Nonreplicative RNA recombination in poliovirus.

Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments of the partners and might have resulted from transesterification reactions, not necessarily involving an enzymatic activity. Some of the crossover loci are clustered. The origin of some of these "hot spots" could be explained by invoking structures similar to known ribozymes. A significant proportion of recombinant RNAs contained the entire 5' partner, if its 3' end was oxidized or phosphorylated prior to being mixed with the 3' partner. All of these observations are consistent with a mechanism that involves intermediary formation of the 2',3'-cyclic phosphate and 5'-hydroxyl termini. It is proposed that nonreplicative RNA recombination may contribute to evolutionarily significant RNA rearrangements.

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.

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.

Poliovirus neurovirulence correlates with the presence of a cryptic AUG upstream of the initiator codon.

Poliovirus mutants with extended (> 150-nt) deletions in the 5'-untranslated region between the internal ribosome entry site and the initiator codon have been selected previously (Pilipenko et al., Cell 68, 119-131, 1992; Gmyl et al., J. Virol. 67, 6309-6316, 1993). These deletions were transferred into the genome of a mouse-pathogenic poliovirus strain and found to be strongly attenuating. The deletions can be considered as covering three structural elements, a stem-loop (domain E) with a conserved cryptic AUG and two spacers, upstream and downstream of it. In an attempt to identify putative essential determinants of neurovirulence in these individual structural elements, appropriate mutants were engineered. The results demonstrated that neither of the above elements is essential for neurovirulence. The results strongly suggested that the presence of a cryptic AUG in the oligopyrimidine/AUG tandem followed, at a sufficient distance, by the initiator codon was necessary to ensure the neurovirulent phenotype of our constructs. On the other hand, the attenuated phenotype appeared to correlate with the occurrence of the initiator AUG as a moiety of the oligopyrimidine/AUG tandem. Possible mechanisms underlying these effects are discussed. Identification of the cryptic AUG as an essential determinant for neurovirulence provides a rational basis for the design of genetically stable attenuated poliovirus variants.

Geographical genotypes (geotypes) of poliovirus case isolates from the former Soviet Union: relatedness to other known poliovirus genotypes.

A 150 nucleotide long region corresponding to adjoining segments of the genes encoding polypeptides VP1 and 2A of 84 poliovirus strains recently isolated from patients with paralytic poliomyelitis over the territory of the former Soviet Union (FSU) were characterized by sequencing and/or PCR amplification using specially designed primers. Eighteen isolates were found to be very closely related to one or another of the three Sabin vaccine strains. Three distinct classes of geographical genotypes (geotypes) were discerned among 42 wild-type (non-Sabin) strains of serotype 1. One such geotype (called A) was widely circulating in 1990-91 in the Caucasian (Azerbaijan and Georgia) as well as Asian (Kyrgyzstan and Turkmenistan) Republics; this geotype exhibited only weak relatedness to known strains isolated outside the FSU. On the other hand, a subset of strains belonging to another geotype (T) of serotype 1, which circulated in 1991 in Tajikistan, demonstrated very close relatedness to contemporaneous strains isolated in Pakistan, India and Jordan. Strains that were somewhat different, but belonging to the same T-geotype, were found also in Moldova and Georgia. Strikingly, the primary structure of the VP1/2A junction of certain T-geotype isolates differed from the corresponding region of Sabin 1 only in 13-15% of positions, thereby not reaching the upper limit accepted for a geotype. This observation raises, though does not prove, the possibility that at least the relevant segment of the T-geotype RNA originated from the vaccine strain. The third geotype of serotype 1 was represented by a single, perhaps imported, isolate. Four distinct subsets of a common geotype (C) were discerned among 24 wild-type isolates belonging to serotype 3. These strains exhibited a broad geographical distribution being found, in particular, in Armenia, Azerbaijan, Georgia, Turkmenistan and Tajikistan; on the other hand, the C-geotype strains exhibited only a relatively distant relatedness to a strain isolated outside of the FSU (in Oman).

[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.

Towards development of an in vitro translation test for poliovirus neurovirulence.

We showed previously, using a cell-free system from Krebs-2 cells, that the genomes of the Sabin type 1 and 3 poliovaccine strains are poorly translated compared to RNAs of their neurovirulent progenitors. Here we show that similar or even greater differences in translation efficiencies of RNAs from attenuated and virulent poliovirus strains are exhibited by extracts prepared from HeLa cells. In addition, RNA of the Sabin type 2 vaccine is shown to be less efficient in translation that RNA of the revertant strain, 117, isolated from a case of vaccine associated paralytic poliomyelitis. Sabin-derived strains of all three serotypes selected for growth in the human gut were typically characterized by the increased translation efficiency of their genomes. The observed augmentation in translation efficiency correlated well with the occurrence of point mutations in the putative 5' cis-acting element controlling translation and neurovirulence. The mutations were shown to restore the base pairings, disrupted in the Sabin strains, in the predicted secondary structure of the control element. The results demonstrate the usefulness of the cell-free translation assay for differentiation of closely related poliovirus strains on the basis of their neurovirulence.

[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.

Frequent isolation of intertypic poliovirus recombinants with serotype 2 specificity from vaccine-associated polio cases.

Five representatives from a collection of 21 Sabin type 2-like poliovirus strains isolated from paralytic poliomyelitis cases in two regions of the USSR have been subjected to limited nucleotide sequencing. All proved to be intertypic recombinants having the genes encoding capsid proteins of Sabin 2 origin and a 3'-end portion of the genome derived from either type 1 (3 isolates) or type 3 (2 isolates) Sabin strains. The crossover points in all the 5 genomes have been mapped to different loci of the P3 region. At least 6 additional isolates from the same collection (and 2 isolates from healthy contacts), appeared to have a type 2/type 1 recombinant genome, as judged by oligonucleotide mapping. The biological significance of frequent occurrence of recombinants among field isolates of vaccine-related strains is discussed.

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.

[The molecular epidemiology of poliomyelitis: the characteristics of the strains isolated from patients in Moscow in 1973-1986]. / Molekuliarnaia épidemiologiia poliomielita: kharakteristika shtammov, vydelennykh ot bol'nykh v Moskve v 1973-1986 gg.

Eleven virus strains isolated from poliomyelitis patients in Moscow in 1973-1986 were analyzed by the method of oligonucleotide mapping of RNA. The genome of the isolates showed considerable similarity to the genomes of Sabin's vaccine strains and mainly to the vaccine strain of antigenic type 2. The conclusion was made that the sporadic cases of poliomyelitis registered in this region were etiologically linked with the vaccine strains of poliomyelitis virus. Only in one case the disease appeared in the recipient of the vaccine, in all other cases the patients were infected through contacts.

[Isolation of recombinant vaccine strains of poliovirus from patients with poliomyelitis]. / Vydelenie rekombinantov mezhdu vaktsinnymi shtammami poliovirusa ot bol'nykh poliomielitom.

Oligonucleotide maps of some poliovirus type 2 strains isolated from polio cases, while being clearly related to that of the Sabin vaccine type 2 strain, exhibited, nevertheless, marked differences from the reference (vaccine) map. Several large oligonucleotides derived from 4 such strains were subjected to enzymatic sequencing. The results strongly suggest that all of them were intertypic recombinants between the Sabin strains. The 5'-parts of the genomes of these strains were derived from the type 2 vaccine whereas the 3'-parts were of type 1 (in 2 strains) or type 3 (in other 2 strains) origin.

Distinct modes of poliovirus polyprotein initiation in vitro.

RNAs of poliovirus type 1 and type 3 were translated in extracts from Krebs-2 cells after annealing with oligodeoxyribonucleotides complementary to different sites in the 5'-untranslated region (5'-UTR). Due to a high level of endogenous RNase H activity in the extracts, such RNAs appeared to be efficiently 5'-truncated prior to translation. The observed levels of initiation on differently truncated templates suggested that a region in the middle of the poliovirus 5'-UTR is essential for the cap-independent initiation of viral polyprotein synthesis. The data reported here, in conjunction with the results from other laboratories, permitted to relate the essential cis-acting control elements to the 5'-UTR secondary structure domains defined previously (E.V. Pilipenko et al., Virology 168, 201-209). However, the removal of these domains activated another mode of polyprotein initiation, which appeared to require another set of translation initiation factors.

[Possible role of translation initiation factors in the regulation of expression of attenuated mutations of poliovirus]. / Vozmozhnoe uchastie faktorov initsiatsii transliatsii v reguliatsii ékspressii attenuiruiushchikh mutatsii poliovirusa.

Initiation of poliovirus RNA translation in reticulocyte lysates is mainly not precise, i.e. it occurs at the sites in the middle of the viral genome but not at the beginning of the polyprotein reading frame. The anomaly is due to the deficiency of translation initiation factors. Partial purification of the protein fraction stimulating the precise translation from the Krebs-2 cells is reported in the paper. This fraction, like the crude lysates factors, was considerably less active with the RNA of attenuated poliovirus strains of type 1 and 3 than with the RNA of virulent strains. The change in interaction of the specific segment of viral RNA with the translation initiation factors is suggested to contribute to the attenuated phenotype of the Sabin poliovirus strains.

Conserved structural domains in the 5'-untranslated region of picornaviral genomes: an analysis of the segment controlling translation and neurovirulence.

A model of secondary structure common for the central part (ca. 400 nucleotides) of the 5'-untranslated regions (5'-UTR) of all the so far sequenced genomes of polioviruses, coxsackieviruses, and rhinoviruses was derived on the basis of evolutionary and thermodynamic considerations. According to the model, this part of the genome comprises three domains, which appear to be involved, at least in the poliovirus genome, in the control of viral neurovirulence and in vitro translation. Some salient features of this model were supported by investigating RNAs of five poliovirus and one coxsackievirus strains with respect to their accessibility to modifications with dimethyl sulfate and sensitivity to single-strand- and double-strand-specific nucleases. In contrast to the previous suggestion, no major changes in the conformation of the Sabin vaccine poliovirus type 3 5'-UTR due to the transition in position 472 were observed. The biological relevance of the conserved primary and secondary structure elements in the picornaviral 5'-UTRs is discussed.