Functional analysis of the two alternative translation initiation sites of foot-and-mouth disease virus.

The effect of deletion of each of the two authentic polyprotein translation initiation sites of foot-and-mouth disease virus on viral protein synthesis and replication was analyzed. Deletion of either the first or the second initiation site led to the expression of only one form of the leader protein, L or L', respectively, but in vitro processing of the viral polyprotein and cleavage of eIF-4 gamma were not affected by either deletion. Whereas RNA in which the first translation initiation site had been deleted led to the production of viruses in transfected BHK cells, deletion of the second translation initiation site abolished virus replication.     

Differential factor requirement to assemble translation initiation complexes at the alternative start codons of foot-and-mouth disease virus RNA

The foot-and-mouth disease virus (FMDV) RNA contains two in-frame AUG codons separated by 84 nt that direct translation initiation of the viral polyprotein. The mechanism of initiation at the IRES-proximal AUG codon (AUG1) has been previously analyzed, whereas no data on factor requirements for AUG2 have been reported. Here, using the method of 48S translation initiation complex reconstitution, we show that eIF1 is indispensable in forming the 48S initiation complex at AUG2. In contrast, it reduces the assembly of this complex at AUG1. Stabilization of a stem-loop between the initiation triplets induces a small decrease in the toeprint intensity at AUG2, accompanied by an increase in the AUG1/AUG2 ratio as well as a moderate reduction of protein synthesis initiated at AUG2 in transfected cells. PTB and ITAF45 exerted an additive positive effect on the 48S complex at AUG2, although a substantial reconstitution on both AUGs occurs on omission of either of these proteins. Relative to the beta-globin mRNA, the 48S complex formation at AUG1 and AUG2 is slow and occurs with the same kinetics as revealed by the "kinetic" toeprint assay. Mutation of AUG1 to AUA does not abrogate protein synthesis in transfected cells, and has no effect on the rate of the 48S complex formation at AUG2. We conclude that the AUG2 initiation region is selected independently of 48S complex formation at the upstream AUG1. The kinetic toeprint assay also shows that cap-dependent assembly of the 48S complex in vitro occurs faster than the FMDV IRES-mediated complex assembly.     

Functional analysis of the internal translation initiation site of foot-and-mouth disease virus.

Mutagenesis of the large untranslated sequence at the 5' end of the genome of foot-and-mouth disease virus revealed that a region of approximately 450 nucleotides preceding the open reading frame of the viral polyprotein is involved in the regulation of translation initiation at two internal start sites. Variations in two domains of this region reduced the translation efficiency up to 10-fold, whereas an intermediate segment seemed to be less essential. A pyrimidine-rich sequence preceding the start codon was most sensitive in that conversion of single pyrimidine residues to purines decreased the translation efficiency strongly. The data are in agreement with a recently proposed general structural model for the internal ribosome entry site of the cardiovirusaphthovirus subgroup of picornaviruses (E. V. Pilipenko, V. M. Blinov, B. K. Chernov, T. M. Dmitrieva, and V. I. Agol, Nucleic Acids Res. 17:5701-5711, 1989). They suggest, however, that this model represents only a core structure for the internal entry of ribosomes and that foot-and-mouth disease virus and other members of the picornaviruses need additional regulatory RNA elements for efficient translation initiation.     

Location of the initiation site for protein synthesis on foot-and-mouth disease virus RNA by in vitro translation of defined fragments of the RNA

An mRNA-dependent reticulocyte lysate has been used to translate foot-and-mouth disease virus RNA in vitro. Polypeptides P16, P20a, and P88, which have been shown to be derived from the 5' end of the RNA by pactamycin mapping experiments with infected cells, were preferentially synthesized in vitro. Removal of VPg, the small protein covalently linked to the 5' end of the genome RNA, had no effect on the translation of the RNA. The two RNA fragments (L and S) produced by specific digestion of the polycytidylic acid [poly(C)] tract with RNase H were also translated in vitro. The L fragment, consisting of RNA to the 3' side of the poly(C) tract and including the polyadenylic acid [poly(A)] tract, directed the synthesis of the same products as those made by full-length RNA. However, no small defined products were produced when the S fragment, which contains the 5' end of the RNA, was translated. These results show that the major initiation site for protein synthesis on foot-and-mouth disease virus RNA is to the 3' side of the poly(C) tract. Furthermore, the use of N-formyl [35S]methionine tRNAfMet as a label for the initiation peptides showed that the major polypeptide labeled in lysates primed with both full-length RNA and the L fragment was P16, i.e., the protein nearest the initiation site for translation as deduced from pactamycin mapping experiments. Fragments of RNA were also translated in vitro. Those containing the poly(C) tract gave products similar to those produced when full-length RNA was translated. The polypeptides synthesized when fragments containing the poly(A) tract were used, however, did not resemble those made from full-length RNA.     

Internal translation initiation on the foot-and-mouth disease virus IRES is affected by ribosomal stalk conformation

A human cell line, in which expression of the ribosomal stalk proteins P1 and P2 has been suppressed by RNAi technology, has been used to test how the loss of these proteins affects IRES-dependent translation. Foot-and-mouth disease virus (FMDV) IRES-dependent translation from a bicistronic construct is about three fold higher in the P1/P2-depleted cells than in control cells in the presence of Lb protease. By contrast, no effect on Hepatitis C virus (HCV) IRES translation was observed. These results emphasize the functional heterogeneity of the IRES and they highlight a functional connection between the ribosomal stalk and picornavirus IRES-dependent translation.     

Feature selection for the prediction of translation initiation sites

Translation initiation sites （TISs） are important signals in cDNA sequences. In many previous attempts to predict TISs in cDNA sequences, three major factors affect the prediction performance： the nature of the cDNA sequence sets, the relevant features selected. and the classification methods used. In this paper, we examine different approaches to select and integrate relevant features for TIS prediction. The top selected significant features include the features from the position weight matrix and the propensity matrix, the number of nucleotide C in the sequence downstream ATG, the number of downstream stop codons. the number of upstream ATGs, and the number of some amino acids, such as amino acids A and D. With the numerical data generated from these features, different classification methods, including decision tree. naive Bayes, and support vector machine, were applied to three independent sequence sets. The identified significant features were found to be biologically meaningful. while the experiments showed promising results.     

Dual initiation sites of protein synthesis on foot-and-mouth disease virus RNA are selected following internal entry and scanning of ribosomes in vivo.

The initiation of protein synthesis on foot-and-mouth disease virus RNA occurs at two sites separated by 84 nucleotides. Immediately upstream from the first of these sites is the internal ribosome entry site (IRES), which directs the translation of this RNA to be cap-independent. The utilization of these two initiation sites has been examined using artificial fusion genes in vivo under a variety of conditions. Additional in-frame AUG codons have been introduced between these two authentic start sites to determine the mechanism by which ribosomes recognize the second start site. The results indicate that following internal entry of ribosomes on the 5' side of the first initiation codon, many fail to initiate protein synthesis at this position and scan along the RNA to the second initiation site. In the presence or absence of the IRES both initiation sites are efficiently used but the utilization of the two sites is slightly biased towards the second initiation site by the IRES. Furthermore, in the presence of the IRES, protein synthesis initiates at both sites independently of the activity of the cap-binding complex.     

Interaction of a cellular 57-kilodalton protein with the internal translation initiation site of foot-and-mouth disease virus.

A cellular 57-kDa protein (p57) that binds specifically to the internal translation initiation site in the 5' untranslated region of foot-and-mouth disease virus RNA was detected in cell extracts of different mammalian species by UV cross-linking. The protein binds to two distinct sites of the translation control region which have as the only common sequence a UUUC motif. The first binding site consists of a conserved hairpin structure, whereas the second binding site contains an essential pyrimidine-rich region without obvious secondary structure. Competition experiments indicate that the complexes with the two binding sites were formed by a single p57 species. The protein binds also to the 5' untranslated region of other picornaviruses. Results from footprint analyses with foot-and-mouth disease RNA suggest the participation of additional cellular factors in the translation initiation complex.     

Antigenic sites on foot-and-mouth disease virus type A10.

A set of monoclonal antibodies was used to isolate nonneutralizable foot-and-mouth disease virus variants, and the RNAs of the variants were sequenced. Cross-neutralization studies and mapping of the amino acid changes indicated two major antigenic sites. The first site was trypsin sensitive and included the VP1 140 to 160 sequence. The second site was trypsin insensitive and included mainly VP3 residues. Two minor sites were located near VP1 169 and on the C terminus of VP1. Comparison with poliovirus type 1 and human rhinovirus 14 showed a similarity in the immunogenicity of comparable sites on the viruses.     

A cellular 57 kDa protein binds to two regions of the internal translation initiation site of foot-and-mouth disease virus

A ribosome-associated 57 kDa protein from rabbit reticulocytes was linked to the internal translation initiation site of foot-and-mouth disease virus by mild UV-irradiation. Binding studies with different RNA fragments revealed that this protein interacts with two distinct sites within the translational control region. One site is located approximately 400 nucleotides upstream from the translational start codon and the second binding site could be confined to 60 nucleotides preceding this codon. Both sequences coincide with hairpin structures at the two opposite ends of a secondary structure model of the internal ribosomal entry site proposed by Pilipenko et al. [(1989) Nucleic Acids Res. 17, 5701-5711].     

Functional importance of RNA interactions in selection of translation initiation codons

RNA base pairing between the initiation codon and anticodon loop of initiator tRNA is essential but not sufficient for the selection of the 'correct' mRNA translational start site by ribosomes. In prokaryotes, additional RNA interactions between small ribosomal subunit RNA and mRNA sequences just upstream of the start codon can efficiently direct the ribosome to the initiation site. Although there is presently no proof for a similar important ribosomal RNA interaction in eukaryotes, the 5' non-coding regions of their mRNAs and 'consensus sequences' surrounding initiation codons have been shown to be strong determinants for initiation-site selection, but the exact mechanisms are not yet understood. Intramolecular base pairing in mRNA and participation of translation initiation factors can strongly influence the formation of mRNA–small ribosomal subunit–initiator tRNA complexes and modulate translational activities in both prokaryotes and eukaryotes. Only recently has it been appreciated that alternative mechanisms may also contribute to the selection of initiation codons in all organisms. Although direct proof is currently lacking, there is accumulating evidence that additional cis -acting mRNA elements and trans -acting proteins may form specific 'bridging' interactions with ribosomes during translation initiation.     

A complex RNA sequence determines 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.     

Stability of poliovirus RNA in cell-free translation systems utilizing two initiation sites

The stability of purified poliovirus RNA in cell-free translation systems prepared from HeLa cells or rabbit reticulocytes has been examined. Degradation of the RNA occurs with a t1/2 of approximately 35 min at 30 degrees C under conditions used for in vitro translation. Degradation is due in part to activity in the cell lysate, and in part to contaminants in the commercial preparations of creatine phosphokinase used in the energy-regenerating system. Addition of crude preparations of initiation factors significantly slows degradation, presumably as a result of protein-RNA interactions which confer resistance to nuclease action. Prior treatment of RNA with methylmercury hydroxide has no effect on degradation rates. On the other hand, endogenous mRNA, present as a messenger ribonucleoprotein particle in extracts from poliovirus-infected HeLa cells, remains completely intact during in vitro translation. These infected cell extracts synthesize the normal complement of viral proteins and utilize two different initiation sites for translation. Treatment of the infected cell extract with micrococcal nuclease destroys the endogenous mRNA. Subsequent addition of exogenous RNA to the same extract results in the formation of a protein-associated RNA particle with sedimentation properties slightly different from the endogenous messenger ribonucleoprotein, and the added RNA is unstable. We conclude that two initiation sites can be utilized on intact poliovirus mRNA, and fragmentation of the RNA is not prerequisite for generation of a second site in this RNA.     

Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus

Equine rhinitis A virus (ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinases Lab and Lb. Here we show that the ERAV 5' nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, stem-loops D to L, in addition to a stem-loop (termed M) downstream of the first putative initiation codon, are required for translation of the second reporter gene. In FMDV, translation of Lb is more efficient than that of Lab despite the downstream location of the Lb AUG codon. The ERAV genome also has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Using the bicistronic expression system, we detected initiation from both AUG pairs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is the major initiation site, although AUG1 can be accessed, albeit inefficiently, in the absence of AUG2. Further mutational analysis indicated that codons downstream of AUG2 appear to be accessed by a mechanism other than leaky scanning. Furthermore, we present preliminary evidence that it is possible for ribosomes to access downstream of the two AUG pairs. This study reveals important differences in IRES function between aphthoviruses.     

Functional involvement of polypyrimidine tract-binding protein in translation initiation complexes with the internal ribosome entry site of foot-and-mouth disease virus.

The synthesis of picornavirus polyproteins is initiated cap independently far downstream from the 5' end of the viral RNA at the internal ribosome entry site (IRES). The cellular polypyrimidine tract-binding protein (PTB) binds to the IRES of foot-and-mouth disease virus (FMDV). In this study, we demonstrate that PTB is a component of 48S and 80S ribosomal initiation complexes formed with FMDV IRES RNA. The incorporation of PTB into these initiation complexes is dependent on the entry of the IRES RNA, since PTB and IRES RNA can be enriched in parallel either in 48S or 80S ribosomal complexes by stage-specific inhibitors of translation initiation. The formation of the ribosomal initiation complexes with the IRES occurs slowly, is temperature dependent, and correlates with the incorporation of PTB into these complexes. In a first step, PTB binds to the IRES, and then the small ribosomal subunit encounters this PTB-IRES complex. Mutations in the major PTB-binding site interfere simultaneously with the formation of initiation complexes, translation efficiency, and PTB cross-linking. PTB stimulates translation directed by the FMDV IRES in a rabbit reticulocyte lysate depleted of internal PTB, and the efficiency of translation can be restored to the original level by the addition of PTB. These results indicate that PTB plays an important role in the formation of initiation complexes with FMDV IRES RNA and in stimulation of internal translation initiation with this picornavirus.     

Unique features of internal initiation of hepatitis C virus RNA translation.

The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40-370 and that deletions from the 3'-end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5'-UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7-H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation. As a probable correlate with this, it was also found that the efficiency of internal initiation was only marginally compromised when the authentic initiation codon was mutated to a non-AUG codon, again in sharp contrast with the picornaviruses. The finding that coding sequences are required for internal initiation has important implications for the design of experiments to test for internal initiation of translation of cellular mRNAs.     

Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids.

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.     

Selection and characterization of RNA aptamers to the RNA-dependent RNA polymerase from foot-and-mouth disease virus

Foot-and-mouth disease virus causes a highly contagious disease of agricultural livestock and is of enormous economic importance. Replication of the RNA genome of the virus, via negative strand intermediates, involves an RNA-dependent RNA polymerase (3Dpol). RNA aptamers specific to this enzyme have been selected and characterized. Some of these molecules inhibit enzymatic activity in vitro, with IC50 values of <20 nM and Ki values of 18-75 nM. Two of these show similarity, both with each other and with regions of the viral genome. Furthermore, truncated versions of one of the aptamers have been used to define the parts of the molecule responsible for its inhibitory activity.