Cell-specific proteins regulate viral RNA translation and virus-induced disease.

Translation initiation of the picornavirus genome is regulated by an internal ribosome entry site (IRES). The IRES of a neurovirulent picornavirus, the GDVII strain of Theiler's murine encephalomyelitis virus, requires polypyrimidine tract-binding protein (PTB) for its function. Although neural cells are deficient in PTB, they express a neural-specific homologue of PTB (nPTB). We now show that nPTB and PTB bind similarly to multiple sites in the GDVII IRES, rendering it competent for efficient translation initiation. Mutation of a PTB or nPTB site results in a more prominent decrease in nPTB than PTB binding, a decrease in activity of nPTB compared with PTB in promoting translation initiation, and attenuation of the neurovirulence of the virus without a marked effect on virus growth in non-neural cells. The addition of a second-site mutation in the mutant IRES generates a new PTB (nPTB) binding site, and restores nPTB binding, translation initiation and neurovirulence. We conclude that the tissue-specific expression and differential RNA-binding properties of PTB and nPTB are important determinants of cell-specific translational control and viral neurovirulence.

Molecular mechanisms of translation initiation in eukaryotes.

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5' end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.

A cell cycle-dependent protein serves as a template-specific translation initiation factor.

Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) and requires both eukaryotic initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). We show here that the requirements for trans-acting factors differ between related picornavirus IRESs and can account for cell type-specific differences in IRES function. The neurovirulence of Theiler's murine encephalomyelitis virus (TMEV; GDVII strain) was completely attenuated by substituting its IRES by that of foot-and-mouth disease virus (FMDV). Reconstitution of initiation using fully fractionated translation components indicated that 48S complex formation on both IRESs requires eIF2, eIF3, eIF4A, eIF4B, eIF4F, and the pyrimidine tract-binding protein (PTB) but that the FMDV IRES additionally requires ITAF(45), also known as murine proliferation-associated protein (Mpp1), a proliferation-dependent protein that is not expressed in murine brain cells. ITAF(45) did not influence assembly of 48S complexes on the TMEV IRES. Specific binding sites for ITAF(45), PTB, and a complex of the eIF4G and eIF4A subunits of eIF4F were mapped onto the FMDV IRES, and the cooperative function of PTB and ITAF(45) in promoting stable binding of eIF4G/4A to the IRES was characterized by chemical and enzymatic footprinting. Our data indicate that PTB and ITAF(45) act as RNA chaperones that control the functional state of a particular IRES and that their cell-specific distribution may constitute a basis for cell-specific translational control of certain mRNAs.

Cross-talk between orientation-dependent recognition determinants of a complex control RNA element, the enterovirus oriR.

The coxsackie B3 virus oriR is an element of viral RNA thought to promote the assembly of a ribonucleoprotein complex involved in the initiation of genome replication. The mutual orientation of its two helical domains X and Y is determined by a kissing interaction between the loops of these domains. Here, a genetic approach was worked out to identify spatial orientation-dependent recognition signals in these helices. Spatial orientation changes (due to linear and rotational shifts) were introduced by appropriate insertions/deletions of a single base pair into one or both of the domains, and phenotypic consequences caused by these mutations were studied. The insertion of a base pair into domain Y caused a defect in viral reproduction that could be suppressed by a base-pair insertion into domain X. Similarly, a defect in viral replication caused by a base-pair deletion from domain X could be suppressed by a base-pair deletion from domain Y. Thus, certain areas of the two domains should cross-talk to one another in the sense that a change of space position of one of them required an adequate reply (change of space position) from the other. Phenotypic effects of the local rotation of one or more base pairs (and of some other mutations) in either domain X or domain Y suggested that the two most distal base pairs of these domains served as orientation-dependent recognizable signals. The results were also consistent with the notion that the recognition of the distal base pair of domain Y involved a mechanism similar to the intercalation of an amino acid residue.

Distinct attenuation phenotypes caused by mutations in the translational starting window of Theiler's murine encephalomyelitis virus.

Upon initiation of translation of picornavirus RNA, the ribosome is believed to bind the internal ribosome entry site of the template and then to form a productive complex with a downstream RNA segment, the starting window. The presence or absence of an AUG triplet within the starting window of the RNA of Theiler's murine encephalomyelitis virus (a picornavirus) is known to modulate its neurovirulence. In this study, mutants of this virus in which the starting windows, lying upstream of the viral polyprotein reading frame, had AUGs with different nonoptimal contexts were engineered. Upon intracerebral inoculation of mice, the mutants proved to be partially attenuated, as judged by a significant increase in the dose causing paralysis in 50% of the animals (PD50). Mutants with similar PD50s might differ from one another by eliciting either a severe, fatal tetraplegy or only mild, recoverable neurologic lesions. Some of the mutants triggered a chronic inflammatory reaction in the white matter of the spinal cord in the absence of detectable viral RNA or antigen. Thus, point mutations changing the context of an AUG within the starting window outside the polyprotein reading frame may differently affect the morbidity and mortality caused by a viral infection and may result in distinct attenuation phenotypes.

Structural requirements of the higher order RNA kissing element in the enteroviral 3'UTR.

The origin of replication ( oriR ) involved in the initiation of (-) strand enterovirus RNA synthesis is a quasi-globular multi-domain RNA structure which is maintained by a tertiary kissing interaction. The kissing interaction is formed by base pairing of complementary sequences within the predominant hairpin-loop structures of the enteroviral 3' untranslated region. In this report, we have fully characterised the kissing interaction. Site-directed mutations which affected the different base pairs involved in the kissing interaction were generated in an infectious coxsackie B3 virus cDNA clone. The kissing interaction appeared to consist of 6 bp. Distortion of the interaction by mispairing of each of the base pairs involved in this higher order RNA structure resulted in either temperature sensitive or lethal phenotypes. The nucleotide constitution of the base which gaps the major groove of the kissing domain was not relevant for virus growth. The reciprocal exchange of the complete sequence involved in the kissing resulted in a mutant virus with wild type virus growth characteristics arguing that the base pair constitution is of less importance for the initiation of (-) strand RNA synthesis than the existence of the tertiary structure itself.

An attenuated variant of the GDVII strain of Theiler's virus does not persist and does not infect the white matter of the central nervous system.

The DA strain of Theiler's virus causes a persistent and demyelinating infection of the white matter of spinal cord, whereas the GDVII strain causes a fatal gray-matter encephalomyelitis. Studies with recombinant viruses showed that this difference in phenotype is controlled mainly by the capsid. However, conflicting results regarding the existence of determinants of persistence in the capsid of the GDVII strain have been published. Here we show that a GDVII virus whose neurovirulence has been attenuated by an insertion in the 5' noncoding region does not persist in the central nervous systems of mice. Furthermore, this virus infects the gray matter efficiently, but not the white matter. These results confirm the absence of determinants of persistence in the GDVII capsid. They suggest that the DA capsid controls persistence by allowing the virus to infect cells in the white matter of the spinal cord.

Kissing of the two predominant hairpin loops in the coxsackie B virus 3' untranslated region is the essential structural feature of the origin of replication required for negative-strand RNA synthesis.

Higher-order RNA structures in the 3' untranslated region (3'UTR) of enteroviruses are thought to play a pivotal role in viral negative-strand RNA synthesis. The structure of the 3'UTR was predicted by thermodynamic calculations using the STAR (structural analysis of RNA) computer program and experimentally verified using chemical and enzymatic probing of in vitro-synthesized RNA. A possible pseudoknot interaction between the 3D polymerase coding sequence and domain Y and a "kissing" interaction between domains X and Y was further studied by mutational analysis, using an infectious coxsackie B3 virus cDNA clone (domain designation as proposed by E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V.I. Agol (Nucleic Acids Res. 20:1739-1745, 1992). The higher-order RNA structure of the 3'UTR appeared to be maintained by an intramolecular kissing interaction between the loops of the two predominant hairpin structures (X and Y) within the 3'UTR. Disturbing this interaction had no effect on viral translation and processing of the polyprotein but exerted a primary effect on viral replication, as was demonstrated in a subgenomic coxsackie B3 viral replicon, in which the capsid P1 region was replaced by the luciferase gene. Mutational analysis did not support the existence of the pseudoknot interaction between hairpin loop Y and the 3D polymerase coding sequence. Based on these experiments, we constructed a three-dimensional model of the 3'UTR of coxsackie B virus that shows the kissing interaction as the essential structural feature of the origin of replication required for its functional competence.

Cis-element, oriR, involved in the initiation of (-) strand poliovirus RNA: a quasi-globular multi-domain RNA structure maintained by tertiary ('kissing') interactions.

The key steps in the replication of the poliovirus genome, initiation of (-) and (+) strands, require two different cis-acting elements, oriR and oriL, respectively. It has been proposed that the spatial organization of these elements is maintained by tertiary ('kissing') interactions between the loops of two constituent hairpins. Here, the putative partners of the kissing interaction within the oriR of the full-length poliovirus RNA were modified by site-directed mutagenesis. The destabilization of this interaction resulted in a severe suppression of the viral RNA synthesis, but the mutant transcripts proved to be infectious. With a single exception, the potential for the kissing interaction within the oriR of the recovered viruses was partially or completely restored due to either true reversions or second-site compensatory mutations. There was a good correlation between the restoration of this potential and the phenotypic properties of the viruses. It was concluded that the kissing interaction in the poliovirus oriR is functionally important. Using the above experimental data, a three-dimensional structure was derived by molecular modeling techniques, which demonstrated the overall feasibility of the proposed interactions and displayed the poliovirus oriR as a quasi-globular multi-domain structure.

Modification of translational control elements as a new approach to design of attenuated picornavirus strains.

The translation machineries of different host cells may exhibit varying requirements for a specific structure of cis-acting control elements in the viral RNA templates. Thus, the appropriately spaced oligopyrimidine/AUG tandem (OAT), a conserved control element in the 5' noncoding region of the picornavirus genomes, is dispensable for the growth of Theiler's murine encephalomyelitis virus (TMEV) in BHK-21 cells, but is essential for the neurovirulence of this virus. Also, the replacement of the cryptic (non-initiator) AUG moiety of the wild-type poliovirus OAT by the initiator AUG affects the viral reproduction in cultured cells only slightly, whereas neurovirulence of the relevant mutants is dramatically suppressed. These observations allow us to propose a rational way to construct novel attenuated viral strains by elimination or severe modification of host-specific regulatory regions in their genomes. The relevant genetic rearrangements may be so extensive that the probability of reversion to the virulent phenotype should be negligible. The feasibility of engineering of highly attenuated and genetically stable TMEV and poliovirus variants is illustrated.

A model for rearrangements in RNA genomes.

Engineered mutants of Theiler's murine encephalomyelitis virus (TMEV) and poliovirus having altered spacing between the oligopyrimidine and AUG moieties of a translational control element are known to generate pseudorevertants with deletions or insertions that tend to restore the wild-type structure of this element. The primary structure of the rearranged region of these pseudorevertants suggests that short direct repeats are strongly preferred as parting and anchoring sites during the jumps of the nascent strand 3' end. When the parting and anchoring sites are separated by a long RNA segment, they can be brought in close proximity by an appropriate folding of the template strand. On the basis of evidence derived from the analysis of the pseudorevertant genomes, it is proposed that a class of RNA rearrangements (some recombinations, deletions, insertions) proceed through the following steps: (i) pausing of the nascent strand caused by misincorporations (or other reasons); (ii) dissociation of the RNA polymerase together with the 3' end of the nascent strand (a kind proof-reading); and (iii) re-annealing of the nascent and template strands (precise or imprecise, but with the 3' base paired) and resumption of the synthesis.

[The critical significance of a conserved single-stranded interdomain linker in the 5'-untranslated region of poliovirus RNA]. / Kriticheskoe znachenie konservativnogo odnotsepochechnogo mezhdomennogo linkera v 5'-netransliruemoi oblasti poliovirusnoi RNK.

Translation of polioviral RNA is initiated by interaction of a small ribosomal subunit with internal segments of the 5'-untranslated region (5'UTR). Several mutations were constructed within 5'UTR segment 425-449. All of them (including a single C444-->U replacement) inhibited in vitro translation, which decreased about 10-fold. Two mutant constructs, pPV12-05 (C444-->U) and pPV12K (containing also an AAUU insert between positions 441 and 442) produced plaques on monolayers of susceptible cells. All the viruses isolated from these plaques exhibited a reversion at position 444; the template activities of the revertant RNAs were restored completely or significantly. The results show the importance of the relevant 5'UTR segment for the initiation of polioviral RNA translation.

Attenuation of Theiler's murine encephalomyelitis virus by modifications of the oligopyrimidine/AUG tandem, a host-dependent translational cis element.

A set of Theiler's murine encephalomyelitis virus mutants with engineered alterations in the conserved oligopyrimidine/AUG tandem (E. V. Pilipenko, A. P. Gmyl, S. V. Maslova, G. A. Belov, A. N. Sinyakov, M. Huang, T. D. K. Brown, and V. I. Agol, J. Mol. Biol. 241:398-414, 1994) were assayed for their growth potential in BHK-21 cells (as reflected in plaque size) and for neurovirulence upon intracerebral inoculation of mice. Tandem-destroying mutations, which included substitutions in the oligopyrimidine moiety and extended insertions into the oligopyrimidine/AUG spacer, exerted relatively little effect on the plaque size but ensured a high level of attenuation. The attenuated mutants exhibited remarkable genetic stability upon growth in BHK-21 cells. However, the brains of rare animals that developed symptoms after the inoculation with high doses of these mutants invariably contained pseudorevertants with the oligopyrimidine/AUG tandem restored by diverse deletions or an AUG-generating point mutation. The AUG moiety of the tandem in the revertant genomes was represented by either a cryptic codon or initiator codon. The results demonstrate that the tandem, while dispensable for the Theiler's murine encephalomyelitis virus growth in BHK-21 cells, is essential for neurovirulence in mice. Thus, the oligopyrimidine/AUG tandem is a host-dependent cis-acting control element that may be essential for virus replication under certain conditions. The functional activity of the tandem was retained when its oligopyrimidine or AUG moieties were made double stranded. A possible role of the tandem in the cap-independent internal initiation of translation on the picornavirus RNA templates is discussed.

Starting window, a distinct element in the cap-independent internal initiation of translation on picornaviral RNA.

Initiation of translation on picornaviral RNA templates occurs via cap-independent ribosome binding to a cis-acting element, internal ribosome entry site (IRES). Mapping of the starting point of translation relative to the IRES was attempted using Theiler's murine encephalomyelitis virus (TMEV) RNA as a model. The possibility that the starting point is determined by the conserved oligopyrimidine upstream of the initiator codon was studied. In contrast to poliovirus, neither the conserved oligopyrimidine nor an AUG at a fixed distance downstream of this oligopyrimidine are required for efficient translation of the TMEV RNA in Krebs-2 extracts or reticulocyte lysates or for viral infectivity; mutants lacking the oligopyrimidine/AUG tandem were stable upon passage in BHK-21 cells. A short template segment, the starting window, was defined, wherefrom ribosomes begin translation or downstream scanning depending, respectively, on the presence or absence of a good-context AUG within this window. Using a collection of the engineered TMEV mutant RNAs, the starting window was mapped to 16-17 nt downstream of the IRES and was found to be approximately a dozen nt long. The efficiency of translation initiation from an AUG linearly increased upon the 5'-->3' displacement of the initiator codon within the window. The competence of the starting window did not appear to depend markedly on its primary structure; however, it was completely inactivated ("closed") with concomitant dramatic inhibition of total protein synthesis upon conversion of the corresponding RNA segment into a double-stranded form.

Functional and genetic plasticities of the poliovirus genome: quasi-infectious RNAs modified in the 5'-untranslated region yield a variety of pseudorevertants.

Poliovirus RNA species with nucleotides 564 to 571 deleted or with a secondary structure domain (positions 564 to 629) replaced by a shorter irregular oligonucleotide have been engineered previously; these RNAs have been considered quasi-infectious (yielding a single late revertant plaque) and dead, respectively (E. Pilipenko, A. Gmyl, Y. Svitkin, S. Maslova, A. Sinyakov, and V. Agol, Cell 68:119-131, 1992). By using large amounts of these RNAs for transfections, revertant clones with a great variety of genetic changes (point mutations, insertions of foreign sequences, short or extended deletions) were isolated. The pattern of these changes supported the notion that an appropriately spaced oligopyrimidine-AUG tandem is important for efficient poliovirus RNA translation. Structural features within and around this tandem modulated the initiation efficiency. The functional and genetic plasticities of the poliovirus genome are briefly discussed.

[Probable compact structure of a cis-active translation element in the 5'-untranslated region of the enterovirus and rhinovirus genome]. / Veroiatnaia kompaktnaia struktura tsis-aktivnogo transliatsionnogo élementa v 5'-netransliruemoi zone genoma énterovirusov i rinovirusaov.

A previously proposed consensus secondary structure model of the internal portion of the 5'-untranslated region of the enterovirus and rhinovirus genomes encompassing the cis-acting translational control element was extended and detailed on the basis of comparative sequence data and biochemical analysis. Using this model as a reference, numerous pairs of apparently single-stranded noncontiguous nucleotide stretches with a potential to form inter-domain tertiary bonds were shown to be conserved among the respective segments of all the known enterovirus and rhinovirus RNAs. It is suggested that at least some of these inter-domain bonds may actually exist, resulting in a compact, quasi-globular, perhaps "two-lobe" folding of the translational control element. Such an organization should have important functional implications.

Towards identification of cis-acting elements involved in the replication of enterovirus and rhinovirus RNAs: a proposal for the existence of tRNA-like terminal structures.

On the basis of a comparative analysis of published sequences, models for the secondary structure of the 3'-terminal [poly(A)-preceding] untranslated region of the entero- and rhinovirus RNAs were worked out. The models for all these viruses share a common core element, but there are an extra enterovirus-specific element and still an additional element characteristic of a subset of enterovirus RNAs. The two latter models were verified for poliovirus and coxsackievirus B genomes by testing with single-strand and double-strand specific enzymatic and chemical probes. A tRNA-like tertiary structure model for the 3'-terminal folding of enterovirus RNAs was proposed. A similar folding was proposed for the 3' termini of the negative RNA strands as well as for the 5' termini of the positive strand of all entero- and rhinovirus RNAs. Implications of these data for template recognition during negative and positive RNA strands synthesis and for the evolution of the picornavirus genomes are discussed.

Prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus RNA.

Initiation of translation on picornavirus RNAs is accomplished through internal binding of ribosomes to a complex cis-acting element. Here we show that efficient function of this element involves two appropriately spaced smaller elements: UUUCC and an AUG. This conclusion emerged from analysis of the genome structures of spontaneous revertants of mutant polioviruses with extended insertions between the UUUCC and AUG motifs. It was confirmed by the results obtained with specially designed constructs. A similarity to the prokaryotic translation initiation mechanism, which involves the Shine-Dalgarno sequence, is emphasized, but in the picornavirus system the position of the UUUCC must be strictly fixed relative to upstream cis-acting elements, and the AUG may not necessarily serve as an initiation codon.

Coupled mutations in the 5'-untranslated region of the Sabin poliovirus strains during in vivo passages: structural and functional implications.

All entero- and rhinovirus RNAs sequenced thus far possess A and U residues at positions corresponding to nucleotides 480 and 525, respectively, of poliovirus type 1. These two nucleotides have been proposed previously to form a base pair. The single exception to this rule appears to be the Sabin type 1 strain, which has a G480. Isolates of the Sabin 1 virus from healthy vaccinees were shown to have either a reversion to A480 or a second-site mutation U525----C, both restoring a potential for efficient base pairing. In vitro translation experiments demonstrated that poliovirus type 1 RNAs with either A480-U525 or G480-C525 are more efficient in promoting translation initiation as compared with the Sabin 1 RNA (G480-U525). The Sabin 2 strain has a U and an A at position 398 and 481, respectively, while its predecessor, strain P712, is shown to have C398 and G481. All the derivatives of the Sabin 2 isolated from vaccine-associated paralytic poliomyelitis cases shown reversion to G481, and most of them reverted also to C398. It is proposed that bases at positions 398 and 481 may be involved in a tertiary interaction. The in vitro template activity of the Sabin type 2 RNA (A481) is significantly lower than that of the isolate RNAs with G481, thus confirming the relation between attenuation and translation efficiency demonstrated previously for the type 1 and type 3 Sabin strains. The C----U change at position 398 exerted only a minor effect on the RNA template activity.

Gross rearrangements within the 5'-untranslated region of the picornaviral genomes.

An analysis of reported nucleotide sequences revealed several cases of gross rearrangements in the 5'-untranslated region (5-UTR) of picornaviral genomes. A large (greater than 100 nt) duplication was discovered in a downstream region of poliovirus 5-UTR involved in the translational control. Properties of the poliovirus mutants with large deletions [Kuge and Nomoto (1987) J. Virol. 61, 1478-1487] show that a single copy of the appropriate repeating unit is compatible with a wild type phenotype of the virus. In contrast to poliovirus and another enterovirus genomes, human rhinovirus RNAs contain only a single copy of this repeating unit. Another similarly large repeat was found in an upstream segment of the bovine enterovirus 5-UTR. A comparison of the primary and secondary structures of cardio- and aphthovirus 5-UTRs demonstrated the existence of a large (ca. 250 nucleotides) insertion/deletion in a region preceding the poly(C) tract. The two latter rearrangements appear to involve elements of the viral genome replication machinery. Possible origin as well as evolutionary and functional implications of these structural peculiarities are discussed.