Translation Of Encephalomyocarditis Virus RNA Research Articles

Translation of Viral mRNA without Active eIF2: The Case of Picornaviruses

Previous work by several laboratories has established that translation of picornavirus RNA requires active eIF2α for translation in cell free systems or after transfection in culture cells. Strikingly, we have found that encephalomyocarditis virus protein synthesis at late infection times is resistant to inhibitors that induce the phosphorylation of eIF2α whereas translation of encephalomyocarditis virus early during infection is blocked upon inactivation of eIF2α by phosphorylation induced by arsenite. The presence of this compound during the first hour of infection leads to a delay in the appearance of late protein synthesis in encephalomyocarditis virus-infected cells. Depletion of eIF2α also provokes a delay in the kinetics of encephalomyocarditis virus protein synthesis, whereas at late times the levels of viral translation are similar in control or eIF2α-depleted HeLa cells. Immunofluorescence analysis reveals that eIF2α, contrary to eIF4GI, does not colocalize with ribosomes or with encephalomyocarditis virus 3D polymerase. Taken together, these findings support the novel idea that eIF2 is not involved in the translation of encephalomyocarditis virus RNA during late infection. Moreover, other picornaviruses such as foot-and-mouth disease virus, mengovirus and poliovirus do not require active eIF2α when maximal viral translation is taking place. Therefore, translation of picornavirus RNA may exhibit a dual mechanism as regards the participation of eIF2. This factor would be necessary to translate the input genomic RNA, but after viral RNA replication, the mechanism of viral RNA translation switches to one independent of eIF2.

Pyrimidine tract binding protein strongly stimulates in vitro encephalomyocarditis virus RNA translation at the level of preinitiation complex formation

Cellular protein p57/58, now known to be identical to polypyrimidine tract binding protein (PTB), has earlier been shown to specifically bind to the internal ribosome entry sites (IRES) of encephalomyocarditis virus (EMCV) and some other picornaviral RNAs. To elucidate its relevance to the internal initiation, the effect of cloned purified PTB on EMCV IRES directed translation was studied in cytoplasmic extracts of Krebs-2 ascites carcinoma cells partially depleted of endogenous PTB. Addition of PTB to such extracts resulted in a strong stimulation of translation of a β-glucuronidase (GUS) reporter cistron fused to the EMCV IRES, but had no effect on translation of capped mRNAs, such as β-globin, and tobacco mosaic virus (TMV) RNAs. PTB was found to exert its effect at the level of 48S pre-initiation complex formation.

Internal translation initiation on poliovirus RNA: further characterization of La function in poliovirus translation in vitro.

Initiation of poliovirus RNA translation by internal entry of ribosomes is believed to require the participation of trans-acting factors. The mechanism of action of these factors is poorly defined. The limiting amount of one of these factors, La protein, in rabbit reticulocyte lysates (RRL) has been postulated to partially explain the inefficient translation of poliovirus RNA in this system. To further characterize La activity in translation and to identify other potential limiting factors, we assayed the ability of La protein as well as purified initiation factors, eIF-2, guanine nucleotide exchange factor (GEF), eIF-4A, eIF-4B, eIF-4F, and eIF-3, to stimulate the synthesis of P1, the capsid precursor protein, in poliovirus type 1 (Mahoney) RNA-programmed RRL. Of the proteins tested, only La, GEF, and to some extent eIF-2 stimulated the synthesis of P1. The enhanced translation of P1 in response to La occurred concomitantly with the inhibition of synthesis of most aberrant polypeptides, resulting from initiation in the middle of the genome. Deletion of the carboxy-terminal half (214 amino acids) of La did not decrease its binding to the poliovirus 5' untranslated region but abrogated the stimulatory and correcting activity in translation. In contrast to La, GEF and eIF-2 stimulated the overall translation and increased the synthesis of aberrant products as well as P1. Neither La, GEF, nor any other factor stimulated translation of encephalomyocarditis virus RNA in RRL. The implications of these findings for the mechanism of internal translation initiation on picornavirus RNAs are discussed.

Dependence of the adenovirus tripartite leader on the p220 subunit of eukaryotic initiation factor 4F during in vitro translation. Effect of p220 cleavage by foot-and-mouth-disease-virus L-protease on in vitro translation.

The adenovirus tripartite leader (TPT) 5' untranslated region (5'UTR) allows translation in poliovirus-infected cells, in which the p220 subunit of eukaryotic initiation factor 4F is degraded. This p220-independent translation was investigated by measuring in vitro translation in a reticulocyte lysate of a reporter gene, chloramphenicol acetyltransferase, coupled to the TPT 5'UTR. The p220 subunit was degraded by translation of a foot-and-mouth-disease L-protease construct. Surprisingly, the TPT 5'UTR was dependent on intact p220, as are other naturally capped mRNA species. Translation of encephalomyocarditis virus RNA was p220 independent, as expected from its ability to support internal, cap-independent initiation. In vitro protein-synthesis experiments with purified initiation factors confirmed the dependence of TPT mRNA translation on eukaryotic initiation factor 4F. The relationship between adenovirus TPT-5'UTR-directed translation and poliovirus-induced host cell shut-off is discussed.

Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation.

The nucleotide sequence of the 5' nontranslated region of encephalomyocarditis virus (EMCV-Rueckert) was determined, and a consensus RNA structural model for this sequence (850 bases) and three other poly(C)-containing cardioviruses (mengovirus, EMCV-B, and EMCV-D) was created through reiterative use of a minimum-free-energy folding algorithm. The RNA elements within this region which contribute to translation of EMCV proteins were mapped in cell-free reactions programmed with cDNA-derived RNA transcripts. The data provide evidence that stem-loop motifs I, J and K, formed by viral bases 451 to 785, are important components of cap-independent translation. In contrast to other reports, a minimal role for stem-loop H (bases 406 to 444) in translational activity is indicated. Small 5' nontranslated region fragments (bases 667 to 797) containing the J and K motifs proved strong competitive inhibitors when added to cell-free reactions programmed with exogenous capped or uncapped mRNAs. The putative sequestering of required translational factors by this segment clearly contributes to translational activity, but also suggests a possible competitive mechanism for the down regulation of host protein synthesis during viral infection.

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 KC1, that the optimum concentration of potassium acetate is higher than for KC1, 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 exception 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 KC1 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 KC1. However, KC1 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 KC1 gave a product pattern closer to that observed in vivo. In particular case of the reticulocyte lysate system, accurate translation therefore requires the use of the KC1 rather than potassium acetate, but the choice of salt was found to be less critical in cell-free extracts from HeLa or L-cells.

A Complex RNA sequence determines the initiation of encephalomyocarditis virus RNA translation

A Complex RNA sequence determines the initiation of encephalomyocarditis virus RNA translation

A complex RNA sequence detemines the internal initiation of encephalomyocarditis virus RNA translation

Translation initiation on EMCV RNA occurs via binding of ribosomes to an internal sequence within the 5' noncoding region. To investigate the organization of the internal ribosome entry site (IRES) we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of EMCV RNA. Three functional regions have been distinguished: a sequence between nts 315-484 and the upper parts of the double-helical structural domains III (nts 488-647) and IV (nts 701-763). The first one greatly enhances translation, but is not absolutely necessary for internal initiation. The other two regions are indispensable to this process. A sequence within domain IV determines inhibition of in vitro translation of mRNAs with 5'-terminal dependent initiation. It is proposed to interact with a translational factor(s) common to the internal and 5'-terminal dependent initiation.

Initiation of encephalomyocarditis virus RNA translation: the authentic initiation site is not selected by a scanning mechanism.

The authentic initiation site on encephalomyocarditis virus (strain R) RNA is the 11th AUG codon (at nt. 834) from the 5' end of the viral RNA, the downstream of the two AUGs in the sequence....ACGAUGAUAAUAUGGCC... In order to assess the role of ribosome scanning in the selection of the correct initiation site, transcripts of a construct comprising the viral 5' noncoding sequence fused to a reporter gene were translated in vitro and the relative frequency of initiation at these two AUG codons, AUG-10 and AUG-11, was assayed. When deletions from the 5' end were made that retained less than 100 nucleotides of the viral 5'-noncoding sequence, initiation was highly cap-dependent and AUG-10 was utilized in preference to AUG-11, consistent with the scanning ribosome model. On the other hand, when the constructs included the entire 574 nt. segment of the 5' noncoding region situated between the poly(C) tract of the virion RNA and nt. 834, initiation was cap-independent and occurred exclusively at AUG-11, with negligible use of AUG-10. These results suggest that structural features of the 574 nucleotide segment cause initiating ribosomes to bind directly to AUG-11, without scanning the immediate upstream sequences that include AUG-10.

Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein.

Translation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal internal entry into the 5'-nontranslated region (5' NTR) rather than by ribosomal scanning. The internal ribosomal entry site (IRES) in the EMCV 5' NTR was determined by in vitro translation with RNAs that were generated by in vitro transcription of EMCV cDNAs containing serial deletions from either the 5' or 3' end of the EMCV 5' NTR. Regions downstream of nucleotide 403 and upstream of nucleotide 811 of EMCV were required for efficient translation. Site-directed mutagenesis revealed that a stem-loop structure (400 nucleotides upstream of the initiation codon) was essential for IRES function. We discovered a 57-kD cellular protein whose specific interaction with this stem-loop appears to be prerequisite for IRES function. A A pyrimidine-rich stretch proximal to the initiation codon was also crucial for efficient translation of EMCV mRNA. We propose that ribosomes bind directly to the initiating AUG without scanning.

An unusual coordinated cleavage event in the processing of encephalomyocarditis virus polypeptides

During the translation of encephalomyocarditis virus RNA in the rabbit reticulocyte lysate system, polypeptides 213 and 2C, encoded by the central region of the genome, appear simultaneously in the absence of any detectable 2BC precursor and at a time when translation has advanced into the region coding for polypeptide 3C. This implies the operation of closely coordinated proteolysis at two sites, with at least one cleaved by the virus-coded protease activity 3C.

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.

Effects of cDNA hybridization on translation of encephalomyocarditis virus RNA.

Cell-free translation of the RNA of encephalomyocarditis virus was examined after hybridization of chemically synthesized cDNA fragments to different sites of the 5' noncoding region of the viral RNA. The following results were obtained. The binding of cDNA fragments to the first 41 nucleotides, to the poly(C) tract (between nucleotides 149 and 263), and to the sequence between nucleotides 309 and 338 did not affect translation of the viral RNA; the binding of cDNA fragments to the sequence between nucleotides 420 and 449 caused a slight inhibition; and the binding of fragments to eight different sites between nucleotides 450 and the initiator AUG codon (nucleotide 834) caused high degrees of inhibition. The results suggest that the first part of the 5' untranslated region, at least to nucleotide 338, may not be required for encephalomyocarditis viral RNA translation; however, the region near nucleotide 450 is important for translation of the viral RNA. The possibility that initiation occurs at an internal site is discussed.

A detailed kinetic analysis of the in Vitro synthesis and processing of encephalomyocarditis virus products

Translation of encephalomyocarditis virus RNA in rabbit reticulocyte lysates has been used to analyse the pathway of proteolytic processing of the primary translation products. A minimum of two distinct proteases is required to account for the results: one for the excision of the capsid precursor protein, Al, from the nascent polyprotein, and the other for all other cleavages including cleavage at the F/C junction. The excision of Al is an extremely rapid reaction which occurs as soon as the cleavage site has been synthesised and is resistant to all the proteolytic inhibitors tested and to high temperature, characteristics which are more consistent with an intramolecular cleavage catalysed by a virus-coded protease than cleavage by an endogenous reticulocyte protease. Once excised, A1 remains stable until translation has reached the middle of the region of the genome coding for C, at which time a number of events occur in rapid succession: (i) F is excised in its mature form probably via an intramolecular cleavage; (ii) a proteolytic activity capable of secondary processing of A1 to A, B, D1, α, λ and ϵ appears; and (iii) a polypeptide of molecular weight about 32,000 appears. This protein (p32) originates from the N-terminal portion of C, and maps in the same position on the genome as p22, the protein previously identified as the virus-coded protease. Polypeptide p32 is derived from C by a single step cleavage generating E as the other product, a processing pathway at least as important, if not more important than the step-wise route via D as an intermediate. Since p32 first appeared at the same time as the start of secondary processing, whilst p22 was first detected much later, it is argued that at least the early stages of processing of the capsid precursor must have been carried out by p32 rather than p22.

Processing of the encephalomyocarditis virus capsid precursor protein studied in rabbit reticulocyte lysates incubated with N-formyl-[35S]methionine-tRNAfMet.

Translation of encephalomyocarditis virus RNA in rabbit reticulocyte lysates in the presence of N-formyl-[(35)S]methionine-tRNA(f) (Met) revealed that a small polypeptide is cleaved from the N-terminus of the capsid protein precursor, preA, by virus-coded protease activity. Therefore, this N-terminal segment comprising the translation initiation site is not conserved in any of the mature capsid proteins.

Leader polypeptides encoded in the 5′-region of the encephalomyocarditis virus genome

The synthesis of encephalomyocarditis (EMC)virus proteins, like proteins of other picornaviruses, is initiated at a site located in the 5’-adjacent region of the viral genome; the product of this synthesis is represented by a giant polypeptide chain, polyprotein, which contains all the amino acid sequences present in the structural and non-structural viral proteins; the final products of translation, ‘mature’ proteins, are generated by sequential proteolytic cleavages of the polyprotein molecule starting while the latter is in the nascent form [I]. Using a variety of techniques, cleavage maps of the EMC virus polyprotein were constructed, which showed the order of different polypeptide chains relative to the N-terminus of the polyprotein molecule [ 11. Most of the details of these maps are now generally accepted, but some points remain unclear or disputable. One of these important points concerns the uppermost N-terminal region of the polyprotein: which particular polypeptide is the first to be translated? The usually considered candidate is the precursor of capsid proteins, polypeptide A, which is assumed to share the N-terminal amino acid sequence with its cleavage products, polypeptides B and Dl . This view was supported by the finding that, upon in vitro translation of EMC virus RNA, formyl[35S]Met-tRNAyet could donate a labelled formylmethionyl residue to the polypeptides (by inference, to their N-terminal position) that comigrated with polypeptides A, B and possibly, Dl [2]. On the other hand, earlier data obtained with the same labelled substrate but using less efficient cell-free translation systerns suggested that formyl-methionine might be incorporated in peptides other than those corresponding to the capsid proteins [3-51. We have attempted to solve the controversy and show here that: (i) The initiator amino acid sequences, as defined by the presence of formyl-methionyl residue, are contained in a precursor polypeptide, preA; (ii) Upon a short treatment of preA with a preparation of EMC virus-specific protease [6], these initiator sequences are cleaved off and emerge in two low-M, closely-related polypeptides, p14 and p12; (iii) After the removal of p 14 (p 12) the remaining moiety of preA corresponds to the precursor of capsid proteins, polypeptide A; (iv) preA, p 14 and p 12 appear to share an N-terminal amino acid sequence. These data suggest that all 3 latter polypeptides are initiated at a common single site on the viral genome and that p14 (p 12) should occupy the N-terminal position on the EMC virus polyprotein cleavage map followed by polypeptide A, a precursor of the capsid proteins.

Stimulation of encephalomyocarditis virus RNA translation in wheat embryo extracts by rabbit reticulocyte ribosomal wash.

Encephalomyocarditis virus RNA induced a low level of amino acid incorporation in wheat embryo extracts, but the incorporation did not result in the formation of any viral proteins. However, if wheat embryo extracts were supplemented with a ribosomal wash fraction from rabbit reticulocytes, a large stimulation of amino acid incorporation and the formation of encephalomyocarditis virus-specific proteins were obtained. Among the proteins synthesized were capsid precursor proteins which can be processed by an encephalomyocarditis virus-coded protease to produce the viral capsid proteins.

Translation of encephalomyocarditis virus RNA in reticulocyte lysates: kinetic analysis of the formation of virion proteins and a protein required for processing

In cell-free extracts derived from rabbit reticulocytes, encephalomyocarditis RNA can be translated completely, and the products can be processed extensively to give encephalomyocarditis virion proteins and several nonvirion proteins, including a genome-coded protein required for processing. The latter is probably a protease. Translation is very efficient. Under typical conditions, each EMC RNA is translated approximately eight times during a 3-h period. Kinetic analyses (time-course experiments, pulse-chase experiments, and pulse-stop experiments) have been used to determine the time of appearance of major products, and these times have been correlated with map positions. The gene for the putative protease is located near the middle of the genome downstream from the virion protein genes. Ribosomes can travel the length of encephalomycarditis RNA within 30 min, but there is a delay in their progress along the RNA at some point soon after they traverse the region coding for virion protein precursors. This delay results in the accumulation of precursors for a period of about 10 min before the putative protease is made and virion proteins (epsilon, alpha, and gamma) are released by proteolysis.

Translation of Encephalomyocarditis Virus RNA in vitro Yields an Active Proteolytic Processing Enzyme

In contrast to other cell-free translation systems, the mRNA-dependent reticulocyte lysate can translate encephalomyocarditis virus RNA efficiently and completely when supplemented with heterologous tRNA. Cleavage of the nascent polypeptide chain occurs, and one of the translation products appears to be a specific proteolytic enzyme which correctly processes the primary products. The identity of the proteins made in vitro was verified by comparison with infected cell proteins on dodecylsulphate/polyacrylamide gels, and by mapping their coding sequences on the viral genome.

Complete translation of encephalomyocarditis virus RNA and faithful cleavage of virus-specific proteins in a cell-free system from Krebs-2 cells

Received 27 December 1977 1. Introduction There are numerous reports describing translation of encephalomyocarditis (EMC) virus RNA in cell- free systems (c.f. [l-l 0] ). The products formed in these systems, however, did not coincide, upon poly- acrylamide gel electrophoresis, with ‘mature’ virus- specific polypeptides found in EMC ,virus-infected cells. The difference between the in vitro and in vivo products seemed to be primarily due to two factors, non-complete translation of the RNA template and inefficient cleavage of precursor polypeptides formed. Indeed, the major products of in vitro synthesis corresponded to high-molecular-weight polypeptides containing amino acid sequences of capsid proteins, which implied that only a 5’-terminal region of EMC virus RNA was predominantly, or exclusively, trans- lated. The reasons for such an incomplete translation were thought to be either premature termination at certain regions of the template or template degrada- tion, or both [5,6,10]. In systems where these problems were partially overcome, the resulting product did contain, as minor components, poly- peptides corresponding to the non-capsid 3’-terminal portion of viral genome but only in a high-molecular- weight uncleaved form [ 1 l] . The present report describes conditions under which the entire genome of EMC virus appears to be translated and proper cleavage of polypeptide pre- cursors takes place in vitro. The combination of these events results in formation of almost all ‘mature’ virus-specific polypeptides. Although the formation