly(A) tail, both of which are required for efficient translation. In yeast and plants, binding of eIF4G to poly(A)-binding protein (PABP) was implicated in poly(A)-dependent translation. In mammals, however, there has been no evidence that eIF4G binds PABP. Using 5' rapid amplification of cDNA, we have extended the known human eIF4GI open reading frame from the N-terminus by 156 amino acids. Co-immunoprecipitation experiments showed that the extended eIF4GI binds PABP, while the N-terminally truncated original eIF4GI cannot. Deletion analysis identified a 29 amino acid sequence in the new N-terminal region as the PABP-binding site. The 29 amino acid stretch is almost identical in eIF4GI and eIF4GII, and the full-length eIF4GII also binds PABP. As previously shown for yeast, human eIF4G binds to a fragment composed of RRM1 and RRM2 of PABP. In an in vitro translation system, an N-terminal fragment which includes the PABP-binding site inhibits poly(A)-dependent translation, but has no effect on translation of a deadenylated mRNA. These results indicate that, in addition to a recently identified mammalian PABP-binding protein, PAIP-1, eIF4G binds PABP and probably functions in poly(A)-dependent translation in mammalian cells.
Abstract: The 5'-terminal untranslated region (5' UTR) of the uncapped hepatitis A virus (HAV) RNA contains two pyrimidine-rich sequences; one about 20 nucleotides (nt) in length in the vicinity of the AUG initiation codon (nt 706-726), and a longer one (about 40 nt) encompassing nt 100 to 140. The latter includes a 13 nt 'core' sequence (positions 126-138 in the HM175 strain) which is 80% identical to the pyrimidine-rich tract of poliovirus type 1 RNA (Mahoney strain). Representative cDNAs of the entire 5' UTR of HAV RNA were inserted in the intercistronic region of the bi-cistronic plasmid pSV-GH/CAT between the genes coding for the human growth hormone (GH) and bacterial chloramphenicol acetyltransferase (CAT). When COS-7 cells were transfected with these constructs they transiently expressed CAT indicating that the 5' UTR of HAV was efficiently directing internal initiation of translation of the reporter gene. Under similar conditions the 5' UTR of poliovirus type 2 (Lansing strain) was 30% more efficient in directing the expression of the CAT gene. Removal of the 'core' sequence from the 5'-distal pyrimidine-rich stretch extending between nt 117 and 131 in the HAV 5' UTR reduced the CAT activity in the lysates of transfected cells by 40%, whereas point mutations engineered in this segment strongly decreased (80% inhibition) the HAV-driven expression of the reporter gene. Limited mutations systematically introduced in the reiterated (U)UUUCCC motifs of the 5'-distal pyrimidine-rich tract identified two major functional domains extending between nt 100-106 and 113-119. Substitutions in these hexanucleotides abrogated internal initiation of translation, whereas similar changes in the neighbouring domains (nt 107-112 and 120-126) had no effect on the expression of the reporter gene, suggesting that the 5'-most pyrimidine-rich tract is indeed part of the structure(s) recognized by ribosomes and associated factors at initiation of translation and that the hexanucleotides 100-106 and 113-119 constitute an important part of it. Although HAV replicates better at 33 degrees C than at 37 degrees C, incubation of transfected cultures at 33 degrees C delayed the expression and slightly reduced the level of CAT activity in the cell lysates, but the overall effect of the mutations remained unchanged.
Abstract: The first enzymatic step in the biosynthesis of steroid hormones occurs in the mitochondrial inner membrane and is dependent on the mobilization of cholesterol from cellular stores. We report on the isolation of a human cDNA which encodes a mitochondrial protein called steroidogenic acute regulatory (StAR) protein, implicated in transport of cholesterol into mitochondria. Nucleotide and predicted amino acid sequence analyses indicate that the human and murine polypeptides are highly conserved, sharing 87% identity with an overall homology of 92%. Analysis of the distribution of StAR mRNA transcripts in human tissues by Northern blotting reveals several mRNA species, the most abundant of which is a 1.8 kb mRNA transcript present in testes, ovaries and kidneys. Using in vitro translated protein, we demonstrate that the StAR gene product can be efficiently imported into exogenously added mitochondria.
