The cap-binding protein complex in uninfected and poliovirus-infected HeLa cells

In poliovirus-infected HeLa cells, the mechanism of protein synthesis initiation factor recognition of m7G cap groups on mRNA is impaired. Translation of capped host cell mRNAs is inhibited, whereas translation of uncapped poliovirus mRNA proceeds exclusively. The site of this defect has been localized to the cap-binding protein complex (CBPC). To elucidate the specific structural and functional defects of the CBPC following poliovirus infection, the CBPC and/or its polypeptide components were purified from uninfected and poliovirus-infected HeLa cells. The CBPC from uninfected cells consisted of tightly associated 24- and 220-kDa polypeptides; minor amounts of polypeptides of 40, 44, and 80 kDa also consistently co-purified with the p24/p220 cores. No evidence of a 50-kDa, eIF-4A-related polypeptide subunit of the CBPC was obtained. The CBPC from poliovirus-infected cells had undergone major structural alterations. The 220-kDa component was absent; antigenically related (100-130 kDa) degradation products were present instead. The 24-kDa component co-purified with the p220 degradation products, but other components were missing. The association of the infected cell CBPC components was quite labile compared with that demonstrated by the components of CBPC from uninfected cells. Differential stimulation of capped, but not uncapped mRNAs in a cell-free translation assay was demonstrated by unmodified CBPC. Conversely, modified CBPC from poliovirus-infected cells differentially stimulated in vitro translation of uncapped poliovirus mRNA but not capped mRNAs. The implications of these results for the mechanism of cap-independent translation are briefly discussed.     

Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells

Extracts from poliovirus-infected HeLa cells were used to study ribosome binding of native and denatured reovirus mRNAs and translation of capped mRNAs with different degrees of secondary structure. Here, we demonstrate that ribosomes in extracts from poliovirus-infected cells could form initiation complexes with denatured reovirus mRNA, in contrast to their inability to bind native reovirus mRNA. Furthermore, the capped alfalfa mosaic virus 4 RNA, which is most probably devoid of stable secondary structure at its 5' end, could be translated at much higher efficiency than could other capped mRNAs in extracts from poliovirus-infected cells.     

mRNAs containing the unstructured 5' leader sequence of alfalfa mosaic virus RNA 4 translate inefficiently in lysates from poliovirus-infected HeLa cells.

Poliovirus infection is accompanied by translational control that precludes translation of 5'-capped mRNAs and facilitates translation of the uncapped poliovirus RNA by an internal initiation mechanism. Previous reports have suggested that the capped alfalfa mosaic virus coat protein mRNA (AIMV CP RNA), which contains an unstructured 5' leader sequence, is unusual in being functionally active in extracts prepared from poliovirus-infected HeLa cells (PI-extracts). To identify the cis-acting nucleotide elements permitting selective AIMV CP expression, we tested capped mRNAs containing structured or unstructured 5' leader sequences in addition to an mRNA containing the poliovirus internal ribosome entry site (IRES). Translations were performed with PI-extracts and extracts prepared from mock-infected HeLa cells (MI-extracts). A number of control criteria demonstrated that the HeLa cells were infected by poliovirus and that the extracts were translationally active. The data strongly indicate that translation of RNAs lacking an internal ribosome entry site, including AIMV CP RNA, was severely compromised in PI-extracts, and we find no evidence that the unstructured AIMV CP RNA 5' leader sequence acts in cis to bypass the poliovirus translational control. Nevertheless, cotranslation assays in the MI-extracts demonstrate that mRNAs containing the unstructured AIMV CP RNA 5' untranslated region have a competitive advantage over those containing the rabbit alpha-globin 5' leader. Previous reports of AIMV CP RNA translation in PI-extracts likely describe inefficient expression that can be explained by residual cap-dependent initiation events, where AIMV CP RNA translation is competitive because of a diminished quantitative requirement for initiation factors.     

Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5'' noncoding region.

Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.     

Presence of the cap-binding protein in initiation factor preparations from poliovirus-infected HeLa cells.

Crude preparations of initiation factors from mock-infected and poliovirus-infected HeLa cells were analyzed for the presence of proteins which could be cross-linked to the 5' cap group of mRNA. A protein having an apparent molecular weight of 26,000, similar to the cap-binding protein in rabbit reticulocytes described by Sonenberg and Shatkin (Proc. Natl. Acad. Sci. U.S.A. 75:4843-4847, 1978), was found in the ribosomal salt wash from both uninfected and infected cells. Cross-linking of this polypeptide was inhibited by the cap analog m7GMP. In addition, cross-linking of a protein having an approximate molecular weight of 60,000 was similarly inhibited by cap analog. The smaller cap-binding protein fractionated in a 0 to 40% ammonium sulfate precipitate of ribosomal salt wash; the larger protein was found in the 40 to 70% ammonium sulfate fraction. Although the cap-binding proteins were present in both mock-infected and poliovirus-infected ribosomal salt wash, only preparations from uninfected HeLa cells were able to restore translation of capped vesicular stomatitis virus mRNA by extracts prepared from poliovirus-infected cells.     

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.     

The ATP requirement for initiation of eukaryotic translation varies according to the mRNA species

The requirement for ATP for initiation of eukaryotic mRNA translation was tested using gel-filtered rabbit reticulocyte lysates incubated with labelled Met-tRNAfMet and exogenous RNA templates, and assaying the formation of labelled 80S initiation complexes in the presence of GTP, or labelled 40S initiation complexes in the presence of a non-hydrolysable analogue of GTP. Initiation complex formation on globin mRNA, or on capped viral RNAs such as papaya mosaic virus RNA and tobacco mosaic virus RNA, was strongly stimulated by ATP. In contrast, initiation complex formation on (uncapped) encephalomyocarditis virus RNA was uninfluenced by the presence or absence of ATP, which may be correlated with the recent evidence for scanning-independent internal initiation on this viral RNA. In addition, initiation complex formation on uncapped cowpea mosaic virus RNA and on poly(A,U,G) was only slightly stimulated by ATP, much less than in the case of the capped RNAs. These results suggest that most of the ATP hydrolysed during translation initiation is consumed in cap-dependent processes, probably in unwinding the mRNA, and relatively little in the actual migration or scanning of 40S subunits along the mRNA.     

Two forms of purified m7G-cap binding protein with different effects on capped mRNA translation in extracts of uninfected and poliovirus-infected HeLa cells

Eukaryotic mRNA cap binding proteins were purified from ribosomal salt wash in the presence of protease inhibitors by sucrose gradient sedimentation and m7GDP-Sepharose affinity chromatography. Rabbit reticulocyte and erythrocyte proteins with sedimentation constants of less than 6 S yielded a approximately 24,000-dalton cap binding protein. It stimulated capped mRNA translation in extracts of uninfected HeLa cells but did not restore capped mRNA function in extracts prepared from poliovirus-infected cells. Restoring and stimulatory activities both were associated with a larger, approximately 8-10 S complex that included the approximately 24,000-dalton polypeptide and several higher molecular mass components. The same two translational activities were also obtained in a slightly smaller approximately 5-7 S complex from uninfected HeLa cells but were absent from poliovirus-infected cell preparations.     

An efficient mammalian cell-free translation system supplemented with translation factors

Development of an efficient cell-free translation system from mammalian cells is an important goal. We examined whether supplementation of HeLa cell extracts with any translation initiation factor or translational regulator could enhance protein synthesis. eIF2 (eukaryotic translation initiation factor 2) and eIF2B augmented translation of capped, uncapped and encephalomyocarditis virus-internal ribosome entry site-promoted mRNAs. eIF4E specifically stimulated capped mRNA translation, while p97, a homologue to the C-terminal two-thirds of eIF4G, increased uncapped mRNA translation. When the HeLa cell extract was supplemented with a combination of eIF2, eIF2B, and p97, the capacity to synthesize a protein from an uncapped mRNA became comparable to that from the capped counterpart stimulated with a combination of eIF2, eIF2B, and eIF4E. A dialysis method rendered the HeLa cell extract capable of synthesizing proteins for 36h, and the yield was augmented when supplemented with initiation factors. In contrast, the productivity of a rabbit reticulocyte lysate was not enhanced by this method. Collectively, the translation factor-supplemented HeLa cell extract should become an important tool for the production of recombinant proteins.     

Translation of capped and uncapped vesicular stomatitis virus and reovirus mRNA'S. Sensitivity to m7GpppAm and ionic conditions.

In an attempt to elucidate the role of the 5'-terminal 7-methylguanosine residue in translation of mammalian mRNAs, vesicular stomatitis virus (VS virus), and reovirus mRNAs containing and lacking this residue, and also Qbeta RNA, were translated in cell-free extracts from reticulocytes and wheat germ under a variety of ionic conditions. Optimal translation of mRNAs lacking a 5' m7G occurred at concentrations of KOAc or KCl which were lower than those optimal for normal "capped" mRNAs. However, this salt dependence was much less marked in the mammalian reticulocyte extract and, at salt concentrations optimal for translation of normal capped mRNAs, reticulocyte lysates translated uncapped with mRNAs at 30 to 60% the normal efficiency. At low K+ concentrations, wheat germ ribosomes bound and translated appreciable amounts of uncapped VS virus mRNA; controls showed that no m7G residue is added to the 5' end of the bound RNA. Analogues of the 5' end, such as m7GpppAm, inhibited translation of both normal and uncapped VS virus RNAs in wheat germ extracts to about the same extent, but the efficiency of its action was reduced at low K+ concentrations. We conclude that there is a reduced importance of the 5' m7G residue in ribosome-mRNA recognition at low K+ concentrations, and that translation of mRNAs in reticulocyte extract is, under any reaction conditions, less dependent on the presence of a 5' "cap" than in wheat germ extracts.     

Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.     

Association of cap-binding protein with eucaryotic initiation factor 3 in initiation factor preparations from uninfected and poliovirus-infected HeLa cells.

Extracts from poliovirus-infected HeLa cells are unable to translate vesicular stomatitis virus or cellular mRNAs in vitro, probably reflecting the poliovirus-induced inhibition of host cell protein synthesis which occurs in vivo. Crude initiation factors from uninfected HeLa cells are able to restore translation of vesicular stomatitis virus mRNA in infected cell lysates. This restoring activity separates into the 0 to 40% ammonium sulfate fractional precipitate of ribosomal salt wash. Restoring activity is completely lacking in the analogous fractions prepared from poliovirus-infected cells. The 0 to 40% ammonium sulfate precipitates from both uninfected and infected cells contain eucaryotic initiation factor 3 (eIF-3), eIf-4B, and the cap-binding protein (CBP), which is detected by means of a cross-linking assay, as well as other proteins. The association of eIF-3 and cap binding protein was examined. The 0 to 40% ammonium sulfate precipitate of ribosomal salt wash from uninfected and infected cells was sedimented in sucrose gradients. Each fraction was examined for the presence of eIF-3 antigens by an antibody blot technique and for the presence of the CBP by cross-linking to cap-labeled mRNAs. From uninfected cells, a major proportion of the CBP cosedimented with eIF-3; however, none of the CBP from infected cells sedimented with eIF-3. The results suggest that the association of the CBP with eIF-3 into a functional complex may have been disrupted during the course of poliovirus infection.     

The adenovirus tripartite leader may eliminate the requirement for cap-binding protein complex during translation initiation.

The adenovirus tripartite leader is a 200-nucleotide 5' noncoding region that is found on all late viral mRNAs. This segment is required for preferential translation of viral mRNAs at late times during infection. Most tripartite leader-containing mRNAs appear to exhibit little if any requirement for intact cap-binding protein complex, a property previously established only for uncapped poliovirus mRNAs and capped mRNAs with minimal secondary structure. The tripartite leader also permits the translation of mRNAs in poliovirus-infected cells in the apparent absence of active cap-binding protein complex and does not require any adenovirus gene products for this activity. The preferential translation of viral late mRNAs may involve this unusual property.     

A viral sequence in the 3''-untranslated region mimics a 5'' cap in facilitating translation of uncapped mRNA.

For recognition by the translational machinery, most eukaryotic cellular mRNAs have a 5' cap structure [e.g. m7G(5')ppp(5')N]. We describe a translation enhancer sequence (3'TE) located in the 3'-untranslated region (UTR) of the genome of the PAV barley yellow dwarf virus (BYDV-PAV) which stimulates translation from uncapped mRNA by 30- to 100-fold in vitro and in vivo to a level equal to that of efficient capped mRNAs. A four base duplication within the 3'TE destroyed the stimulatory activity. Efficient translation was recovered by addition of a 5' cap to this mRNA. Translation of both uncapped mRNA containing the 3'TE in cis and capped mRNA lacking any BYDV-PAV sequence was inhibited specifically by added 3'TE RNA in trans. This inhibition was reversed by adding initiation factor 4F (eIF4F), suggesting that the 3'TE, like the 5' cap, mediates eIF4F-dependent translation initiation. The BYDV-PAV 5'UTR was necessary for the 3'TE to function, except when the 3'TE itself was moved to the 5'UTR. Thus, the 3'TE is sufficient for recruiting the translation factors and ribosomes, while the viral 5'UTR may serve only for the long distance 3'-5' communication. Models are proposed to explain this novel mechanism of cap-independent translation initiation facilitated by the 3'UTR.     

Potassium salts influence the fidelity of mRNA translation initiation in rabbit reticulocyte lysates: unique features of encephalomyocarditis virus RNA translation.

It is widely assumed that in vitro translation of mRNA is more efficient in the presence of potassium acetate rather than KCl, that the optimum concentration of potassium acetate is higher than for KCl, and that uncapped RNAs exhibit a lower optimum salt concentration than capped mRNAs. When these assumptions were examined using several different mRNA species in four batches of rabbit reticulocyte lysate, some notable exceptions were found. The translation of encephalomyocarditis virus (EMCV) RNA exhibited a salt optimum unusually high for an uncapped mRNA, and was very much more efficient and accurate with KCl rather than potassium acetate. It was also unique in being strongly activated by low concentrations (5-10 mM) KSCN in the presence of 90 mM potassium acetate. For the translation of other uncapped RNAs (poliovirus RNA, cowpea mosaic virus (CPMV) M RNA and bacteriophage MS2 RNA) amino acid incorporation at the optimum potassium acetate level was significantly greater than could be achieved using KCl. However, KCl was found to be restrictive and potassium acetate permissive for the synthesis of abnormal products thought to arise from initiation at incorrect sites, with the result that KCl gave a product pattern closer to that observed in vivo. In the particular case of the reticulocyte lysate system, accurate translation therefore requires the use of KCl rather than potassium acetate, but the choice of salt was found to be less critical in cell-free extracts from HeLa or L-cells.     

Inhibition of noncapped mRNA translation by the cap analogue, 7-methylguanosine-5'-phosphate.

The cap analogue, 7-methylguanosine-5′-phosphate (pm⁷G), inhibits the translation of the noncapped STNV (satellite tobacco necrosis virus) RNA and CPMV (cowpea mosaic virus) RNA in the $\text{in vitro}$ wheat germ protein synthesizing system. While the translation of some capped mRNAs is inhibited more strongly by the analogue, other capped mRNAs have a level of sensitivity similar to that of the noncapped RNAs. Evidence is presented demonstrating that the effect of the analogue is exerted at a cap binding site even when it is inhibiting noncapped mRNAs. These results therefore indicate that the cap binding site of the translational system is either part of or is closely linked to another mRNA binding component, this component being specific for a site on the mRNA other than the 5′ cap. The observations also suggest caution in the use of pm⁷G inhibition to indicate the presence of a 5′ cap on a particular mRNA.     

La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease.

Uncapped reovirus mRNA extracted at late times from infected L-cells is preferentially translated in extracts from infected L-cells. However, translation of this uncapped, late, reovirus mRNA in extracts from infected cells is sensitive to inhibition by the cap analog m7GTP . These results imply that reovirus infection does not induce a transition from cap-dependent to cap-independent translation. Nevertheless, the results of in vitro translational competition experiments between L-cell mRNA and late viral mRNA were consistent with the view that reovirus does induce an alteration in the cap-dependent translational apparatus of L-cells. The reduced efficiency of translation of a variety of capped mRNAs in extracts from infected cells is also consistent with this notion. We further conclude that a factor exists in reovirus-infected L-cells that specifically stimulates translation of uncapped reovirus mRNAs.