Cap-binding Protein Complex Research Articles

Isolation and structural characterization of cap-binding proteins from poliovirus-infected HeLa cells.

In poliovirus-infected HeLa cells, poliovirus RNA is translated at times when cellular mRNA translation is strongly inhibited. It is thought that this translational control mechanism is mediated by inactivation of a cap-binding protein complex (comprising polypeptides of 24 [24-kilodalton cap-binding protein], 50, and approximately 220 kilodaltons). This complex can restore the translation of capped mRNAs in extracts from poliovirus-infected cells. We have previously shown that the virally induced defect prevents interaction between cap recognition factors and mRNA. Here, we show that the cap-binding protein complex (and not the 24-kilodalton cap-binding protein) has activity that restores the cap-specific mRNA-protein interaction when added to initiation factors from poliovirus-infected cells. Thus, the activity that restores the cap-specific mRNA-protein interaction and that which restores the translation of capped mRNAs in extracts from poliovirus-infected cells, copurify. The results also indicate, by an alternative assay, that the cap-binding protein complex is the only factor inactivated by poliovirus. We also purified cap-binding proteins from uninfected and poliovirus-infected HeLa cells. By various criteria, the 24-kilodalton cap-binding protein is not structurally modified as a result of infection. However, the 220-kilodalton polypeptide of the cap-binding protein complex is apparently cleaved by a putative viral (or induced) protease. By in vivo labeling and m7GDP affinity chromatography, we isolated a modified cap-binding protein complex from poliovirus-infected cells, containing proteolytic cleavage fragments of the 220-kilodalton polypeptide.

Poliovirus protease does not mediate cleavage of the 220,000-Da component of the cap binding protein complex.

Poliovirus infection of HeLa cells results in a rapid shutoff of host protein synthesis but does not inhibit the translation of poliovirus mRNA. It has been suggested that this virus-induced translational control is mediated by the inactivation of a cap binding protein (CBP) complex, and it has been shown that the 220,000-Da component(s) (p220) of the CBP complex is cleaved in infected HeLa cells to form antigenically related peptides of 100,000-130,000 Da. To determine whether the known viral protease (peptide 3C) was the mediator of the cleavage of p220, we used immunoblot techniques to analyze partially purified infected HeLa cell extracts for cleavage activity. We report here that p220 cleavage activity does not copurify with viral peptide 3C or with any precursors containing 3C sequences. We also show that cleavage of p220 can be demonstrated in vitro in HeLa cell extracts under conditions where the functional activity of the poliovirus protease is inhibited by specific antibody.

Human rhinovirus 14 infection of HeLa cells results in the proteolytic cleavage of the p220 cap-binding complex subunit and inactivates globin mRNA translation in vitro.

One of the characteristics of picornavirus infection of cells in tissue culture is a specific inhibition of utilization of host cell mRNA for protein synthesis. In this study we show that human rhinovirus 14 is similar to poliovirus in that the inhibition of host cell translation that occurs during infection correlates with the proteolytic cleavage of an Mr 220,000 subunit of the cap-binding protein complex.

Preferential stimulation of rabbit α globin mRNA translation by a cap-binding protein complex

A cap-binding protein complex (Edery et al. (1983) J. Biol. Chem. 258, 11398–11403) is shown here to stimulate preferentially the translation of endogenous α versus β globin mRNA in a rabbit reticulocyte lysate. Several initiation factors (eIF-2, eIF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4E and eIF-5) and elongation factor 1 were found to have no such discriminatory effect. These results are in contrast to several previous reports and demonstrate that the only factor capable of relieving translational competition between α and β globin mRNAs is the cap-binding protein complex.

Demonstration in vitro that eucaryotic initiation factor 3 is active but that a cap-binding protein complex is inactive in poliovirus-infected HeLa cells.

Protein synthesis initiation factor preparations from poliovirus-infected HeLa cells have reduced ability to initiate translation on capped mRNA. The defect in initiation factors has been variously attributed to inactivation of eucaryotic initiation factor 3 (eIF3), eIF4B, or a cap-binding protein (CBP) complex. We have developed a series of in vitro protein synthesis assays to show that eIF3 is active but a CBP complex activity is inactivated after poliovirus infection. eIF3 activity, when determined in the presence of purified CBP complex, is present in sucrose gradients of factors from both infected and uninfected cells. CBP complex activity, determined in the presence of eIF3 from poliovirus-infected cells, is present in uninfected cells only and comigrates on sucrose gradient with an activity which restores the ability of crude initiation factors from infected cells to translate capped globin mRNA. This is the first demonstration by a fractionated translation system that an activity which is attributable to CBP complex is inactivated in poliovirus-infected cells. The results also indicate that eIF3 is undetectable or has greatly reduced activity in the absence of CBP complex.

Functional characterization of eukaryotic mRNA cap binding protein complex: effects on translation of capped and naturally uncapped RNAs.

We examined the effects of a eukaryotic mRNA cap binding protein (CBP) complex purified by cap analogue affinity chromatography [Edery, I., Humebelin, M., Darveau, A., Lee, K.A. W., Milburn, S., Hershey, J.W.B., Trachsel, H., & Sonenberg, N. (1983) J. Biol. Chem. 258, 11398 11403], on translation of several capped and naturally uncapped mRNAs in extracts prepared from poliovirus-infected or mock-infected HeLa cells. The CBP complex has activity that restores capped mRNA (globin, tobacco mosaic virus, and others) function in extracts from poliovirus-infected HeLa cells. Translation of two naturally uncapped RNAs (poliovirus and mengovirus RNAs), the translation of which is not restricted in extracts from poliovirus-infected cells, is also not stimulated by the CBP complex. Translation of several capped eukaryotic mRNAs (vesicular stomatitis virus, reovirus, and tobacco mosaic virus) in extracts from mock-infected cells is inhibited when the potassium ion concentration is increased. However, translation of capped AMV-4 RNA, which has negligible secondary structure at its 5' end, is resistant to this inhibition. Furthermore, the CBP complex reverses the high salt induced inhibition of translation of the former mRNAs. Since mRNA secondary structure is more stable at elevated salt concentrations, these data are consistent with a model in which the CBP complex has a role in melting mRNA secondary structure involving 5'-proximal sequences, to facilitate ribosome binding.

Involvement of eukaryotic initiation factor 4A in the cap recognition process.

Antibodies against eukaryotic initiation factor 4A (eIF-4A) were used to study the involvement of this factor in recognizing the 5' cap structure of eukaryotic mRNA. We demonstrate that an approximately 50-kilodalton polypeptide present in rabbit reticulocyte ribosomal high salt wash which can be specifically cross-linked to the 5' oxidized cap structure of reovirus mRNA (Sonenberg, N. (1981) Nucleic Acids Res. 9, 1643) reacts with an anti-eIF-4A monoclonal antibody. We also show that antibodies against eIF-4A react with a 50-kilodalton polypeptide present in a cap-binding protein complex obtained by elution from a m7GTP-agarose affinity column. Comparative peptide analysis of eIF-4A and the 50-kilodalton component of the cap-binding protein complex indicates a very strong similarity between the two polypeptides.

Role of mRNA competition in regulating translation: further characterization of mRNA discriminatory initiation factors.

Host and reovirus mRNAs compete with one another for translation in infected cells. Kinetic analysis has suggested that the site of competition is a message discriminatory initiation factor which must bind to the mRNA before it can interact with the 40S ribosomal subunit. The present communication describes an in vitro assay which can detect message discriminatory activities. A competitive situation is established by using reovirus and globin mRNAs, and then the specificity with which this competition is relieved by added components is measured. Among the various initiation factors surveyed with this assay, two have the properties expected of the mRNA discriminatory factor. These are eukaryotic initiation factor 4A and a "cap binding protein" complex. Inasmuch as the cap binding protein complex contains a subunit similar or identical to the initiation factor eIF-4A, it seems likely that only one form of the latter factor may be active in vivo. In vitro, both factors relieve competition among both capped and uncapped reovirus mRNAs according to similar hierarchies. These results suggest that some feature other than the m7G cap, such as nucleotide sequence or secondary structure, is recognized by the discriminatory factor.

Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex.

Following poliovirus infection of HeLa cells, the synthesis of cellular proteins is inhibited but translation of poliovirus mRNA proceeds. The defect in the recognition of host cell mRNA may be due to a change in a cap recognition complex which, when added to an infected cell lysate, restores the ability to translate capped mRNAs. We employed immunoblotting techniques to examine initiation factors in crude lysates from uninfected and poliovirus-infected HeLa cells. Using an antiserum against eucaryotic initiation factor 3, we detected an antigen of approximate molecular weight 220,000 in uninfected cell lysates but not in infected cell lysates. Antigenically related polypeptides of 100,000 to 130,000 daltons, presumably degradation products, were detected in the infected cell lysate. The time course for degradation of the 220,000-dalton polypeptide correlates with that for inhibition of cellular protein synthesis in vivo. A portion of the population of 220,000-dalton polypeptides apparently associates with initiation factor eIF3 but is readily dissociated in buffers containing high salt. Affinity-purified antibodies against the polypeptide recognize a protein of the same size in a purified preparation of a cap binding protein complex obtained by cap-affinity chromatography. We postulate that the 220,000-dalton polypeptide is an essential component of the cap recognition complex and that its degradation in poliovirus-infected cells results in the inhibition of host cell translation. These results are in the first demonstration of a specific structural defect in an initiation factor resulting from poliovirus infection.