Structure containing cellular mRNA in picornavirus infections

The fate of cellular mRNA upon infection of Krebs-2 ascites carcinoma cells with encephalomyocarditis (EMC) virus was investigated. The cell mRNA was discovered in a structure with a sedimentation coefficient of about 100S and a buoyant density of 1.50--1.519 g/cm3 during active virus-specific synthesis (3.0--4.0 hr post infection). The template activity of the 100S structure in a cell-free protein-synthesizing system and of mRNA isolated from it was studied and the nature of synthesized products was analyzed. It was shown that the 100S structure seems to be translationally inactive. On the contrary, the RNA isolated from its is functionally active.     

4,5-S-RNAI in virus-specific polyribosomes from ascitic Krebs 2 carcinoma cells infected with encephalomyocarditis virus

Polyribosomes of Krebs 2 ascite carcinoma cells non-infected and infected with encephalomyocarditis (EMC) virus contain a heterogeneous population of low molecular weight small RNAs. Analysis of the RNAs by polyacrylamide gel electrophoresis did not reveal any qualitative differences in the small RNA sets within the composition of polyribosomes from virus-infected and non-infected cells. However, the content of one of the small RNAs was markedly elevated in polyribosomes from virus-infected cells. As can be followed from partial determination of its primary structure, this small mRNA is identical to 4,5S-RNAI previously detected in the nuclei of Novikov hepatoma cells of the rat. The data obtained suggest that 4,5S-RNAI can be involved in the regulation of protein synthesis in virus-infected cells.     

Ability of translation factors from infected encephalomyocarditis viruses of ascites carcinoma cells to maintain the protein-synthetizing activity of cellular polyribosomes

A highly effective protein-synthesizing system fully dependent on translation factors was isolated from the ascite Krebs-2 carcinoma. The effect of EMC-infection on the ability of these factors to maintain the translation of polyribosomes containing cell mRNA was studied. It was shown that the activity of translation factors of infected cells does not differ from that of non-infected cells in terms of their ability to maintain the protein-synthesizing activity of cell polyribosomes in vitro.     

Initiation of endogenous messenger RNA translation on hen oviduct polysomes.

The eIF-2A fraction of reticulocyte ribosomal salt wash is capable of maximally stimulating the translation of endogenous messenger RNA by hen oviduct polysomes. The factor increases the initiation of protein synthesis 2--3-fold when measured by the factor-dependent synthesis of NH2-terminal peptides. The addition to these polysomes of elongation factor, EF-1, also increases protein synthesis but at a distinctly different rate and Mg2+ concentration optimum than the eIF-2A fraction. Moreover, there is no stimulation of NH2-terminal peptide synthesis with EF-1 alone. In contrast, all the known initiation factors are required for the translation of exogenous globulin mRNA on oviduct polysomes. Reticulocyte polysomes isolated by an identical procedure to that used for oviduct polysomes or by standard methods also require all the initiation factors for the translation of either endogenous mRNA or exogenous ovalbumin mRNA. Addition of 7-methylguanosine 5'-monophosphate does not inhibit the factor-dependent stimulation of oviduct polysomes except at high concentrations (1.0 mM) indicating that the sites with which 7-methylguanosine 5'-monophosphate normally competes are already occupied. These findings suggest that the messenger RNA remains bound to the oviduct polysomes or initiation factors. Hence the addition of exogenous factors which are involved with mRNA recognition and binding to the ribosome are not required. It has been previously shown that eIF-2A is capable of binding in vitro the initiatior tRNA to an existing Ado-Urd-Gua-40 S complex and initiating protein synthesis when such a complex is present. These present studies indicate that such an initiation complex may exist within the oviduct cell on membrane-associated polysomes. Under these circumstances eIF-2A mediates binding of the initiator tRNA and initiates protein synthesis.     

Translation of Sindbis virus 26S mRNA does not require intact eukariotic initiation factor 4G

The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.     

Translational control of protein synthesis after infection by vesicular stomatitis virus.

Four hours after infection of BHK cells by vesicular stomatitis virus (VSV), the rate of total protein synthesis was about 65% that of uninfected cells and synthesis of the 12 to 15 predominant cellular polypeptides was reduced to a level about 25% that of control cells. As determined by in vitro translation of isolated RNA and both one- and two-dimensional gel analyses of the products, all predominant cellular mRNA's remained intact and translatable after infection. The total amount of translatable mRNA per cell increased about threefold after infection; this additional mRNA directed synthesis of the five VSV structural proteins. To determine the subcellular localization of cellular and viral mRNA before and after infection, RNA from various sizes of polysomes and nonpolysomal ribonucleoproteins (RNPs) was isolated from infected and noninfected cells and translated in vitro. Over 80% of most predominant species of cellular mRNA was bound to polysomes in control cells, and over 60% was bound in infected cells. Only 2 of the 12 predominant species of translatable cellular mRNA's were localized to the RNP fraction, both in infected and in uninfected cells. The average size of polysomes translating individual cellular mRNA's was reduced about two- to threefold after infection. For example, in uninfected cells, actin (molecular weight 42,000) mRNA was found predominantly on polysomes with 12 ribosomes; after infection it was found on polysomes with five ribosomes, the same size of polysomes that were translating VSV N (molecular weight 52,000) and M (molecular weight 35,000) mRNA. We conclude that the inhibition of cellular protein synthesis after VSV infection is due, in large measure, to competition for ribosomes by a large excess of viral mRNA. The efficiency of initiation of translation on cellular and viral mRNA's is about the same in infected cells; cellular ribosomes are simply distributed among more mRNA's than are present in growing cells. About 20 to 30% of each of the predominant cellular and viral mRNA's were present in RNP particles in infected cells and were presumably inactive in protein synthesis. There was no preferential sequestration of cellular or viral mRNA's in RNPs after infection.     

Interrelationship between protein synthesis and mRNA metabolism in rat liver cells]

It is demonstrated that RNA isolated from polyribosomes and postmitochondrial fraction of rat liver cells and bound to nitrocellulose filters (Milliport) represent mRNA. RNA taken from the nitrocellulose filters sedimented in sucrose concentration gradient with a wide peak within the range of 18--6S, attaining a maximum at 12S. The (A+U)/(G+C) ratio of this RNA was equal to 1.04. On the other hand, the same ratio for rRNA was 0.64. Specific radioactivity of polysomal mRNA containing poly-A sequences, was significantly lower at 14-hour labelling with 14C-orotate than at 4-hour labelling (control). Inhibitors (cycloheximide, puromycin, ethionine, actinomycin D) stabilized polysomal mRNA. Specific radioactivity of postmitochondrial fraction mRNA was higher at 14-hour labelling than at 4-hour labelling. Specific radioactivity of postmitochondrial fraction mRNA during protein synthesis blocking by different inhibitors was comparable to those of control animals. It is hypothesized that active translation is necessary for the initiation of rat liver mRNA degradation.     

Poliovirus translation: a paradigm for a novel initiation mechanism

All eukaryotic cellular mRNAs, and most viral mRNAs, are blocked at their 5' ends with a cap structure (m7GpppX, where X is any nucleotide). Poliovirus, along with a small number of other animal and plant viral mRNAs, does not contain a 5' cap structure. Since the cap structure functions to facilitate ribosome binding to mRNA, translation of polio-virus must proceed by a cap-independent mechanism. Consistent with this, recent studies have shown that ribosomes can bind to an internal region within the long 5' noncoding sequence of poliovirus RNA. Possible mechanisms for cap-independent translation are discussed. Cap-independent translation of poliovirus RNA is of major importance to the mechanism of shut-off of host protein synthesis after infection. Moreover, it is likely to play a role in determining poliovirus neurovirulence and attenuation.     

Structural basis for competitive inhibition of eIF4G-Mnk1 interaction by the adenovirus 100-kilodalton protein

Translation of most cellular mRNAs involves cap binding by the translation initiation complex. Among this complex of proteins are cap-binding protein eIF4E and the eIF4E kinase Mnk1. Cap-dependent mRNA translation generally correlates with Mnk1 phosphorylation of eIF4E when both are bound to eIF4G. During the late phase of adenovirus (Ad) infection translation of cellular mRNA is inhibited, which correlates with displacement of Mnk1 from eIF4G by the viral 100-kDa (100K) protein and dephosphorylation of eIF4E. Here we describe the molecular mechanism for 100K protein displacement of Mnk1 from eIF4G and elucidate a structural basis for eIF4G interaction with Mnk1 and 100K proteins and Ad inhibition of cellular protein synthesis. The eIF4G-binding site is located in an N-terminal 66-amino-acid peptide of 100K which is sufficient to bind eIF4G, displace Mnk1, block eIF4E phosphorylation, and inhibit eIF4F (cap)-dependent cellular mRNA translation. Ad 100K and Mnk1 proteins possess a common eIF4G-binding motif, but 100K protein binds more strongly to eIF4G than does Mnk1. Unlike Mnk1, for which binding to eIF4G is RNA dependent, competitive binding by 100K protein is RNA independent. These data support a model whereby 100K protein blocks cellular protein synthesis by coopting eIF4G and cap-initiation complexes regardless of their association with mRNA and displacing or blocking binding by Mnk1, which occurs only on preassembled complexes, resulting in dephosphorylation of eIF4E.     

The simulation of thymidine kinase enzyme kinetics in cultured cells using the computer language cellsim

Thymidine kinase is an enzyme that occurs in cells actively synthesizing DNA. In studies of synchronized cell populations, it has been shown that the enzyme activity disappears during the G1 phase of the cell cycle and reappears during the S and G2 phases. Its reappearance is consistent with the synthesis of the mRNA for this enzyme during the S and G2 phases and its immediate translation into active enzyme by the protein synthesis machinery within the cell. The disappearance of the enzyme is consistent with the cessation of mRNA synthesis by mitotic cells. We have now tested this concept by computer simulation of a growing cell population in which a specific mRNA is generated while cells are in the S and G2 phases of the cell cycle. The computer simulation was done using the simulation language Cellsim designed for modeling populations of cells. The Cellsim program which we developed allowed each cell to make about 1 mRNA molecule per min during the S and G2 phases. Every 3 min each mRNA molecule generated a protein enzyme molecule. The mRNA had a half-life of about 9 min, and the enzyme had a half-life of about 150 min. When these molecular parameters were coupled to the cell cycle parameters for Chinese hamster fibroblasts, the resulting curve of enzyme production with time closely matched the observed kinetics of enzyme activity seen in synchronized cells. The only part of the curve that did not fit was the rapid drop in enzyme activity which was seen as the population of mitotic cells was permitted to enter G1. This drop in activity was not seen in mitotic cells blocked with Colcemid where mRNA synthesis must be lacking. Earlier studies have shown that the Gl cells do not contain any inhibitor of enzyme activity. It therefore appears that the enzyme molecule is more unstable during the G1 phase than in any of the other phases of the cell cycle.     

Purification of histone messenger ribonucleoprotein particles from HeLa cell S-phase polysomes. Characterization of associated proteins

We have purified HeLa histone mRNA from polysomes of S-phase cells which had been synchronized by hydroxyurea treatment. This mRNA was shown to direct the in vitro synthesis of all five histones which amount to at least 90-95% of its total translational activity. Polysomal histone mRNP was also purified and identified by cell-free translation and hybridization to a clone of histone DNA from E. esculentus. The protein moiety of this mRNP contained three prominent species of molecular weight 86,000, 73,000 and 53,000 daltons. The presence of the 73,000 species previously assessed to be bound to poly(A) is discussed in view of the fact that histone mRNA does not contain a pail. As globin mRNA, histone mRNA as well as histone mRNP were translated with equal efficiency in cell-free extracts from either S-phase or hydroxyurea blocked HeLa cells.     

Effect of high salt treatment on influenza B viral protein synthesis in MDCK cells

Based on the information that high salt inhibits the initiation of cellular mRNA translation which depends on the function of the 5'-terminal structure of mRNA, we compared the effect of high salt on translation of host cellular mRNAs and influenza viral mRNAs, both of which are of 5'-terminal structure. Brief exposure of influenza B virus-infected MDCK cells to high salt medium resulted in a dose-dependent inhibition of viral polypeptide synthesis as well as of cellular polypeptide synthesis, but it had less effect on synthesis of viral polypeptides, particularly nonstructural protein (NS). Under these conditions the Na+ content of the infected cells was significantly increased. A similar salt effect on in vitro translation of viral and cellular mRNAs extracted from infected cells was also observed. There was no significant difference in sensitivity to hypertonic block of in vivo translation of influenza viral mRNAs and vesicular stomatitis virus mRNAs, the latter of which possess a virus-directed structure at the 5'-terminus.     

The complete structure of the chloroplast 70S ribosome in complex with translation factor pY

Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo‐EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid‐specific ribosomal proteins. These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid‐specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light‐ and temperature‐dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A‐site and P‐site, where it protects the decoding centre and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non‐rotated state, in which the intersubunit bridges to the large subunit are stabilized.     

Rotavirus Nonstructural Protein NSP3 is not required for viral protein synthesis

Initiation is the rate-limiting step in protein synthesis and therefore an important target for regulation. For the initiation of translation of most cellular mRNAs, the cap structure at the 5' end is bound by the translation factor eukaryotic initiation factor 4E (eIF4E), while the poly(A) tail, at the 3' end, is recognized by the poly(A)-binding protein (PABP). eIF4G is a scaffold protein that brings together eIF4E and PABP, causing the circularization of the mRNA that is thought to be important for an efficient initiation of translation. Early in infection, rotaviruses take over the host translation machinery, causing a severe shutoff of cell protein synthesis. Rotavirus mRNAs lack a poly(A) tail but have instead a consensus sequence at their 3' ends that is bound by the viral nonstructural protein NSP3, which also interacts with eIF4GI, using the same region employed by PABP. It is widely believed that these interactions lead to the translation of rotaviral mRNAs, impairing at the same time the translation of cellular mRNAs. In this work, the expression of NSP3 in infected cells was knocked down using RNA interference. Unexpectedly, under these conditions the synthesis of viral proteins was not decreased, while the cellular protein synthesis was restored. Also, the yield of viral progeny increased, which correlated with an increased synthesis of viral RNA. Silencing the expression of eIF4GI further confirmed that the interaction between eIF4GI and NSP3 is not required for viral protein synthesis. These results indicate that NSP3 is neither required for the translation of viral mRNAs nor essential for virus replication in cell culture.     

Inhibition of viral mRNA translation in interferon-treated L cells infected with reovirus.

Murine L cells were treated with interferon (IFN) concentrations which reduced by 75 to 80% the synthesis of viral mRNA after infection with reovirus. Protein synthesis was not inhibited in these cells up to 6 h after infection, but a large fraction of the viral mRNA was not associated with polyribosomes and sedimented at about 50S. In contrast, most of the reovirus mRNA was associated with polyribosomes in control infected cells. This mRNA was of similar size to non-polyribosomal mRNA from IFN-treated cells when analyzed by Northern blot hybridization with a cloned cDNA for the s2 reovirus mRNA, indicating that the non-polyribosomal mRNA was not appreciably degraded. Viral mRNA was labeled with [3H]uridine and the non-polyribosomal mRNA was isolated from IFN-treated cells. This mRNA could quantitatively bind to 80S initiation complexes when incubated in a rabbit reticulocyte cell-free system. These findings indicated that the non-polyribosomal RNA was translatable, but that its binding to functional initiation complexes was inhibited in IFN-treated cells by a discriminatory mechanism, which did not affect translation of cellular mRNA. Previous experiments showed that mRNA is blocked in 48S complexes when the alpha subunit of initiation factor eIF-2 is phosphorylated by the double-stranded RNA-dependent protein kinase induced by IFN. A localized activation of this kinase could explain the block of viral mRNA in 48S complexes. By labeling the phosphoproteins of IFN-treated cells with 32P, eIF-2 (alpha P) was shown to cosediment with non-polyribosomal mRNA, presumably in 48S complexes.     

On the association of mRNA with the cytoskeleton in uninfected and adenovirus-infected human KB cells

Triton-insoluble cytoskeletons were prepared from uninfected and adenovirus-infected KB cells. Gradient analysis showed that all cellular polyribosomes were present in the cytoskeletons. After disaggregation of the polyribosomes, in vivo or in vitro, most of the messenger RNA (mRNA) remained associated with the cytoskeletal framework. Translation experiments showed that most mRNA species were present in a bound (cytoskeletal), as well as in an unbound state. However, whereas some mRNA species were predominant as unbound mRNP particles, other mRNA species were almost exclusively found in polyribosomes associated with the cytoskeletal framework. Incubation of cytoskeletons in an mRNA-dependent reticulocyte cell-free system revealed synthesis of the same set of polypeptides as took place when using whole cells. Furthermore, the gradual shift from translation of cellular to translation of viral mRNA species during late phase of productive infection with adenovirus could also be followed when cytoskeletons were translated in the cell-free system. These results support the hypothesis that Triton X-100 extraction does not remove actively translating mRNA from the cells, thus suggesting a functional relationship between mRNA translation and mRNA binding to a cytoskeletal framework.     

Hepatitis C virus internal ribosome entry site-dependent translation in Saccharomyces cerevisiae is independent of polypyrimidine tract-binding protein, poly(rC)-binding protein 2, and La protein

Translation initiation of some viral and cellular mRNAs occurs by ribosome binding to an internal ribosome entry site (IRES). Internal initiation mediated by the hepatitis C virus (HCV) IRES in Saccharomyces cerevisiae was shown by translation of the second open reading frame in a bicistronic mRNA. Introduction of a single base change in the HCV IRES, known to abrogate internal initiation in mammalian cells, abolished translation of the second open reading frame. Internal initiation mediated by the HCV IRES was independent of the nonsense-mediated decay pathway and the cap binding protein eIF4E, indicating that translation is not a result of mRNA degradation or 5'-end-dependent initiation. Human La protein binds the HCV IRES and is required for efficient internal initiation. Disruption of the S. cerevisiae genes that encode La protein orthologs and synthesis of wild-type human La protein in yeast had no effect on HCV IRES-dependent translation. Polypyrimidine tract-binding protein (Ptb) and poly-(rC)-binding protein 2 (Pcbp2), which may be required for HCV IRES-dependent initiation in mammalian cells, are not encoded within the S. cerevisiae genome. HCV IRES-dependent translation in S. cerevisiae was independent of human Pcbp2 protein and stimulated by the presence of human Ptb protein. These findings demonstrate that the genome of S. cerevisiae encodes all proteins necessary for internal initiation of translation mediated by the HCV IRES.